UNIVERSITÀ DEGLI STUDI DI TRIESTE
XXXIII CICLO DEL DOTTORATO DI RICERCA IN
AMBIENTE E VITA
Global changes in harsh environments: effects on plant species and communities from a trait- analysis perspective. Settore scientifico-disciplinare: BIO/03
DOTTORANDO STEFANO VITTI
COORDINATORE PROF. GIORGIO ALBERTI
SUPERVISORE DI TESI DOTT. VALENTINO CASOLO
CO-SUPERVISORE DI TESI DOTT. FRANCESCO BOSCUTTI
ANNO ACCADEMICO 2019/2020 - 2 - STATEMENT OF ORIGINAL CONTRIBUTION
This research thesis is an original contribution to the field of ecology and it is submitted as the final outcome of my Ph.D. project. It focused on linking plant responses to global climate changes to harsh environmental conditions, which determine remarkable species responses, from cell to community level, by means of physiological and morphological plant responses. I joined the Plant Biology Laboratory, Dipartimento di Scienze AgroAlimentari, Ambientali ed Animali (DI4A), University of Udine, Italy. My supervisor, Dr. Valentino Casolo, and co-supervisor Dr. Francesco Boscutti, always care for my work professionally, and their participation is revealed to be of fundamental relevance.
This dissertation contains the papers, and other related works, I produced during my Ph.D. program. According to my academic discipline (BIO/03 - ENVIRONMENTAL AND APPLIED BOTANY), it is divided into main works (Chapter 1, 2, 3, 4) and other minor works (n.2), as follow:
Chapter 1 Vitti, S., Pellegrini, E., Casolo, V., Trotta, G., & Boscutti, F. (2020). Contrasting responses of native and alien plant species to soil properties shed new light on the invasion of dune systems. Journal of Plant Ecology, rtaa052. doi: 10.1093/jpe/rtaa052; Chapter 2 Boscutti, F., Vitti, S., Casolo, V., Roppa, F., Tamburlin, D., & Sponza, S. (2019). Seagrass meadow cover and species composition drive the abundance of Eurasian wigeon (Mareca penelope L.) in a lagoon
- 3 - ecosystem of the northern Adriatic Sea. Ecological Research, 34(2), 320– 327. doi: 10.1111/1440-1703.1070; Chapter 3 Population density modulates the response of morphological traits and non-structural carbohydrates in Vaccinum myrtillus when exposed to different rain regimes. Original draft, not submitted. Chapter 4 Trifilò, P., Kiorapostolou, N., Petruzzellis, F., Vitti, S., Petit, G., Lo Gullo, M.A., Nardini, A. and Casolo, V. (2019). Hydraulic recovery from xylem embolism in excised branches of twelve woody species: Relationships with parenchyma cells and non-structural carbohydrates. Plant Physiology and Biochemistry, 139, 513–520. doi: 10.1016/j.plaphy.2019.04.013.
In addition to the above manuscripts, as previously said, other outputs have been presented at national - international conferences (for details see Appendix):
• Vitti, S., Boscutti, F., Pellegrini, E., & Casolo, V. (2018). Study of plant traits, soil features and plant invasion in sandy beach of Grado and Marano lagoon (northern Adriatic Sea). 113° Congresso della Società Botanica Italiana - V International Plant Science Conference (IPSC). abstracts keynote lectures, communications, posters. Università di Salerno, Fisciano (SA). 978-88-85915-22-0. abstracts keynote lectures, communications, posters. Fisciano (SA): Università di Salerno. • Vitti, S., Boscutti, F., Casolo, V., & Sponza, S. (2019). Seagrass - waterbirds interactions in a lagoon ecosystem of the northern Adriatic Sea. 114° Congresso della Società Botanica Italiana - VI International Plant Science Conference (IPSC). abstracts keynote
- 4 - lectures, communications, posters. Università di Padova, Padova. 978-88-85915-23-7. abstracts keynote lectures, communications, posters. Padova: Università di Padova;
- 5 - ACKNOWLEDGEMENTS
My most important thank goes to my supervisor, Dr. Valentino Casolo, and my co-supervisor, Dr. Francesco Boscutti, for being always helpful, professional, respectful, and full of wise advice, giving me a warm welcome to their research group. They taught me much about botany and ecology, also permitting me to discover fantastic unknown places during field activities.
I thank the staff of Friuli Venezia Giulia Region for the administrative support, Dr. Glauco Vicario, and Dr. Stefano Sponza for field logistic support.
I thank my office-mate, Antonio and Marco, particularly for recreational moments. They are really funny guys.
Last but not least, I thank my family for morale and mental support, and thanks to Sara, the person who was most able to understand me in difficult moments. With her, I understand that, after all, the obstacles exist to be overcome.
- 6 - - 7 - FOREWORD
Climate is the average of the weather conditions at a particular point on the Earth, and it is expressed in terms of expected temperature, rainfall, and wind conditions based on historical observations (Riedy 2016).
The Earth’s climate has always changed and the history of life on Earth is closely associated with environmental change on multiple spatial and temporal scales (Davis 2001).
The success of human societies depends mainly on the living components of systems (Pecl et al. 2017). In the case of environmental conditions changes, for marine, freshwater, and terrestrial species, the first response is often a shift in location, to stay within ecologically optimal environmental conditions (Chen et al. 2011; Lenoir and Svenning 2015). At the cooler extremes of their distributions, species are moving poleward, whereas range limits are contracting at the warmer range edge, where temperatures are no longer tolerable. On land, species are also moving to cooler, higher elevations; in the ocean, they are moving to colder water at greater depths (Pecl et al. 2017).
At a time when the world is anticipating unprecedented increases in human population growth and demands, the ability of natural ecosystems to deliver ecosystem services is being challenged by the largest climate-driven global redistribution of species, which is expected to influence climate feedbacks via changes in albedo, biologically driven sequestration of carbon from the atmosphere to the deep sea (the “biological pump”), and the release of greenhouse gases (Prentice et al. 2015).
- 8 - Species are affected by climate in many ways, including range shifts, changes in relative abundance, and changes in activity timing and microhabitat use (Williams et al. 2008; Bates et al. 2014) and the geographic distribution of any species depends upon its environmental tolerance, dispersal constraints, and biological interactions with other species (Peterson et al. 2011).
The race to save biodiversity is being lost, and it is being lost because the factors contributing to its degradation are more complex and powerful than those forces working to protect it (Wood et al. 2000).
- 9 - TABLE OF CONTENT
STATEMENT OF ORIGINAL CONTRIBUTION ______- 3 - AKNOLEDGEMENTS ______- 6 - FOREWORD ______- 8 - INTRODUCTION ______- 13 - Climate change and effects of plants in a changing precipitation scenario ______- 13 - Biological invasion: invasion mechanisms in harsh environments ______- 15 - Effects of biological interactions ______- 17 - THESIS’ AIMS ______- 19 - THESIS’S STRUCTURE ______- 20 - CHAPTER 1______- 23 - Contrasting responses of native and alien plant species to soil properties shed new light on the invasion of dune systems ______- 23 - CHAPTER 2______- 61 - Seagrass meadow cover and species composition drive the abundance of Eurasian wigeon (Mareca penelope L.) in a lagoon ecosystem of the northern Adriatic Sea ___ - 61 - CHAPTER 3______- 87 - Population density modulates the response of morphological traits and non-structural carbohydrates in Vaccinum myrtillus when exposed to different rain regimes _____ - 87 - CHAPTER 4______- 120 - Hydraulic recovery from xylem embolism in excised branches of twelve woody species: relationships with parenchyma cells and non-structural carbohydrates ______- 120 - CONCLUSIONS ______- 155 - APPENDIX ______- 159 - Study of plant traits, soil features and plant invasion in sandy beach of Grado and Marano lagoon (Northern Adriatic Sea) ______- 160 - Seagrass - waterbirds interactions in a lagoon ecosystem of the Northern Adriatic Sea_ - 164 - REFERENCES ______- 168 -
- 10 - “It is not the strongest of the species that survives, nor the most intelligent that survives. It is the one that is the most adaptable to change.”
Charles Robert Darwin (12 February 1809 – 19 April 1882)
INTRODUCTION
Climate change and the effects of plants in a changing precipitation scenario
Although it appeared on Earth relatively recently, man has affected most of the biosphere by means of its activities. Since the beginning of the industrial revolution, in England in the late 18th century, pollution due to human activities has been steadily increasing across the world. After the Second World War, there was an acceleration of this phenomenon: technological development has led to the introduction of new materials and production processes, and many of which have led to new eco- toxicological risks. Climate change is the persistent change in the weather pattern, and one of the major drivers of climate change is global warming (Mgbemene 2011), mainly caused by the emission of greenhouse gases such as carbon dioxide
(CO2), methane (CH4), and nitrous oxide (N2O), chlorofluorocarbons and other chemicals into the atmosphere (Hecht 2007). Climate change is widely acknowledged as a primary environmental problem of the Planet (Zhai and Helman 2019). According to a study of the Intergovernmental Panel on Climate Change (IPCC), in the time interval between 1970 and 2004, greenhouse gas emissions on Earth rose 70% (IPCC 2007). The climate has been consequently warming, with a temperature increment of 0.85 (0.65–1.06) °C throughout 1880–2012 (IPCC 2013), leading to several alterations in each climate region: in the Arctic region, warming results generate dramatic melting of glaciers and ice sheets (Yan et al. 2014) and the reductions in
- 13 - Arctic snow and ice have contributed to the Arctic amplification, which corresponds to a warming rate more than twice that in the other regions (Manabe and Wetherald 1975); in the Amazon region, more than 20% of the rainforests has been destroyed in the past decades (Davidson et al. 2012), causing changes in hydroclimatic regimes (von Randow et al. 2004; D’Almeida et al. 2007; Lawrence and Vandecar 2015). In the ‘80s deforestation caused thermally triggered atmospheric circulations(Roy 2002), with an increase in regional cloudiness (Negri et al. 2004) and precipitation frequency (Chagnon 2005); in West Africa (Niger, Senegal, and Gambia) climate changes influenced evapotranspiration rates, temperatures and rainfall regimes (Abdelkrim 2013), with a consequent impact on water availability given by a reduction in rainfall (Mahé and Olivry 1995). In Europe, the Mediterranean basin is one of the most vulnerable climate change hotspots (Giorgi 2006), indeed it responds quickly to atmospheric forcings (Lionello 2012). In terms of the thermal regime, according to the IPCC’s model, the base scenario from 1980-2000 was used to estimate an increase in average surface temperatures (2.2-5.1 °C) in this century (2080- 2100). For the same period, the models indicate rainfall regime and estimate that precipitation over lands might vary between -4% and -27%. Many studies existing on the consequences that climate change could produce on species distribution and habitat. All this research indicates that a serious alteration of biological and ecological patterns in both marine and terrestrial biomes is already taking. As above reported, the Arctic region has warmed, in the last decades and the warming ratio is more than twice of the rest of the Earth (IPCC 2013; Post et al. 2019): as the Arctic warms, vegetation is responding. One of the most recent reports from the IPCC (Intergovernmental Panel on Climate Change
- 14 - et al. 2015) state that, tundra vegetation changes in response to the warming effect, and this change (called ‘greening of the Arctic’) was recognized as the world’s most important ecological responses to global climate change, on large-scale (Myers-Smith et al. 2020). Arctic vegetation is mainly composed of life forms more adapted to harsh conditions, e.g. dwarf shrubs, that replaced trees at high latitudes or high elevations. This vegetation replacement is highlighted by the treeline; it can be interpreted as the boundary of the forest, although it is usually not a distinct line (Grace 2002). It is so ecologically relevant to be monitored in every part of the Earth (Tranquillini 1979; Beneke and Davis 1980; Alden et al. 1993; Holtmeier 2000). In consideration of this, the study of plant response, both from a morphological and physiological point of view, to several and extreme environmental stresses may be crucial in forecasting further climate scenarios (Filippi et al. 2020).
Biological invasion: invasion mechanisms in harsh environments
The homogenizing force of globalization has triggered a massive spread of species abroad of their native ranges (van Kleunen et al. 2015). Many plant species have become naturalized in new areas. Some of these species have become invasive (Giulio et al. 2020). Invasive species can threaten biological diversity by reducing genetic variation and eroding gene pools (Hulme 2007), causing impacts on plant biodiversity and ecosystem functioning (Vilà et al. 2010; Blackburn et al. 2019). Finally, biological invasions complicate the conservation of biodiversity and ecosystem integrity all around the world (Vilà et al. 2010).
- 15 - Plant invasions are known to be favored by humans, both directly (species introductions) and indirectly (anthropogenic alterations of the environment) (Thuiller et al. 2006; Pysek et al. 2010). In Europe, the increase of human activities, especially in coastal areas, caused habitat loss (Heslenfeld et al. 2008) and also the introduction of many alien plants (Campos et al. 2004a; Carboni et al. 2010). In terms of biodiversity of species and habitat, coastal areas are globally considered among the most valuable but endangered environments, due to their susceptibility to global changes (Defeo et al. 2009). Open sandy coasts comprise up to 40% of the world's coastline (Bird 1996) which is subject to a very high level of human utilization. Sandy coasts can be divided into mainland and barrier island coasts, with some operating physical processes being common to both types of coasts (e.g. storm erosion, coastline recession, spit/barrier breaching due to elevated water levels) and some others being specific to one coast type or the other (e.g. seasonal closure of small tidal inlets on mainland sandy coasts; barrier rollover on barrier island coasts) (Ranasinghe 2016). Among coastal systems, dune has been already proved to be particularly prone to biological invasion (Campos et al. 2004a; Giulio et al. 2020), which leads to major shifts in biodiversity, ecosystem integrity, functions, and services (Vilà et al. 2011; Simberloff et al. 2013). Projected climate change driven variations in storm surge, mean sea level, wave conditions, and river flow will affect the coastal zone in many ways (Nicholls and Cazenave 2010; Field et al. 2014; Cazenave and Cozannet 2014). As the coastal zone is the highly populated and developed land zone in the world (Small 2003), any negative physical impacts of climate change on the coastal zone are certain to have socio-economic impacts on a global
- 16 - scale (Kron 2013; Hinkel et al. 2013; Johnson et al. 2015; Brown et al. 2016). Coastal dunes, occupying transition zones between terrestrial and marine ecosystems, constitute one of the most dynamic landscapes on earth (Carranza et al. 2008; Carboni et al. 2009). In particular, Mediterranean coastal dunes host a highly specialized flora (European Commission. Directorate General for the Environment. 2016) along a well-known vegetation zonation which is similar to the Mediterranean coastline (Acosta et al. 2009). During recent decades along the Mediterranean basin, outbound tourism, the expansion of urban areas, and the spread of agriculture have strongly shaped coastal landscapes (Hesp and Martínez 2007; Malavasi et al. 2013).
Effects of biological interactions
To predict an ecological response to climate change we base our studies on direct climatic effects on species (Suttle et al. 2007) and the impacts of recent climate change on life on Earth are already evident. Both biotic factors, i.e. species properties and interactions, and abiotic factors, i.e. climate and soil characteristics, may affect ecosystem structure and their processes (Loreau 2001) and shape community composition (Bray et al. 2019). Global warming can lead to a modification in abiotic conditions that influence plant performance, with a particular inclination of alpine and arctic ecosystems to be affected (Callaghan and Jonasson 1995) and it may alter soil moisture and nutrient availability. Species interactions link them in space and time and lead to the development of ecological communities (CaraDonna et al. 2020), and this phenomenon
- 17 - can vary through time at multiple scales (McMeans et al. 2015; Trøjelsgaard and Olesen 2016).
- 18 - THESIS’ AIMS
This Ph.D. work aimed to investigate the effects of global changes, analyzing the effects of changing climatic factors, to plant ecosystems achieving precise information to understand and better explain global changes in harsh environments, searching for a link between plant metabolism and communities. Another aim was to add knowledge on how plants respond to extreme climatic treatments. The contribution of the following reported article gives help in understanding how changing climatic factors and environmental parameters can affect plant ecosystems.
Two different approaches were applied: i) morphological and physiological plant traits analysis, ii) and plant communities’ analysis
Under ongoing climate changes, the biological invasion is one of the major threats to world biodiversity, and ecosystem integrity, functioning, and services (Vilà et al. 2010; Simberloff et al. 2013); also non-structural carbohydrates (NSCs) play key roles in plant responses to drought and frost stress (Tomasella et al. 2019).
- 19 - THESIS’S STRUCTURE
This dissertation is composed of 4 chapters: Chapter 1 Vitti, S., Pellegrini, E., Casolo, V., Trotta, G., & Boscutti, F. (2020). Contrasting responses of native and alien plant species to soil properties shed new light on the invasion of dune systems. Journal of Plant Ecology, rtaa052. doi: 10.1093/jpe/rtaa052; this is the last submitted contribute to plant ecology and alien species. Plant communities mainly, and also soil cores, were analyzed along the sea – inland - saltmarsh gradient. Chapter 2 Boscutti, F., Vitti, S., Casolo, V., Roppa, F., Tamburlin, D., & Sponza, S. (2019). Seagrass meadow cover and species composition drive the abundance of Eurasian wigeon (Mareca penelope L.) in a lagoon ecosystem of the northern Adriatic Sea. Ecological Research, 34(2), 320– 327. doi: 10.1111/1440-1703.1070; this is a contribution to plant – animal interaction, where we studied the interactions between primary producers and consumers, which plays an important role in the conservation of sensitive ecosystems such as lagoons. Chapter 3 Population density modulates the response of morphological traits and non-structural carbohydrates in Vaccinum myrtillus when exposed to different rain regimes. Original draft, not submitted. We analyzed the relationships between NSC, plant traits, rain exclusion, and shrub density to determine the response of alpine Vaccinium myrtillus stands to climate variation. Chapter 4 Trifilò, P., Kiorapostolou, N., Petruzzellis, F., Vitti, S., Petit, G., Lo Gullo, M.A., Nardini, A. and Casolo, V. (2019). Hydraulic recovery from xylem embolism in excised branches of twelve woody species: Relationships with parenchyma cells and non-structural
- 20 - carbohydrates. Plant Physiology and Biochemistry, 139, 513–520. doi: 10.1016/j.plaphy.2019.04.013; the last one is a contribution to plant physiology, from a collaboration between Prof. Patrizia Tifilò, University of Messina, and my supervisor, Dr. Valentino Casolo. We investigated the embolism repairation ability on 12 broadleaved species differing in vulnerability to xylem embolism.
In addition to the above manuscripts, other outputs have been produced (for details see Appendix).
- 21 - - 22 - CHAPTER 1
Contrasting responses of native and alien plant species to soil properties shed new light on the invasion of dune systems
Stefano Vitti1,2,*, , Elisa Pellegrini2,3, Valentino Casolo2, Giacomo Trotta2 and Francesco Boscutti2, 1Department of Life Sciences, University of Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy, 2Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, via delle Scienze 91, Udine 33100, Italy, 3Department of Biology, University of Copenhagen, Universitetsparken 4, 2100 København Ø, Denmark *Corresponding author. E-mail: [email protected] Handling Editor: Bin Zhu Citation: Vitti S, Pellegrini E, Casolo V, et al. (2020) Contrasting responses of native and alien plant species to soil properties shed new light on the invasion of dune systems. J Plant Ecol XX:XX–XX. https://doi.org/10.1093/jpe/rtaa052
Abstract Aims Among terrestrial ecosystems, coastal sandy dunes are particularly prone to alien plant invasion. Many studies related the invasion of dune habitats to anthropic causes, but less is known about the role of soil properties and plant traits in plant invasion. In this study, we tested the relationships between soil features and alien plant invasion in dune systems,
- 23 - focusing on the interplay between soil nutrients, soil salinity and plant functional traits. Methods Study sites were sandy barrier islands of the Marano and Grado lagoon (northern Adriatic Sea). One hundred plots of 16 m2 have been randomly selected in three habitats (foredune, backdune and saltmarsh). In each plot, we recorded all plant species occurrence and abundance and we collected a soil core. For each soil sample, soil texture, conductivity (as proxy of soil salinity), organic carbon and nitrogen content were analysed and related to the species number and cover of native and alien plants. Variation of main reproductive and vegetative functional traits among habitats was also analysed for both alien and native species. Important Findings Soil properties were strongly related to overall plant diversity, by differently affecting alien and native species pools. In backdune, the most invaded habitat, a high soil conductivity limited the number of alien species, whereas the content of soil organic carbon increased along with alien plant abundance, suggesting also the occurrence of potential feedback processes between plant invasion and soil. We found a significant convergence between native and alien plant functional trait spectra only in backdune habitat, where environmental conditions ameliorate and plant competition increases. Our findings suggest that in harsh conditions only native specialized plants can thrive while at intermediate conditions, soil properties gradient acts in synergy with plant traits to curb/facilitate alien plant richness. Keywords: invasive alien species, functional traits, soil nutrients, plant communities, dune system
- 24 - Introduction Coastal areas are globally considered among the most valuable but endangered habitats due to their susceptibility to global changes (Defeo et al., 2009). Among coastal systems, dune has been already proved to be particularly prone to biological invasion (Campos et al., 2004; Giulio et al., 2020), which leads to major shifts in biodiversity, ecosystem integrity, functions and services (Vilà et al., 2010; Simberloff et al., 2013). In dune systems, many studies focused on the relationships between invasive plants and regional environmental variables (Marcantonio et al., 2014; Malavasi et al., 2014; Tordoni et al., 2018; Marzialetti et al., 2019). However, while land-use and climate are considered pivotal at large-scale (i.e. landscape), variations of soil and stand structure might prevail when considering the local spread of alien species in semi-natural habitat (Ohlemüller et al., 2006). Soil features are supposed to directly affect the success of exotic plants in introduced habitats (Carvalho et al., 2010). Among soil properties, nutrient content and organic matter are known as important determinants of plant community diversity (Chapin III et al., 1986). For instance, high levels of soil nitrogen increase the abundance of invasive alien plants and decrease plant diversity (Vitousek et al., 1997). On the other hand, some invaders can trigger cascading effects on ecosystem proprieties by altering nutrient cycles (Boscutti et al., 2020). In coastal systems, soil salinity is the major driver of species distribution (Donnelly and Pammenter, 1983; Gorham, 1992; Lortie and Cushman, 2007). Nonetheless, the effect of salinity on the distribution of invasive species was studied only for few specific alien taxa, focusing on their phenotypic response to salt stress (Ishikawa et al., 1991; Caño et al., 2016). Abiotic site conditions can influence ecosystem processes both directly, by determining environmental stress conditions, and indirectly, through the
- 25 - functional response of plants (Boscutti et al., 2018a; Pellegrini et al., 2018; Bu et al., 2019). In turn, the range of functions provided by a plant community is thought to largely depend on the diversity of functional traits (Dı́az and Cabido, 2001), expressed as global variability of functional traits (functional spectrum). For these reasons, plant functional traits have been proved to be pivotal in elucidating plant invasion success (Rejmanek and Richardson, 1996; Boscutti et al., 2018b). In particular, plasticity of plant traits are supposed to affect the success of alien plants (Davidson et al., 2011) by producing a divergence/convergence of plant traits in response to the invaded habitat conditions (e.g. Marchini et al. 2019). In fact, invasive species are supposed to be more tolerant to environmental stresses (Alpert et al., 2000; Antunes et al., 2018), showing a general higher phenotypic plasticity than native species (Feng et al., 2007; Raizada et al., 2009). In this observational study, we aimed at parsing the relationships between native and alien plants and soil properties (i.e. soil conductivity, soil nutrients) in three main habitats of coastal dune systems, i.e. foredune, backdune and saltmarsh. In addition, the variation of important reproductive and vegetative functional traits for both alien and native species were considered. In particular, we hypothesized that: (i) low soil conductivity (i.e. less salt content) favours the abundance and richness of alien species in each considered habitat; (ii) soil nutrients increase along with alien species abundance; (iii) alien and native species functional spectra differ according to the considered habitat and its level of invasion.
- 26 - Materials and methods Study site and plant communities The study sites were the barrier islands of the Marano and Grado lagoon (from 45°42’10.5’’N 13°9’17.8’’ E to 45°40’49.8’’N 13°21’31.2’’ E), located in the northern part of the Adriatic Sea (Friuli Venezia Giulia region, Italy) (Fig. 1.1a-b). The lagoon is included in the Natura 2000 network, recognized both as Special Area of Conservation (SAC) and Special Protection Area (SPA). The mean annual rainfall is 974 mm. The driest month is July. The mean lowest temperature is in January; with a value of 3.1°C; the mean highest temperature is in July, with a value of 29.0°C. Wind average speed ranges from 7.8 to 10.4 km/h. The four main barrier islands of the Lagoon considered for the present study were: (from W to E) Martignano (length 1.8 km, maximum width 0.8 km), Sant’Andrea (length 5.3 km, maximum width 0.7 km), Bocca d’Anfora (length 3.1 km, maximum width 0.7 km) and Banco d’Orio (length 4.9 km, maximum width 0.2 km). Each barrier island represents a dynamic system produced by the interaction between tidal movements and alongshore sediment transport, where both dune and halophile systems coexist. In this study, three main habitats were surveyed, namely: foredune, backdune and saltmarsh, which reflect the zonation of the vegetation along the environmental sea–inland gradient (Fig. 1.1c). Foredune, the nearest habitat to the shoreline, represent the first colonized part of the sandy shore, encompassing highly dynamic communities, strongly shaped by winter wash over events. Backdune is more stable, less subjected to winter storms and shows a greater ecological complexity when compared to foredune. On the lagoon side, the influence of tide produces the establishment of saltmarsh habitats. Saltmarsh are a major, widely distributed, intertidal habitat which differs from dunes for soil salinity and soil texture (larger presence of clay),
- 27 - and consequently on soil nutrient availability. Despite many saltmarshes are estuarine, they can also be found associated with barrier islands, usually due to wash over events that brake the sand barrier and leads to the deposit of fine soil particles just behind dunes (Fontolan et al., 2012).
Sampling design Along the islands shore we identified a total of 10 areas (ca. 5 ha each). Each area represented the described ecological gradient including the 3 main habitats of our interest (i.e. foredune, backdune and saltmarsh). Ten points were randomly selected within each of the 10 previously selected areas (see Fig. 1.1c as example), giving overall 100 points: 32 points for foredune, 40 for backdune and 28 for saltmarsh. In each point, plant communities and soil were surveyed.
Data collection Plant community At each point, a sample area (plot) of 16 m2 (4x4 m) was established. Within each plot we recorded the occurrence and abundance (cover percentage) of the vascular plant species. Species nomenclature follows Bartolucci et al. (2018) and Galasso et al. (2018) for natives and aliens, respectively. Native and alien status of the species were assigned according to Buccheri et al. (2018). Out of the considered literature, we selected 5 functional traits supposed to be highly sensitive to the analysed environmental gradient and putatively related to the success of invasive species in the studied area. The selected functional traits were specific leaf area (SLA), seeds mass, mean length of flowering period, mean flowering month and root depth. Data were derived from Landolt et al. (2010) and Kleyer et al. (2008).
- 28 - Soil Within each plot, a soil sample was collected using a cylindrical tube (height: 12 cm, width: 3.5 cm, volume: 115.5 cm3), transported to the lab and stored at 4°C. Soil samples (n=100) were afterwards homogenized and divided into two aliquots. The first aliquot was air-dried, sieved at 2 mm and ball-milled for the further chemical analysis of soil organic carbon (C) and nitrogen (N), while the second aliquot was stored at 4°C in plastic bags for the analyses of conductivity and granulometry. Soil organic carbon and nitrogen content were measured on a set of subsamples using a CHNS Elemental Analyser (Vario Microcube © Elementar). Before analysis, all soil samples were treated with HCl to remove the carbonate fraction. Conductivity was measured in 5:1 extract using about 10 g of dry soil and 50 mL of water. The solution was shacked for 2 hours and filtered using a Whatman n°42 filter paper. Conductivity was measured in the filtered solution using the CM35+ portable conductivity meter (Crison). The Bouyoucos method was applied to determine granulometry. A small amount of soil was used to determine soil humidity. About 50 g of corresponding dry soil was treated with 100 ml of sodium hexametaphosphate (SHMP). The extract (1:2) was shacked for 2 hours and then pour in a Bouyoucos’ cylinder, where density was measured with the hydrometer ASTM 152H after 4 minutes and 2 hours (silt plus clay and only clay, respectively).
Data analysis All statistical analyses were performed with the statistical software R 3.4.4 (R Team, 2019).
- 29 - For each plot, we calculated species richness (number of species) and abundance (sum of species cover) of the overall, native and alien pools of species, respectively. Since the ranges of native and alien species richness and abundance were very different, we standardized species richness and abundance (z-scores) within each status level. Differences between habitat in terms of overall and status-pooled species richness were tested using linear mixed-effects models (LMMs), considering the id of the 5 ha surveyed area (i.e. area id) and sub replicates for the status (i.e. plot id) as random effects. A Tukey pairwise test was then applied to detect significant differences between habitat and status (native or alien) interaction (p<0.05). LMMs were applied with the “nlme” package (Pinheiro et al., 2019), pairwise comparisons were performed with the ‘multcomp’ R package (Hothorn et al., 2008). We used Multi-Model Inference (Barton, 2015) to evaluate the influence of soil on standardized species richness and abundance of aliens and natives, respectively, within the different habitats (Burnham and Andreson 2002). Preliminarily, we analysed the correlation (Pearson test) between all the soil features measured (see online Supplementary Material, Fig. S1.1). As the correlation between all soil granulometry term (i.e. sand, silt and clay content %) and soils conductivity was high (r > |0.75|, p < 0.001), we used only soil conductivity in further analyses to avoid collinearity issues (Dormann et al., 2013). We used LMMs to estimate model parameters as model residuals did not violated any linear model assumption. We further tested the performance of Generalized Mixed-Effects Models with Poisson distribution but models residuals were worst. Models included standardized species richness or abundance as response variable and species status (i.e. aliens or natives), habitat type, and main soil features (i.e. soil carbon and nitrogen content, soil conductivity) and their interaction with habitat and status as fixed effects.
- 30 - The random effects of the 5 ha surveyed area (i.e. area id) and sub replicates for the status (i.e. plot id) were included. Given the non-linear relationship between independent variable and dependent variables, the models were linearized by logarithmic transformation as best solution after considering the inclusion of a quadratic term. Multi-model inference compared the fit of all possible models obtained by the combination of the variables. We used Akaike’s information criterion (AIC) to choose the best fitting model. The best fit is indicated by the lowest AIC value (AIC MIN). In a set of models each model i can be ranked using its difference in AIC score to the best- fitting model (Δ AICi = AICi- AICi MIN). A model in the set can be considered plausible if its Δ AIC is below 2 (Burnham and Anderson 2002).The multi-model inference based on AIC was executed using the ‘MuMIn’ package (Barton, 2015). The LMMs were applied using the “nlme” package (Pinheiro et al., 2019). Functional traits variation was assessed using a multivariate approach testing the differences between habitat, species status and their interaction in terms of traits homogeneity. Homogeneity of traits was tested calculating the distance between centroids (‘variation’ of beta diversity) and testing for homogeneity of multivariate dispersion between habitats and species status (i.e. alien vs native). This method produces an independent dissimilarity value for each sample, distance to group centroid (Anderson et al., 2006). Differences in mean trait distances were tested using PERMANOVA on the distance matrices ran with 999 permutations. Analyses of traits variation was performed using the “vegan” R package (Oksanen et al., 2019), considering the Euclidean distance applied to standardized traits values (z-scores) as distance metric.
- 31 - Results Plant diversity, habitat and alien plant invasion The total number of species within the 100 surveyed plots was 97 (73 native and 24 alien) (Appendix S1.1). The most common native species were Cakile maritima Scop., occurring in 38% of the overall number of plots, Elymus acutus (DC.) M.A. Thiébaud (29 %), Limonium narbonense Mill. (28 %) and Limbarda crithmoides (L.) Dumort. (28%). Among the alien species, the most frequent were Sporobolus pumilus (Roth) P.M. Peterson & Saarela (50 %), Xanthium italicum Moretti (35 %), Ambrosia psilostachya DC. (34%) and Oenothera biennis L. aggr. (30 %). The average species number found in each plot was 7.8 ± 3.1 (mean ± standard deviation). Species richness was significantly different among habitats (F2-88 = 8.34, p < 0.001), where foredune (7.7 ± 3.23) and backdune (9.2 ± 2.7) had significant higher values than saltmarsh (5.7 ± 2.3) (p < 0.05). A significant interaction was found between habitat and status (i.e. alien vs native) (F2-97 = 18.7, p < 0.001). Differences between native and alien standardized species richness (hereafter species richness) were significant in saltmarsh and backdune where native species showed higher values (Fig. 1.2).
Relationships between plant invasion and soil features Multi-Model Inference analysis showed that species richness of alien and native plants were related to soil features by only one plausible model (Δ AIC < 2), which included soil conductivity, soil nitrogen content and the interactions with species status and habitat for the conductivity, and with habitat, for the soil nitrogen (Table 1.1; R2=0.51). Soil conductivity affected alien and native species number in relation to the habitat (Fig. 1.3a-f). In foredune, plant species number was positively related
- 32 - with soil conductivity, native species showed a stronger increase respect to aliens (Fig. 1.3a). In backdune, native species number increased when conductivity increased, whereas alien species number decreased (Fig. 1.3b). In saltmarsh, the number of specie was not correlated with soil conductivity (Fig. 1.3c). High values of nitrogen content in the soil increased the overall number of species in foredune, while decreasing it in saltmarsh (Fig. 1.3d). Finally, in backdune, the number of both native and alien species was not affected by soil nitrogen (Fig. 1.3e). We also analysed the effect of conductivity, soil organic carbon and total nitrogen on the abundance of species (species overall cover) in relation to each status and habitat. Multi-model inference analysis showed that only one model was plausible (Δ AIC < 2) and it included all considered interactions (Table 1.2), explaining the 48% of the total variation in species abundance. High soil conductivity favored native species abundance in both foredune and backdune (Fig. 1.4a, b), whereas plant cover was not related to soil conductivity in saltmarsh (Fig. 1.4c). Soil organic carbon content was positively related to alien overall cover in both foredune and backdune, whereas native plant abundance decreased with increasing content of soil organic carbon, but only in backdune (Fig. 1.4e). In saltmarsh, species abundance was not affected by soil organic carbon content (Fig. 1.4f). Soil nitrogen showed negligible effects on species cover in foredune and saltmarsh (Fig. 1.4g, i). In backdune there was a considerable increase of native species cover, whereas alien cover slightly decreased in nitrogen rich- soils (Fig. 1.4h).
- 33 - Functional convergence We found a significant difference between native and alien plant functional trait spectrum (distance) (PERMANOVA: r2 = 0.09, p = 0.001), in different habitats (r2 = 0.14, p = 0.001) and their interaction was significant as well (r2 = 0.06, p = 0.001). Interestingly, we found a functional convergence (similarity) between alien and native species pool only in backdune habitat, which was also the most invaded. On the other hand, functional traits variation (dispersion) did not differ between species status, habitat and their interactions (p > 0.05) (see online Supplementary Material, Fig. S1.2).
Discussion Our findings suggest that plant invasion of dune systems is related to soil properties, whose nutrients and conductivity (i.e. salinity) showed contrasting relationships with plant diversity and abundance in the studied habitats across the sea-inland gradient. Most invaded habitat was backdune, where its highest overall biodiversity was also sustained by the high level of invasion. In backdune, abundance and richness of alien species were more affected by soil features than in the other habitats, also probably due to a less intensive disturbance regime (weaker wash over influence). In general, soil conductivity determined the species richness of both alien and native species, while soil organic carbon and nitrogen were related to their abundance. In backdune, a significant functional convergence was also found, which is probably consistent to a greater habitat stability which also contributed to ameliorate the general ecological conditions of these communities. Here, in less salty soil, the overlap of studied traits (reproductive and growth traits) between native and alien species suggests that competition of aliens consist in substituting native species rather than filling the empty ecological space (niche differentiation).
- 34 - Plant diversity, habitat and alien plant invasion Alien species represented the 25% of the total species richness in the studied dune system, much higher than the frequency of alien taxa at regional (16%) and national (12%) scale (Galasso et al., 2018); hence confirming that coastal dunes are one of the most invaded habitat by neophytes at the European level (Chytrý et al., 2008). Saltmarsh showed an overall low plant diversity, similar to what found by Kunza and Pennings (2008), but also harbored a low number of alien species. In this habitat, flooding, sediment anoxia and salt create extremely harsh conditions (Redelstein et al., 2018), difficult to cope for generalist plants. Only few adapted species thrive in this habitat and their response to the environment can indirectly ameliorate pant community condition and overall plant diversity (Redelstein et al., 2018; Pellegrini et al., 2018). We found harsh condition of saltmarsh to limit plant invasion. In contrast, backdune had concurrently a high number of alien and native plants. In this habitat, the interplay between stability and disturbance of habitat (wash over events) might explain the high values of invasion. In fact, when dune habitats are subject to naturally induced disturbance (e.g. winter storms) they harbor a large number of alien species (Del Vecchio et al., 2015). On the other hand, the particularly extreme conditions given by natural events (e.g. seashore nearness, high wind speed, sand storm, shore erosion) occurring in foredune (Perumal and Maun, 2006; Ciccarelli, 2014) allow only few characteristic native species to colonize the habitat, e.g. Cakile maritima and Salsola kali (Debez et al., 2004). Backdune showed concurrently high richness of both native and alien species. This could be related to a weaker influence of direct stressors (distance from the sea) which ameliorate the environment conditions, becoming adequate also for generalist species coming from other
- 35 - habitats such as inland agricultural land-use and semi-natural grasslands (Marcantonio et al., 2014).
Relationships between plant invasion and soil features Among the analysed soil features, soil conductivity (i.e. salinity), organic carbon and nitrogen content showed significant relationships with plant species abundance and diversity, with contrasting trends between alien and native plants, especially in backdune habitat. Soil conductivity have been proved to affect plant distribution in coastal dunes (Ishikawa et al., 1995) and altering the interactions between native and alien species (Wang et al., 2006). Moreover, many alien species are supposed to be less competitive than native species in relation to salt stress (Mesléard et al., 1993; Borgnis and Boyer, 2016). Salt content create harsh condition principally by reducing water availability and increasing osmotic stress. On sandy soils, this is exacerbated by the high soil porosity and thus water drainage. In these conditions, native adapted species thrive better than alien (Antunes et al., 2018). In contrast, a previous study showed that alien species richness increased in sites with high rainfall, as a consequence of a higher soil water availability and salt leaching. Soil nitrogen content increased along with the overall number of species found in foredune, whereas it was not influent in the other habitats. Despite several studies linked a reduction of plant diversity to nutrient concentration and hence ecosystem productivity a consistent pattern among terrestrial ecosystem is lacking (Tilman et al., 1997; Fridley, 2001). In foredune, the average nitrogen content was very low. In such conditions, it is plausible that an increase of nutrients availability allow to a higher number of species to colonize the bare soil, whose occurrence can also indirectly increase the soil organic matter (positive feedback).
- 36 - We found that nitrogen and organic carbon soil content (i.e. overall representing also organic matter content) were mainly involved in explaining plant abundance of alien and native species. The relationships between nitrogen and carbon soil content and species abundance were stronger in backdune, while in the harsher environments the other drivers (e.g. waterlogging, salinity, wave action, storms disturbance) seem to overrule on such effects. In backdune, a high content of nitrogen increased along with the abundance of native species, but not with the abundance of alien species. It is thought that plant invasion is favored by high content of nitrogen, triggering positive feedback between plant invasion and carbon and nitrogen cycles in invaded ecosystems (Liao et al., 2008). Our findings, instead, support the idea that in very poor soils native rather than alien species can intercept such nutrient resources and increase their abundance. However, we cannot exclude the aftereffect of the overall plant abundance (biomass) accumulating in a more stable habitat less subjected to wash over events. In contrast, soil organic carbon was positively related to the increase of alien species abundance in backdune. The content of organic carbon had a great effect on other soil properties and structure, altering microbial activity (Sparling, 1992) and water cycle. Our study suggests that a high content of soil organic carbon might ameliorate soil structure and nutrients-water availability (the more organic matter is present in the soil, the more water is kept and nutrients less leached) thus favoring the growth of generalist alien species. Nonetheless it is also true that a higher abundance of plants, and in particular of some invasive alien species might be the cause of the increase in soil organic carbon (Boscutti et al., 2020), which can accumulate in backdune, less subjected to wash over. In the light of the observational nature of the study it was not possible to definitely define a cause-effects
- 37 - mechanism between soil and plants, opening future experimental perspectives. Functional convergence Plant trait analysis is a methodological approach to better understand the processes linked to alien species invasion (Richardson et al., 2000; Richardson and Pyšek, 2006). Stressed plant communities are mainly ruled by habitat filtering, whereas functional convergence is mainly related to this ecological process (Cornwell et al., 2006). When different plant species are co-existing in the same community, it is usual for species to show some morphological and functional similarities (Grime, 2006; de Bello et al., 2009). Environmental filtering can contribute to community’s similarity and the result of this process is functional redundancy for species traits inside a community (Cornwell et al., 2006). Our findings suggest the presence of an overlap of niche between native and alien species and a potential substitution of native species in the most invaded habitat, namely the backdune. Kowarik (2008) explains that changes in environmental factors can generate a more efficient niche invasion by alien species, rather than favor native species. In fact, this mechanism is known to be more effective where environmental conditions are not extremely harsh (Carboni et al., 2010). In these conditions, we can hypothesize that less specialized alien species are favored to invade dune ecosystem due to ecosystem characteristic which present a higher similarity to human-disturbed habitats (e.g. urban sites or agricultural) where the alien species commonly thrive (Kowarik, 2008). In foredune and saltmarsh, instead, the analyses of trait variability suggest that alien and native species have separated functional spectra, suggesting that here alien species are filling empty ecological niches rather that replace native ones. Given the uniqueness of the environmental niches, plant species
- 38 - evolved to specialized species and only few alien species are suitable to colonize these areas (Marcantonio et al., 2014).
Conclusions Our findings suggest that main soil properties and plant functional traits are related to the plant invasion across the shore-saltmarsh gradient in barrier islands. The initial hypotheses were supported by our results, showing that soil conductivity curb both abundance and specific richness of alien species, favoring the presence of native species. Moreover, nitrogen and organic carbon in soil were related to plant with particular regard to plant abundance, underpinning plausible feedback mechanisms between plant and soil which understanding would need specific experimental approach. The magnitude of the effect is habitat specific: while backdune are the most sensitive habitat, foredune and saltmarsh were mostly unaffected by plant invasion and regardless to soil feature gradients. Finally, the higher invasion of backdune was consistent with a functional convergence between alien and native species pool. Our results highlight that even though coastal systems are reported to be among the most invaded habitats by neophytes (Chytrý et al., 2008), some plant communities may be much more affected by invasion than others, representing real conservation priorities. In these habitats, further experiments concerning the changes/manipulation of soil conductivity and soil nutrients could shed new perspectives in limit ecosystem deterioration in terms of overall biodiversity and invasiveness.
- 39 - Funding This work was supported by Regione Autonoma Friuli Venezia Giulia and University of Udine [grant number Morphological and environmental study of the Marano and Grado Lagoon CUP D26D14000230002].
Acknowledgements We thank the staff of Friuli Venezia Giulia Region, Marano Lagunare municipality for field logistic support and for their assistance. In particular Dr. Glauco Vicario and Dr. Stefano Sponza for the valuable logistic assistance. The authors have no conflicts of interest.
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- 47 - Tables
Table 1.1: Results of the linear mixed-effects models relating standardized species richness with habitat (i.e. foredune, backdune and saltmarsh), species status (i.e. alien and native), soil conductivity (cond), soil nitrogen content
(N) and the interactions between species status, habitat and soil conductivity, nitrogen content and habitat type. Degrees of freedom (DF), F-value and p- value are reported. In bold are indicated the significant outcomes (p<0.05).
DF F-value p-value habitat 2-76 13.372 <.0001 status 1-87 0.001 0.984 log(cond) 1-76 0.619 0.433 log(N + 0.1) 1-76 0.293 0.589 habitat:status 2-87 27.200 <.0001 habitat:log(cond) 2-76 4.820 0.011 status:log(cond) 1-87 4.950 0.029 habitat:log(N + 0.1) 2-76 3.712 0.029 habitat:status:log(cond) 2-87 3.535 0.033
- 48 - Table 1.2: Results of the linear mixed-effects models relating the standardized species abundance with habitat (i.e. foredune, backdune and saltmarsh), species status (i.e. alien and native), soil conductivity (cond), soil nitrogen content (N), soil organic carbon (C) and the interactions between species status, habitat and soil conductivity, nitrogen content, organic carbon content and habitat type. Degrees of freedom (DF), F-value and p-value are reported. In bold are indicated the significant outcomes (p<0.05).
DF F-value p-value log(cond) 0.743 0.391 1-73 Habitat 6.833 0.002 2-73 Status 0.001 0.981 1-81 C 0.003 0.955 1-73 log(N + 0.1) 0.498 0.482 1-73 log(cond):habitat 0.384 0.682 2-73 log(cond):status 92.082 <.0001 1-81 habitat:status 8.792 0.001 2-81 habitat:C 1.552 0.219 2-73 status:C 1.968 0.164 1-81 habitat:log(N + 0.1) 0.866 0.425 2-73 status:log(N + 0.1) 3.564 0.062 1-81 log(cond):habitat:status 0.550 0.579 2-81 habitat:status:C 1.313 0.274 2-81 habitat:status:log(N + 2-81 2.389 0.098 0.1)
- 49 - Figures
Figure 1.1: location of the study area in the lagoon of Marano and Grado in the northern Adriatic Sea (a), the barrier islands and 10 sampling areas (b), and an example of 10 points distribution inside each sampling area (San
Andrea island) (c).
- 50 - Figure 1.2: differences in standardized species richness between status
(native=solid line, alien=dashed line) within the considered habitats.
Confidence intervals (95 %) are also shown (shaded).
- 51 - Figure 1.3: effects of soil conductivity (a, b, c) and soil nitrogen (d, e, f) on standardized plant species richness of native (solid line) and alien (dashed line) status within habitats (vertical columns). Confidence intervals (95 %) are also shown (shaded).
- 52 - Figure 1.4: effects of soil conductivity (a, b, c), organic carbon content in soil (d, e, f) and nitrogen content in soil (g, h, i) on standardized species cover
(abundance) between status (native=solid line, alien=dashed line) and within habitats (vertical columns). Confidence intervals (95 %) are also shown
(shaded).
- 53 - Supplementary Material
Figure S1.1: correlation plot between soil features. Statistically highly correlated values in are marked in bold (r > |0.75|, p < 0.001).
- 54 - Figure S1.2: Principal Coordinates Analysis (PCoA) applied to functional traits of alien and native species pool in backdune habitat (B_Alien and
B_Native), visible in blue oval shape, which was the most invaded habitat.
F_Alien and F_Native are related to foredune habitat; S_Alien and S_Native are related to saltmarsh.
- 55 - Appendix S1.1: Species list. For each taxon the status (alien= A, native= N) and relative frequency (in percentage) in each habitat and overall are reported.
Frequency (%) Species Status Foredune Backdune Saltmarsh Overall Abutilon theophrasti Medik. A 3.1 0 0 1 Ailanthus altissima (Mill.) A 0 2.5 0 1 Swingle Amaranthus retroflexus L. A 3.1 0 0 1 Ambrosia psilostachya DC. A 25 62.5 3.6 34 Amorpha fruticosa L. A 3.1 27.5 0 12 Aristolochia rotunda subsp. N 0 2.5 0 1 rotunda L. Arthrocaulon macrostachyum N 0 0 17.9 5 (Moric) Piirainen & G.Kadereit Arundo donax L. A 0 5 0 2 Asparagus maritimus (L.) Mill. N 0 12.5 0 5 Atriplex patula L. N 9.4 2.5 0 4 Atriplex rosea L. N 40.6 10 7.1 19 Avena barbata Pott ex Link N 0 7.5 0 3 Bryonia dioica Jacq. N 0 2.5 0 1 Cakile maritima subsp. N 93.8 20 0 38 maritima Scop. Calamagrostis arenaria (L.) N 12.5 2.5 0 5 Roth Calamagrostis epigejos (L.) N 0 10 3.6 5 Roth Carex extensa Gooden N 3.1 17.5 21.4 14 Cenchrus longispinus (Hack.) A 3.1 0 0 1 Fernald Centaurea tommasinii A.Kern. N 0 5 0 2 Cerastium pumilum Curtis N 0 5 0 2 Chenopodium album L. N 21.9 2.5 0 8 Convolvulus sepium L. N 0 2.5 0 1 Convolvulus soldanella L. N 0 10 0 4 Crepis foetida L. subsp. A 0 10 0 4 rhoeadifolia (M.Bieb.) Čelak. Cuscuta cesattiana Bertol. N 40.6 12.5 0 18 Cycloloma atriplicifolium A 3.1 0 0 1 (Spreng.) J.M.Coult.
- 56 - Frequency (%) Species Status Foredune Backdune Saltmarsh Overall Cynodon dactylon (L.) Pers. N 6.3 0 0 2 Cyperus capitatus Vand. N 9.4 7.5 0 6 Cyperus esculetus L. A 25 7.5 0 11 Dactylis glomerata L. N 3.1 30 0 13 Diplotaxis tenuifolia (L.) DC. N 0 2.5 0 1 Elymus acutus (DC.) M.A. N 40.6 25 21.4 29 Thiébaud Elymus farctus (Viv.) N 25 15 0 14 Runemark ex Melderis Erigeron annuus (L.) Desf. A 0 2.5 0 1 Erigeron canadensis L. A 12.5 42.5 0 21 Eryngium maritimum L. N 18.8 7.5 0 9 Festuca fasciculata Forssk. N 12.5 10 0 8 Fumaria officinalis L. N 0 2.5 0 1 Galatella pannonica (Jacq.) Galasso, Bartolucci & N 0 0 28.6 8 Ardenghi Helianthemum nummularium N 0 2.5 0 1 (L.) Mill. Helianthus annuus subsp. A 6.3 0 0 2 annuus L. Juncus acutus subsp. acutus L. N 3.1 7.5 17.9 9 Juncus littoralis C.A.Mey. N 0 2.5 3.6 2 Juncus maritimus Lam. N 0 5 32.1 11 Lagurus ovatus L. N 9.4 2.5 0 4 Lepidium virginicum subsp. A 0 2.5 0 1 virginicum L. Limbarda crithmoides (L.) N 21.9 10 57.1 27 Dumort. Limonium bellidifolium N 0 5 3.6 3 (Gouan) Dumort. Limonium narbonense Mill. N 0 2.5 96.4 28 Limonium virgatum (Willd.) N 0 5 7.1 4 Fourr. Medicago littoralis Loisel. N 0 2.5 0 1 Medicago marina L. N 3.1 0 0 1 Oenothera biennis L. aggr. A 18.8 60 0 30 Panicum capillare L. A 12.5 25 0 14 Parapholis incurva (L.) N 12.5 2.5 0 5 C.E.Hubb. Petrorhagia saxifraga (L.) Link N 0 7.5 0 3
- 57 - Frequency (%) Species Status Foredune Backdune Saltmarsh Overall Phleum arenarium L. subsp. N 9.4 10 0 7 caesium H.Scholz Phragmites australis subsp. N 3.1 10 17.9 10 australis (Cav.) Trin. ex Steud. Plantago coronopus L. N 0 12.5 0 5 Poa annua L. N 0 2.5 0 1 Poa trivialis L. N 0 5 0 2 Polypogon maritimus Willd. N 9.4 2.5 0 4 Polypogon viridis (Gouan) N 0 2.5 0 1 Breistr. Portulaca oleracea L. N 3.1 0 0 1 Poterium sanguisorba L. N 6.3 25 0 12 Puccinellia festuciformis (Host) N 0 2.5 32.1 10 Parl. Raphanus raphanistrum L. N 0 2.5 0 1 Robinia pseudoacacia L. A 0 2.5 0 1 Rubus ulmifolius Schott N 0 20 0 8 Salicornia fruticosa (L.) L. N 0 2.5 92.9 27 Salicornia perennans Willd. N 0 0 10.7 3 Salsola kali L. A 68.8 12.5 0 27 Sanguisorba officinalis L. N 0 7.5 0 3 Scabiosa triandra L. N 0 27.5 0 11 Scirpoides holoschoenus (L.) N 0 10 0 4 Soják Sedum sexangulare L. N 0 15 0 6 Senecio inaequidens DC. A 3.1 10 0 5 Setaria italica (L.) P.Beauv. N 0 2.5 0 1 subsp. viridis (L.) Thell. Silene vulgaris subsp. N 3.1 30 0 13 angustifolia (Moench) Garcke Soda inermis Fourr. N 18.8 10 0 10 Sorgum halepense (L.) Pers. A 0 2.5 0 1 Spergularia media (L.) C.Presl N 3.1 0 3.6 2 Sporobolus maritimus (Curtis) N 0 2.5 10.7 4 P.M.Peterson & Saarela Sporobolus pumilus (Roth) A 56.3 62.5 25 50 P.M.Peterson & Saarela Stachys recta L. N 0 2.5 0 1 Suaeda maritima (L.) Dumort. N 3.1 5 14.3 7 Symphyotrichum lanceolatum (Willd.) A 0 2.5 0 1 G.L.Nesom
- 58 - Frequency (%) Species Status Foredune Backdune Saltmarsh Overall Teucrium chamaedrys (L.) N 0 2.5 0 1 Trachomitum venetum (L.) N 6.3 27.5 0 13 Woodson Trifolium repens L. N 0 0 3.6 1 Trigonella alba (Medik.) N 0 17.5 0 7 Coulot & Rabaute Trigonella officinalis (L.) N 0 2.5 0 1 Coulot & Rabaute Tripidium ravennae (L.) N 0 2.5 0 1 H.Scholz Verbascum phlomoides L. N 0 5 0 2 Xanthium italicum Moretti A 75 27.5 0 35 Yucca gloriosa L. A 3.1 2.5 0 2
- 59 - - 60 - CHAPTER 2
Seagrass meadow cover and species composition drive the abundance of Eurasian wigeon (Mareca penelope L.) in a lagoon ecosystem of the northern Adriatic Sea
Francesco Boscutti1 | Stefano Vitti1,2 | Valentino Casolo1 | Flavio Roppa3 | Daniel Tamburlin1 | Stefano Sponza4 1Department of Agricultural and Environmental Sciences, Plant Biology Unit, University of Udine, Udine, Italy 2Department of Life Sciences, University of Trieste, Trieste, Italy 3Via San Giovanni D'Antro 2/2, Udine, Italy 4Department of Mathematics and Geoscience, University of Trieste, Trieste, Italy
Correspondence Stefano Vitti, Department of Life Sciences, University of Trieste, Via Weiss 2, 34128 Trieste, Italy. Email: [email protected]
Abstract A comprehensive understanding of the interactions between primary producers and consumers plays an important role for the conservation of sensitive ecosystems such as lagoons. In this light, we studied the relationships between the flocks' size of Mareca penelope and the
- 61 - distribution of three seagrass species (Cymodocea nodosa, Zoostera marina and Nanozostera noltei) occurring in the Marano and Grado lagoon (Northern Adriatic Sea). Twelve bird monitoring areas were monthly surveyed for 3 years whereas seagrass distribution data were collected for the whole lagoon in the following years. The overall number of individuals of M. penelope was related to seagrass meadow extension and species cover by using a multiscale approach in four circle buffers (with radius of 500, 750, 1,000 and 1,250 m). Among the considered scales, the 750 m radius scale showed the best performance. The overall number of M. penelope increased where the occupied area by seagrass meadows was larger. Results also showed that when C. nodosa mean percentage cover increased the number of M. penelope decreased, while if N. noltei mean percentage cover increased also M. penelope number increased. Z. marina showed a negligible influence for all the tested scales. Our findings demonstrate that M. penelope populations depend not only on the extension of seagrass meadows but also on their species assembly, evidencing that M. penelope seem to prefer N. noltei stands, avoiding meadows with high abundance of C. nodosa.
KEYWORDS herbivorous birds, lagoon, Mareca penelope, primary producers, seagrasses
Introduction Coastal lagoons are water bodies under the influence of both the marine and the terrestrial domain, which encompass complex ecosystems located at the interface between land and sea. Lagoon environments can be considered as harsh ecosystems exposed to hard environmental stressors (e.g. waterlogging, high salt concentration), due to their shallow and confined
- 62 - waters subjected to tide cycles (Marín-Guirao et al. 2005). Such ecological amplitude positively reverberates on the richness of the environmental mosaic, which is pivotal to harbor high levels of biodiversity for all biota (Sadoul 1997). In many lagoons the primary production is mainly sustained by seagrasses (Moncreiff et al. 1992; Erftemeijer et al. 1993; Ziegler and Benner 1999); therefore, maintaining biodiversity and biocomplexity of seagrass meadows, and other related coastal ecosystems, has important conservation and management implications (Duffy 2006). Although widely distributed, seagrasses have experienced a large-scale decrease in the last decades in most of worldwide populations (Borum et al. 2004; Waycott et al. 2009; Short et al. 2011). In fact, their distribution, productivity, and community composition has been altered by global changes such as variations in sea level, salinity, temperature, atmospheric CO2, and UV radiation (Short and Neckles 1999; Orth, Carruthers, et al. 2006; Unsworth et al. 2014). Herbivorous waterbirds are a crucial component of lagoon ecosystems playing important roles in food web and other ecosystem functions; in particular, as primary consumers, they greatly contribute in determining plant community structure (Jiménez-Ramos et al. 2017). Waterbirds are considered to be indicators of the quality and the importance of wetlands regarding both biodiversity and ecosystem functions (Sadoul 1997; Ma et al. 2009; Farinós Celdrán et al. 2013); in particular, changes in the structure and function of waterbird communities have been linked to changes in the biological integrity of wetlands (Farinós Celdrán et al. 2013; Pöysä et al. 2017). Ducks (genus Anas) are one of the most common inhabitants of wetland ecosystems in temperate areas of the Northern Hemisphere (Bouchaala et al. 2017). In the European wetlands, Eurasian wigeon (hereafter wigeon) Mareca penelope L. has a crucial role in food chain both
- 63 - as prey, for higher levels, and, as primary consumer, transferring energy up the food web (Jiménez-Ramos et al. 2017). Seagrasses are largely studied as primary source sustaining wigeon populations (Nacken and Reise 2000). Moreover, seagrasses are also fundamental in the frame of the overall marine biodiversity. Indeed, structure and species composition of seagrass beds are influent factors for other photosynthetic organisms (Orth, Harwell, et al. 2006) and animal communities (Orth et al. 1984; Somerfield et al. 2002). Several authors already have studied the relationships between wigeon and seagrasses (Valentine and Heck Jr 1999; Nacken and Reise 2000; Borum et al. 2004; Bouchaala et al. 2017). For example, in Northern Sea, Vermaat and Verhagen (1996) showed that the rapid decline in biomass from mid- September onwards could be attributed to grazing by herbivorous migratory waterbirds and it was estimated that wigeon can remove 65 g Ash-Free Dry Weight (AFDW) per bird day−1. Other studies correlated wigeon’s occurrence to seagrass meadow distribution, some of these performed in Mediterranean Sea (Orth et al. 1984; Vermaat and Verhagen 1996; Valentine and Heck Jr 1999; Nacken and Reise 2000). Nevertheless, such studies seldom explored the preferences of wigeon for seagrass meadows constituted by different species. We used an observational approach to verify the relationships between the wigeon abundance and the distribution/cover of three seagrass species occurring in a large lagoon ecosystem of the Northern Adriatic Sea. In particular, we hypothesized that: (i) wigeon flock increased in relation to seagrasses overall cover; (ii) wigeon flock showed diverse responses in relation to each seagrass species abundance.
- 64 - Material and methods Study site The study site was the Marano and Grado lagoon (45°40’40’’ N 13°03’50’’ E to 45°46’30’’ N 13°27’20’’ E; Fig. 2.1a). It is located in the north part of the Adriatic Sea, in Friuli Venezia Giulia region, Italy. The Tagliamento (West) and Isonzo (East) rivers limit the lagoon that has an extension of ca. 160 km2. The lagoon is morphologically classified as a leaky lagoon (Kjerfve 1994) based on the degree of water exchange with the adjacent coastal ocean. It is strongly influenced by tides. At present, three sandbars and two barrier islands separate the Lagoon of Marano and Grado from the Adriatic Sea (Ferrarin et al. 2010). It is divided into two basins (Boscutti et al. 2015): the Marano basin and the Grado basin. Main differences between these two basins consists in shallow water body, few areas above sea level, conspicuous inputs of fresh waters, due to rivers flowing into the lagoon across the principal channels in Marano basin and several islands, salt marshes, shallower waters, a complex channel network in Grado basin. The lagoon encompasses a complex mosaic of ecosystems such as (i) shallow brackish water, (ii) mudflats and sandflats without vegetation, (iii) mudflats and sandflats colonized by Salicornia spp., (iv) salt marshes, (v) coastal sand dunes and (vi) Spartina swords (Spartinion maritimae) (Regione Friuli Venezia Giulia 2018). Marano and Grado lagoon includes two natural reserves, both established in 1996 and managed by the municipality of Marano Lagunare namely “Foci dello Stella” (13.77 km2) and “Valle Canal Novo” (1.21 km2). The entire lagoon is also within the Natura 2000 network, recognized both as Special Area of Conservation (SAC) and Special Protection Area (SPA). The Marano and Grado lagoon is one of the most important resting and wintering sites for waterbirds in Adriatic and in Italy; the coastal area of Friuli Venezia Giulia is of international value for seven
- 65 - waterbird species, and of national value for 33 species (Regione Friuli Venezia Giulia 2018).
Study species and data collection Eurasian wigeon Wigeon is a surface feeding waterbird species; on the wintering ground it feeds mainly on submerged vegetation and sometimes it feeds in absence of water (Larsen 2008). However, it can also eat a larger proportion of benthic invertebrates (Dessborn et al. 2011). In Europe the species is evaluated as Least Concern. The species has an extremely large range and the world population trend appears to be stable. Differently in the EU27 the population is estimated to have decreased by 30-49% during the last 19.2 years (three generations), and it is therefore classified as Vulnerable (IUCN 2016). In the study area wigeon is one of the most common wintering waterbird species. During winter and fall seasons (from September to March) it feeds and rests in the study area with a population of ca. 22,000 individuals (Sponza et al. 2009). The wigeon is hunted in the whole lagoon except for the areas included in the natural reserves (Fig. 2.1b). Grado and Marano lagoon is of international importance for the species, as it hosts at least 1% of the total population of the reference flyway connecting the Western Siberia and the North-Eastern Europe to the Black Sea and Mediterranean regions (Regione Friuli Venezia Giulia 2018). Abundance data of wigeon were collected during the ANSER project (Sponza et al. 2009), which lasted from October 2005 to May 2008. Monitoring of the species was carried out each month, during closed hunting days, using three high tides transects and covering the entire lagoon on the same day or, if not possible, on subsequent days. Data were integrated with further observations in areas of particular interest. The European wigeon
- 66 - population was assessed on 12 areas within 3 transect each (Fig. 2.1b). Centroids points of observation areas were used due to the uncertainty of precisely georeferencing a mobile flock, often composed of thousands of birds. All the flocks observed were composed by wigeons only or in any case wigeon was the dominant species. Data were preliminary summarized as (i) the total number of individuals of wigeon per site and survey and (ii) the mean number of individuals of wigeon per site. For geographical coordinates and summarized counts data of each site, see Table 2.1.
Seagrasses Monitoring of seagrasses was conducted in between spring and fall of the years 2009-2010. During the monitoring, a distribution map of seagrass meadows was realized for the whole Marano and Grado lagoon, with a cartographic scale of 1:5,000. Distribution map was realized via a photointerpretation using digital aerial photos with 0.5 X 0.5 m resolution (taken in 2007 – high tide) and digital aerial photos with 0.2 X 0.2 m resolution (taken in 2003 – low tide) (Boscutti et al. 2015). Single species abundance was recorded in 570 sample areas within a regular grid applied to the whole lagoon (Boscutti et al. 2015). While in Boscutti et al. 2015 the authors disregarded the inner lagoon and brackish water sample areas, we have here included all the sample areas from their original database, thus covering the whole lagoon. In each sample area, presence of each seagrass species and visual estimation of species cover were recorded. Three seagrass species were recorded, namely Zoostera marina L., Nanozostera noltei (Hornem.) Toml. & Posl. and Cymodocea nodosa (Ucria) Asch.
Data analysis Multiple-scale analysis
- 67 - In order to test the relationships between wigeon and seagrasses distribution, a multiscale approach was conducted around the 12 representative points (centroids). Considering the count range of Eurasian wigeon and the scale of seagrasses meadows monitoring grid, to establish the best overlapping patch, four different scales were analysed by using concentric buffers with radius of 500 m (ca. 0.78 km2), 750 m (ca. 1.77 km2), 1,000 m (ca. 3.14 km2) and 1,250 m (ca. 4.91 km2), respectively. The overall cover of seagrass meadows (km2) was calculated using QGIS within each buffer for all the analysed scales (500 m to 1,250 m). Afterwards, the mean cover (expressed as percentage) for each seagrass species (i.e. Z. marina, N. noltei, C. nodosa) was calculated considering all the seagrasses sample areas included in each buffer (Fig. 2.1c). The calculated variables used for the subsequent analyses were reported in Electronic Supplementary Material (ESM 1) with the values for each investigated scale. Statistical analysis All statistical analyses were performed in R 3.4.4 statistical software (R Core Team 2017). Preliminarily, all the calculated indices of size for the wigeon flocks (i.e. overall number of individual, mean number of individuals for each survey) were analysed using Pearson correlation test. To match the assumptions of normality and homoscedasticity, the variables of mean and overall number of individuals were treated with logarithmic transformation. The linear correlation coefficient (Pearson R) between number of individuals and mean number of individuals was 0.95 (p < 0.001). Hence, we further considered only the overall number of individual as dependent variable for the following models. The considered seagrass meadow variables (i.e. meadow extension, species cover) were analysed by Pearson test in order to avoid collinearity. All the variables were not correlated (r < 0.7, p > 0.05) and kept in the analyses.
- 68 - The relationships between the overall number of wigeon individuals and seagrass variables (i.e. meadow extension, each species cover) were tested with a Multi-Model Inference (MMI) approach within an information theoretic framework (Burnham et al. 2002). This technique compares the fit of all possible models (including the null model) obtained by the combination of the variables with the "dredge" function in the MuMIn package (Barton 2015) of the R software, indicating the best model based on the AIC criterion (Akaike's Information Criterion) (Burnham et al. 2002; Zuur et al. 2009). The best fit is indicated by the lowest AIC value (AIC MIN). In a set of models each model i can be ranked using its difference in AIC score to the best-fitting model (delta AICi = AICi- AICi MIN). The models considered as potentially more appropriate were those that had a delta AIC value lower than 2 (Burnham et al. 2002). The outcomes of the models used to assess the analyses results were: the coefficient of determination (R2), model degrees of freedom (df), Akaike's Information Criterion (AIC), delta AIC (difference between AIC of the i-th model and lowest AIC of the whole set of models) and model weight (number between 0 and 1, reflects the relative strength of the i-th model compared to the other candidate models) (Zuur et al. 2009). The analyses were repeated for all the investigated scales (i.e. 500 m, 750 m, 1,000 m, 1,250 m). All the scale and models were compared using the model outcomes, in order to identify the best scale of analysis and, within each scale, the best model. Normality and homogeneity of residuals variance were evaluated by diagnostic plots and tests. In order to match these assumptions, the overall number of wigeon was treated with logarithmic transformation.
- 69 - Results Among the considered scales, the 500 m radius and 1,250 m radius showed similar statistical scores, having lower performances for all the considered statistical parameters. The 1,000 m radius scale showed intermediate statistical scores whereas the 750 m radius scale had the best performances (Table 2.2). In particular, at this scale all the selected models had higher coefficients of determination (R2), model weights and lower AIC values. Within this scale the best model explains 81% of the total variance showing the lowest AIC with a model weight of 0.46. The overall number of Eurasian wigeon individuals was mostly related with area occupied by seagrass meadows and the mean percentage cover of Cymodocea nodosa and Nanozostera noltei. In particular, the number of observed individuals of wigeon increased where the occupied area by seagrasses was larger (F1-8= 20.82, p= 0.002) (Fig. 2.2a). Results showed also that when C. nodosa mean percentage cover increased the number of wigeon decreased (F1-8= 6.90, p= 0.030) (Fig. 2.2b), while if N. noltei mean percentage cover increased wigeon number increased (F1-8= 6.11, p= 0.038) (Fig. 2.2c). Zostera marina showed a negligible influence in all the tested scales (Table 2.2). For all the investigate scales the null models showed a low weight (weight <0.001) and were not considerable as plausible (delta AIC > 11).
Discussion
The presence of wigeon was affected by both seagrass meadows extension and species composition. In fact, not all the species showed a significant and/or positive relationship with the abundance of wigeon, suggesting some possible preference mechanisms. These relationships were stronger when analysed at the scale of 750 m radius.
- 70 - As expected the overall number of individuals of wigeon increased where the seagrass meadows were larger. This confirms the finding of other studies (Hamza et al. 2015; Balsby et al. 2017), where it is reported that dense seagrass populations favor waterbirds, especially in large intertidal areas, characterized by high numbers of tidal channels, elevated amounts of mud and organic materials in the sediment. In such situations, coverage of seagrass hosted a great diversity of species, sustaining also wigeon flocks. Seagrasses are pivotal for herbivorous birds feeding especially during migration periods (Short et al. 2001). For the sustenance of big flocks there is need of a sufficient vegetal biomass due to the high number of individuals and their daily intake of calories. Mayhew (1988) showed that an individual of wigeon need to ingest at least 366 g of grass day-1, with a consumption of 660-715 kJ/bird-1 day-1. As supposed, the abundance of wigeon was also strongly affected by meadow species composition. Surprisingly, we found that each of the three plants species occurring in the area showed to have different relationships with wigeon. In fact, while Cymodocea nodosa and Nanozostera noltei showed opposite trends, Zostera marina did not affect significantly the size of flocks of wigeon. N. noltei resulted the most important species for the suitability of wigeon flocks. This result is consistent with other studies and research which proves that wigeon feeding during winter is based on seagrasses and particularly on the genera Zostera and Nanozostera (Owen and Williams 1976). In lagoon systems, N. noltei prefers low depth water (Boscutti et al. 2015), where wigeon can easily reach the vegetation. On the other hand, we cannot exclude a positive feedback between the abundance of wigeon and the growth of seagrasses. In fact, as it was already demonstrated in other studies (Rodríguez-Pérez and Green 2006), wildfowl (e.g. ducks, swans and coots)
- 71 - and Greater Flamingo (Phoenicopterus roseus) can positively stimulate macrophyte growth with their grazing activities. Similarly, wigeons may favor the growth of N. noltei. The abundance of C. nodosa also affected wigeon flock size, in fact, when its cover increased the overall number of individuals decreased. This trend could be explained by two hypotheses linked to a selective feeding of wigeon: (i) a different species recognition and (ii) a different species palatability. A lower palatability of C. nodosa in comparison with N. noltei might also be important. In fact, the species differ in terms of morphological and nutritional traits. A large survey on leaf-fracture properties and nutritional traits in nine Australian seagrass species revealed that these two parameters are negatively and tightly inter-correlated in seagrasses, indicating that species with high mechanical resistance tend to exhibit a low leaf nutritional value (de los Santos et al. 2012). In the Mediterranean Sea, C. nodosa and N. noltei often co-occur but showing important differences in structure, physiology, and growth dynamic (Kraemer and Mazzella 1999). Jiménez-Ramos et al. 2018 demonstrated that structural traits, such as carbon content, were linked to the lower consumption of C. nodosa by a generalist herbivore (purple sea urchin Paracentrotus lividus), in comparison to the N. noltei consumption. Moreover, wigeon usually feed grazing both the leaves and rhizomes of seagrasses (Jacobs et al. 1981). In this light, the robust rhizomes of C. nodosa can also explain a lower palatability of this species. Finally, Z. marina did not seem to affect the number of individuals of wigeon. Even though this species is potentially pleasant to many waterbird species (Nienhuis and van Ierland 1978), results showed only a weak negative relationship with wigeon. This result could be linked either to the depth of growing of Z. marina, which in situ prefers deep waters (Boscutti et al. 2015). In a similar context, it was suggested that Z. marina was less
- 72 - consumed than another smaller Zostera species (Zostera japonica) due to its shorter accessibility during the tidal cycle and the lower energy content of leaves (Baldwin and Lovvorn 1994). The 750 m radius scale had the best performances (Table 2.2) and in particular at this scale all the selected models had higher coefficients of determination (R2). This scale of investigation might be the best solution for future studies in wintering sites. Our findings suggest that this range of investigation would be the most effective during monitoring actions and for analyses of other ecological relationships between wigeon and seagrasses (e.g. trophic relationships, palatability studies). Moreover, this scale seems to be a proper scale also when considering future nature protection measures of seagrass meadows or planning of management practice within protected areas.
Conclusion
Our findings confirmed that, in lagoon ecosystems, wigeon wintering populations shows co-occurrence on the abundance of seagrass meadows. Interestingly, the wigeon seems to favor N. noltei, avoiding C. nodosa. These results stress the importance to consider seagrass meadows protection and/or management in order to enhance resources availability for bird fauna, especially in protected areas where it might positively reverberate on the whole ecosystem diversity and functioning.
Acknowledgements
For seagrass data collection, we thank the staff of Protezione Civile of Marano Lagunare and Grado municipalities, Giuseppe Milocco and Giuliano
- 73 - Felluga, for field logistic support; the staff of Friuli Venezia Giulia Region - Central Direction Environmental and Energy (Service of waste management and contaminated sites), in particular Eng. Paolo Tonello; and Nathalie Zamparutti for the valuable field survey help. For wigeon data collection, we thank Mauro Cosolo, Paolo Utmar, Nicola Ventolini and the staff of the ‘Office for Studies on Wild Fauna’ of Friuli Venezia Giulia Region. The ANSER Project was financed by Interreg IIIA Transfrontaliero Adriatico.
Funding
The ANSER Project was financed by Interreg IIIA Transfrontaliero Adriatico Conflict of Interest: The authors declare that they have no conflict of interest.
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- 81 - Tables
Table 2.1 Total number of individuals and mean (± SD) number of individuals per survey of Eurasian wigeon Mareca penelope observed for each site. Latitude and longitude are expressed in WGS84 coordinate system.
Overall ID Mean of Individuals mean Site Latitude Longitude number of Code individuals standard deviation individuals 1 Pantani 45°42’14.4’’N 13°05’49.6’’E 78 13 22 2 Secca Muzzana 45°45’00.0’’N 13°07’37.2’’E 919 65 174 3 Marinetta 45°42’46.8’’N 13°09’36.0’’E 4150 830 1329 4 Toppi 45°43’30.0’’N 13°11’31.2’’E 27063 3382 5216 5 Fontane 45°43’26.4’’N 13°13’04.8’’E 3060 765 922 6 Anfora N 45°43’44.4’’N 13°15’25.2’’E 49370 4488 3606 7 Montaron 45°43’30.0’’N 13°19’15.6’’E 1452 484 632 8 Morgo E 45°42’14.4’’N 13°19’58.8’’E 20601 5150 5664 9 Tratauri 45°41’02.4’’N 13°20’42.0’’E 4690 670 631 10 Bivio Natissa 45°42’50.4’’N 13°20’56.4’’E 530 265 332 11 Pampagnola 45°41’24.0’’N 13°22’30.0’’E 1762 440 715 12 Barbana N 45°42’18.0’’N 13°24’18.0’’E 217 72 72
- 82 - Table 2.2 Results of Multi-Model Inference analysis showing the relationships between the total number of individuals of Eurasian wigeon Mareca penelope and seagrass meadow area (seagrass area expressed in km2), and species cover (Cymodocea nodosa, Zostera marina, Nanozostera noltei) expressed as percentage. In this table are reported only models with a delta AIC < 2. For each model are reported related values, variable coefficients, coefficient of determination (R2), degrees of freedom (df), Akaike's Information Criterion (AIC), delta AIC and model’s weight
Variable coefficient
for seagrass Cymodocea Zostera Nanozostera Delta Buffer Intercept R2 df AIC Weight area nodosa marina noltei AIC 5.246 3.82 0.048 0.72 4 41.5 0.00 0.32
500 5.444 5.32 -0.015 0.042 0.75 5 42.6 1.07 0.19 5.261 3.80 -0.002 0.049 0.72 5 43.5 1.98 0.12
5.714 3.43 -0.038 0.036 0.81 5 39.1 0.00 0.46 750 5.969 4.04 -0.050 -0.015 0.036 0.83 6 39.9 0.80 0.31
5.752 2.00 -0.030 0.036 0.79 5 40.4 0.00 0.38
1,000 5.321 1.41 0.040 0.72 4 41.5 1.13 0.21 5.861 2.14 -0.034 -0.009 0.037 0.79 6 42.1 1.72 0.16 5.521 1.50 -0.027 0.040 0.76 5 41.8 0.00 0.26
1,250 5.688 1.93 -0.046 -0.041 0.056 0.79 6 41.9 0.07 0.25 5.102 1.02 0.048 0.71 4 41.9 0.16 0.24
- 83 - Figures
Fig. 2.1 Location of (a) the study area lagoon of Marano and Grado in the Northern Adriatic Sea, (b) the 12 representative points of the areas selected and occupied by Eurasian wigeon Mareca penelope and (c) Seagrass sample points and concentric buffer areas around the site 5, as an example of the multiscale analysis approach used for each representative point. Black dotted line indicates Natural Reserve.
- 84 - Fig. 2.2 Relationship between the sum of Eurasian wigeon Mareca penelope individuals (log-transformed) and cover of (a) seagrass (km2), (b) Cymodocea nodosa and (c) Nanozostera noltei for a buffer area of 750 m. The confidence interval (95%) of the regression line is represented in green.
- 85 - - 86 - CHAPTER 3
Population density modulates the response of morphological traits and non-structural carbohydrates in Vaccinum myrtillus when exposed to different rain regimes
Abstract Drought events continue globally to increase in frequency and intensity. In alpine ecosystems precipitation regime is also changing, generating uncertain responses in plant acclimation of the Alpine region to new water availability conditions. In this work, we aim to understand the effects of rainfall regime alterations and plant competition on the populations of Vaccinium myrtillus, by analyzing morphological and physiological traits in view to forecast further scenarios about shrub responses to climate change. Investigations were carried out in alpine heaths above the treeline, and we investigated the relationships between plant traits and plant-plant interaction (i.e. shrub cover). The sampling design included five randomized blocks, each including a high density and low-density shrub vegetation cover and two regimes of rain precipitation (100 vs 40 %). The obtained results showed that the reduction of precipitation affected the considered plant traits, and only one parameter that did not shows relevant difference was stem tissue density. The experiment confirmed that the reduction of the rain regime has a concrete effect on populations of V. myrtillus by modifying its morphological features.
Keywords Carbohydrates, non-structural carbohydrates, Vaccinium myrtillus, rain control, rainout shelters, Specific Leaf Area, functional traits, plant growth
- 87 -
Introduction Phenotypic plasticity gives the opportunity to a plant individual to grow in various ecological conditions (Schlichting 1986); it is an essential tool for plant acclimation trough ongoing change of climate conditions and, particularly, displays when plant population grows across an environmental gradient In the view of a global increasing temperature, that leads to a shifting of the treeline it is pivotal to evaluate how changes in rain regimes can interfere with this phenomenon, particularly in the view of shrub expansion. Alpine and arctic shrubs are highly sensitive to temperature variation (Sturm et al. 2001; Dial et al. 2007; Blok et al. 2011; Myers-Smith et al. 2015) and the extension of their communities are considered to be evidence of climate change (Archer et al. 1995; Sturm et al. 2001). Among plant traits, carbohydrate transport and storage have been associated with stress given by elevation (Mooney and Billings 1965; Wallace and Harrison 1978). However NSC metabolism is involved also in drought response (Camisón et al. 2020), a phenomenon observed also in alpine shrubs (Ganthaler and Mayr 2015). In particular, drought is a condition characteristic of Mediterranean mountains (Magaña Ugarte et al. 2018) and above treeline, where vegetation grows on xeric soil such as alpine limestone grassland. Furthermore, recently has been reported modulation of non- structural carbohydrates (NSCs) due to plant competition in bilberry (Casolo et al, 2020). In the global scenario of climate change, including an increasing number of drought events added to global climate change and to understand how climate change affects the alpine ecosystem, it has a pivotal role to simulate precipitation regimes under field conditions (Kundel et al. 2018).
- 88 - The work aim is to understand the possible effects of precipitation regime and specific competition on the populations of Vaccinium myrtillus L. in alpine shrub belt over the tree limit in the frame of phenotypic plasticity by analyzing and comparing NSC content related to morphological and growth parameters. In this work, we tested the hypothesis that trait plasticity and plant – plant interactions are fundamental variables driving the response of alpine V. myrtillus stands to climate variation, particularly because the intensity of plant-plant interaction has been proved to have a pivotal role for principal metabolic compounds in shrubs (García-Cervigón et al. 2012; Casolo et al. 2020). We investigated the relationships between NSCs, plant traits (i.e., ramet age), rain exclusion, and shrub density (i.e., shrub cover), deciding to analyze total soluble NSCs and starch. We expect that plant traits and NSCs would change in response to the rain exclusion regime, with a different shaping between high shrub density areas (hereafter HD) and low - density ones (hereafter LD).
Materials and methods Study Area The study area was Malga Pieltinis, Sauris, located on the south-east side of Mount Pieltinis (2,027 m) with north-northeast (NNE) exposure, and southwest (SW) of the Malga Pieltinis on a plateau at 1742 m. The area belongs to the region of Eastern Alps, Friuli Venezia Giulia region (Italy). According to Rivas-Martínez et al. (2011) classification, the study area is characterized by a hyper-humid semi-continental oceanic, orotemperate climate. Meteorological data were obtained from the Sauris station, as it is the closest to the study area and is located at an altitude of 1,212 m above
- 89 - mean sea level. Precipitation varies significantly according to the season, with an average annual rainfall of 1,543 mm. The wettest seasons are spring and autumn, while during the summer they decrease without causing drought phenomena. Snowfall, which reaches an average height of 62 cm on the ground in February, is concentrated in the period from December to April with over 117 days of average snow cover, of which 103 of continuous average snow cover. The average annual temperature is 8.7 °C
Experimental design Five blocks were randomly placed, each block with high and low – density vegetation and control area availability. The experiment included five randomized blocks, each one including an HD and LD vegetation stands in both conditions with and without rain exclusion treatment (Fig. 3.1). Many field studies that try to experimentally alter rainfall regimes primarily use rainout shelters to exclude ambient precipitation from an experimental area (Kundel et al. 2018). Two rain interceptors or precipitation exclusion systems (hereafter rainout shelters), were positioned on each site. The rainout shelter has a 60% cover surface built with polycarbonate elements C shaped. One was placed on an HD area and the other on an LD area. The density of dwarf shrubs was estimated as percentage of cover: > 80% HD; < 30% LD. Shelters defined a sample area (plot) of 4 m2 (2x2 m). In the same way, two HD and LD control areas with the same area, without rain shelters were defined. Concerning environmental parameters, we used data loggers (Campbell Scientific CR1000) connected to environmental sensors (Delta-T SM 150) for soil moisture and temperature detection. In this study, each area was equipped with two sensors into the ground, and data were logged every 30 seconds; about rainfall, we used a rain gauge equipped with a data logger.
- 90 - The data collection covers an interval of 3 and a half months; climatic data were acquired for an interval of 5 months.
Plant traits collection Samples of Vaccinium myrtillus were collected in two different periods: before (mid - June 2018) and after treatment (September 2018) for carbohydrates detection; the random collection has been made on a sunny day, between 11:30 and 12:30 astronomical time, to avoid the risk of interference due to the variability of diurnal carbon assimilation fluctuations. Morphological traits evaluation was performed in mid - June and mid - August. The leaf area was calculated by taking 10 leaves from each area. The leaves were spread on sheets and then scanned. In order to proceed with the calculation of the area, the freeware ImageJ software was used, which allows you to calculate the area from the scanned images. To determine the annual shoot length, 10 blueberry branches were taken from each area and the lengths in mm were measured on the fresh branches. The density of annual shoots, i.e. the dry weight value (mg) of a terminal twig divided by its fresh weight when saturated with water (g), was measured with the pycnometer method. Samples for ramets, leaves, and shoots have been transported in ice to the laboratory and suddenly microwaved at 700 W x 3 minutes to stop enzymatic oxidative activities.
NSCs analysis and morphological traits Starch and soluble NSC content were measured following the method used by Trifilò et al. (2019). Samples were grounded to obtain < 150 µm powder. After this, sample material (15 ± 1 mg) was put in a 1.5 ml Eppendorf.
- 91 - Eppendorf vial with 300 µL of 80 % (v/v) ethanol and vortexed. The Eppendorf was placed in a water-bath at 80 °C for 30 min. After centrifugation for 3 min (12,000 rpm) in Eppendorf centrifuge, the supernatant was transferred into another Eppendorf vial. The same operation was repeated as above and the supernatants were pooled together, then dried overnight at 55 °C. Finally, 500 µL of 50mM TRIS-HCl pH 7.5 was added at room temperature to the pellet and, after new centrifugation, used to re- suspend the dried carbohydrates resulting from ethanol extraction. The final concentration of soluble NSC in the sample was then expressed as mg [glucose] g−1 [DW]. For starch extraction, 1 mL of acetate buffer (0.4M
NaCH3COO, pH 4.6) was added to the pellet residue and treated at 100 °C for 1 h to allow starch gelation. Starch measurement was performed using the insoluble fraction. This starts with an overnight at 55 °C, using 100 U of α-Amylase (EC 3.2.1.1, Sigma-Aldrich,St. Louis, MO, USA) and 25 U of Amylogucosidase (EC 3.2.1.3, Sigma-Aldrich, St. Louis, MO, USA) per each sample. Suddenly, samples boiling prevents sample degradation. The final lecture consists of 2 µl of each sample’s final supernatant in a plate reader (1420 Multilabel Counter Victor3, PerkinElmer), with 300 µl final volume of assay buffer (Tris-HCl with MgCl2 5mM, NADP+ 125 µM, and MgATP 1 mM). A note quantity of Amylose was used to obtain a calibration curve to be compared with samples. The final concentration of starch in the sample was then expressed as mg [starch] g−1 [DW]. Morphological parameters we studied were Specific Leaf Area (SLA), leaf area, stomatal density, length/dry weight ratio, annual shoot length, annual shoot density, shoots length, and the weight of the samples. Samples were then oven-dried at 70 °C for 24 h and grounded to obtain a fine powder. SLA is an index of leaf thickness, and we determined it using a public domain Java image processing and analysis program, called ImageJ (Ferreira and
- 92 - Rasband 2010). SLA has been observed to be lower under drought conditions (Marcelis et al. 1998). From samples, we also determined their shoots porosity, measured by pycnometer method (Jensen et al. 1969). The samples were blotted gently with the tissue paper until free water does not easily transfer to the blotting paper. The weight of empty and water - filled pycnometer was measured, as well as the shoots, on an analytical balance. After that, shoots were inserted into water - filled pycnometer. The pycnometer with water and shoots was weighed on an analytical balance, and then shoots were removed from it.
Statistical analysis All statistical analyses were performed in R 3.4.4 statistical software (R Team 2019). The collected data were aggregated as means of the measured values for each assay area (plot). Linear Mixed-Effects Models were applied to identify the effects of the considered treatments, i.e. rain exclusion, shrub density, and their interaction. Block id was included in the models as a random effect. In the models, for the repeated pre- and post-experiment data, the variable of the pre-experiment works was also included. Where necessary, the variables were treated with a logarithmic transformation to ensure the assumptions of homogeneity of variance and normality of the residuals. The models were calculated using "nlme" package (Pinheiro et al. 2019) from R 3.4.4 statistical software. All the models obtained were evaluated in terms of significance (p-value <0.05).
- 93 - Results Leaf traits The leaf area was significantly different between the stands under - considered experimental factors (rain exclusion and shrub density), while the interaction was not significant (Tab. 3.1). The leaf area of individuals with rain exclusion treatment was on average lower (Fig. 3.3). The HD populations of V. myrtillus had leaves with a greater leaf area than those with LD. Finally, there was a significant relationship between the leaf area recorded before the treatment (June) and the values recorded after the rain exclusion, showing a consistent pattern of leaf growth in the plots during the experiment. In HD stands the SLA showed to be significantly lower in covered plots (C) than in control precipitation regime while it was not different for LD (Fig. 3.2). The interaction between rain regime and stand density being significant reflected this result and SLA was not affected by rain exclusion nor by shrub density while showing a significant interaction between the treatments (Tab. 3.1). It was observed that the stomatal density showed relevant changes in relation to shrub density and its interaction with rain exclusion (Tab. 3.1). It did not vary as a function of rain exclusion and period of sampling; however, this parameter affected the stomatal density in HD stands (Fig. 3.4) Indeed, stomata density showed to be significantly higher different in LD stands. This effect was more pronounced in NC plots (Fig. 3.4), leading to a highly significant interaction between rain exclusion and shrub density (Tab. 3.1).
Shoot traits Annual shoot tissue density showed to be directly linked to population density (Tab. 3.2), with annual shoots of plants from HD less porous
- 94 - compared to LD. This parameter does not show any statistical significance with other variables. The annual shoots length after the experiment was affected by the initial conditions (Tab. 3.2). Moreover, plants from HD areas had significantly longer annual shoots than ones from LD stands. Furthermore, this difference was greater in NC stands (Fig. 3. 5). In fact, even if non-significant, the shoots length of plants exposed to rain exclusion were smaller. The difference, in the length/dry weight ratio of the annual shoots, between both HD and LD zones and rainfall regime was significant (Tab. 3.2). This ratio is higher in shoots from LD areas and rain-excluded ones. In particular, in HD rain-excluded stands there was also a non-significant increase in the ratio. The different behavior of annual shoot length/dry weight ratio between rain regimes, in stands with different shrub density, is highlighted by the significant interaction between these two parameters (Fig. 3.6).
Non-structural carbohydrates The measurement in starch and soluble NSC content, was put in correlation with other variables: i) sampling period (June and September); ii) rain exclusion (cover: C, not cover: NC); iii) shrub density (high density: HD, low density: LD). Starch did not show significant differences between studied variables except for the season (Tab. 3.3; Fig. 3.7). Starch content shows five - folds higher values in June if compared with September. Differently, with respect to starch, soluble NSCs do not show statistical differences due to season even if the same trend was observed (Tab. 3.4). Conversely, soluble NSCs in underground stems from LD stand was significantly higher (Fig. 3.8).
- 95 - Other relevant results in response to soluble NSC content with respect to other variables were generated by an interaction using a Linear Model (LM) or linear regression when other plant traits were taken into consideration. The interaction involves stem length, rain exclusion, and shrub density, as shown in Tab. 3.5. Figure 3.9 shows the differences within the LD population, where soluble NSCs content decrease in underground stems from stands exposed to rain exclusion increase, while in HD plants, soluble NSC content increases regardless of the treatment.
- 96 - Discussion
Our study revealed that NSC, starch, and morphological traits have different responses to drought events. In general, the results showed an effect on V. myrtillus traits of the reduction of precipitation that was mainly dependent on the original shrub density. All the morphological traits of plants show to respond to studied variables.
Leaf traits
The leaf area does not show an inverse trend compared to SLA, but decreases in both LD and HD stands when rain is excluded. The effect of rain exclusion is more pronounced in HD populations also as regards the annual shoots length and the length/dry weight ratio, while those with LD show minimal variations, and with an opposite trend. There was an increase in SLA values under the limitation of rainfall, being SLA a trait involved in drought responses (Valladares and Sánchez-Gómez 2006). Stomatal density, one of the primary morphological regulators that control the response of stomatal conductance under climatic stresses (Franks et al. 2015), decreases when rainfall is limited. It showed to be statistically relevant in the interaction between rain exclusion and shrub density. As also shown by Buckley (2005), leaf stomata may be affected by many environmental variables, including water status. On large leaves, we can observe a low stomatal density. With the increase of the leaf area, the stomata number can increase in a lesser ratio, so there will be a consequent decrease in the stomatal density index, but a higher level of transpiration. However, plants of HD populations under cover condition, have a higher stomata density, which is balanced by a lower SLA. This could be given by a condition in HD stands, where the plant canopy is denser and more occupied
- 97 - (e.g. more stems and more leaves) if compared to LD stands canopy. Although our results showed that stomatal density was not significantly associated with leaf area per plant (data are not shown), it was negatively correlated with specific leaf area (Fig. 3.5), indicating that enhanced leaf thickness may produce more guard cells for a given leaf area. Enlarged leaf thickness and the associated increased stomatal density may also be useful in enhancing the plasticity to a certain degree under moderate drought (Galmés et al. 2007). We have to specify that there we are not aware of existing studies on models that explain how plants compete for water (Craine and Dybzinski 2013). However, high SLA has recently been related to susceptibility to drought-induced mortality (Greenwood et al. 2017) and is considered as a trait implicated in drought responses (Valladares and Sánchez-Gómez 2006). The shrub density was decisive in the modification of the morphological features of the annual shoots. In particular, in HD stands, it was found that the length of the shoots is significantly higher but the ratio between dry matter and length is lower than in LD stands. This suggests that when water availability is lower by the rain exclusion, in HD stands, shoot elongation is slowed down and the tissue matter content decreased (shoot weight), suggesting a compensative mechanism used by the individuals for the competition for light. Therefore, it seems that the effect of precipitation has manifested itself mainly in relation to density. In particular, the leaves characteristics have been modified more by less frequent rainfall only in high - density stands.
Starch serves as an important reserve to balance growth and storage (Wiley et al. 2013). Starch content shows five - folds higher values in September when compared with June. This result is in accordance with results shown in
- 98 - Liu et al. (2019), where was found that starch accumulation in branches is linked to a decrease in mean annual precipitation, and is also in line with Zhang et al. (2008) and Duan et al. (2014), affirming that starch content tends to increase under stressful conditions and decline in favorable conditions. NSCs are not only used for translocating or storage of carbohydrates but also regulated by the metabolism in the plant (Li et al. 2001, 2002; Li, Xiao, Wang, et al. 2008; Li, Xiao, Shi, et al. 2008). The amount of soluble NSCs in underground stems from LD stand was significantly higher if compared with HD stands. This may occur because, during the summer period, plants need more nutrients and inside HD stands intra-specific competition is higher due to its number of individuals (high density of plants), and summer water deficit, or drought, represent an additional obstacle in which is characteristic of Mediterranean mountains (Gimenez-Benavides et al. 2007; García- Cervigón et al. 2012). However, because of the ongoing climate changes, also in the Alps rain regime show to be reduced or at least modified. LD stands, having fewer individuals that compete for soil resources, can take advantage of this. Other results in soluble NSC content were generated by the interaction that involves stem length, rain exclusion, and shrub density. This shows the differences in the LD population, where the NSC content changes considerably as the length of the stem varies. With rain exclusion treatment, the NSC content decreases as the length of the stem increases; on the other hand, where the plants are not covered, the NSC content tends to increase, thus exhibiting an opposite behavior to the previous one.
- 99 - Conclusions In the present study, we analyzed the effects of drought stress on the morphological and physiological features of V. myrtillus and its plant adaptation strategies to rainfall reduction. The obtained results showed that most of the studied plant traits were not significantly affected by the reduction of water availability: in fact, in HD populations only 2 traits from 5 (SLA and stomatal density) showed significant differences, while in LD populations none of the traits showed significant differences. The experiments confirmed the initial hypotheses. It has been verified that rain exclusion has a moderate effect on the HD populations of V. myrtillus by modifying its morphological features. Furthermore, the LD stands revealed higher plasticity in adaptation to a reduction of rain, with respect to the HD ones. We can consider the possibility that the phenomenon of reduced precipitation occurs over a long period. In this case, the consequences for the populations of V. myrtillus could be related to the density of its populations and its spatial distribution. Therefore, a gradual thinning out of the populations could occur, in favor of other species more resistant to drought, or the simple reduction of the average size of the individuals would favor other species. This study shows the importance to always keep a comprehensive approach when studying this particularly sensitive environment.
- 100 -
Figures
Fig 3.1 Sampling design. This picture shows the experimental block.
- 101 -
Fig 3.2 Bar plot for Specific Leaf Area (SLA) index in different shrub density conditions and rain exclusion conditions. Different letters indicate significant differences (p<0.05).
- 102 -
Fig 3.3 Bar plot for leaf area index in different shrub density conditions and rain exclusion conditions. Different letters indicate significant differences (p<0.05).
- 103 -
Fig 3.4 Bar plot for stomatal density index in different shrub density conditions and rain exclusion conditions. Different letters indicate significant differences (p<0.05).
- 104 -
Fig 3.5 Bar plot for annual shoots length in different shrub density conditions and rain exclusion conditions. Different letters indicate significant differences (p<0.05).
- 105 -
Fig 3.6 Bar plot for length/dry weight ratio in different shrub density conditions and rain exclusion conditions. Different letters indicate significant differences (p<0.05).
- 106 -
Fig 3.7 Average content of starch at the beginning and end of the treatment carried out on V. myrtillus underground stems (p <0.05).
- 107 -
Fig 3.8 Average of soluble NSC content on different density stands of V. myrtillus underground stems. in high density (HD) and low density (LD) populations (p <0.05).
- 108 -
Fig 3.9 Relationship between soluble NSC amount, stem length, and rain exclusion. The confidence interval of the regression line is represented in light blue.
- 109 -
Tables
Tab 3.1 Results of the linear mixed-effects models applied to the specific leaf area (SLA) index, leaf area, and stomatal density. Degrees of freedom (DF), F value and P value are reported. In bold are indicated the significant outcomes (p < 0.05).
SLA
PARAMETERS numDF denDF F-value p-value Intercept 1 9 9258.53 <.0001 Rain exclusion 1 9 0.07 0.7976 Shrub density 1 9 0.23 0.6377 SLA 06 9 2.93 0.1209 Rain exclusion:shrub density 1 13.60 0.0050
Leaf area
PARAMETERS numDF denDF F-value p-value Intercept 1 11 334.51 <.0001 Raine xclusion 1 11 6.19 0.0301 Shrub density 1 11 20.39 0.0009 Leaf area 06 1 11 23.98 0.0005 Rain exclusion:shrub density 1 11 0.07 0.7955
Stomatal density
PARAMETERS numDF denDF F-value p-value Intercept 1 6 3144.07 <.0001 Rain exclusion 1 6 0.83 0.3964 Shrub density 1 6 17.83 0.0055 Stoma 06 1 6 0.43 0.5322 Rain exclusion:shrub density 1 6 29.16 0.0017
- 110 -
Tab 3.2 Results of the linear mixed models applied to annual shoots density, annual shoots length and length/dry weight ratio. Degrees of freedom (DF), F value and P value are reported. In bold are indicated the significant outcomes (p < 0.05).
Annual shoots density
PARAMETERS numDF denDF F-value p-value Intercept 1 12 8169.522 <.0001 Rain exclusion 1 12 0.621 0.4460 Shrub density 1 12 9.882 0.0085 Rain exclusion:shrub density 1 12 0.032 0.8601
Annual shoots length
PARAMETERS numDF denDF F-value p-value Intercept 1 11 2341,1155 <.0001 Rain exclusion 1 11 1.3771 0.2654 Shrub density 1 11 28.1648 0.0002 Stem length 06 1 11 11.0899 0.0067 Rain exclusion:shrub density 1 11 0.3340 0.5749
Length/dry weight ratio
PARAMETERS numDF denDF F-value p-value Intercept 1 11 652.4247 <.0001 Rain exclusion 1 11 6.6364 0.0258 Shrub density 1 11 28.0081 0.0003 Stem length/dry weight 06 1 11 0.2043 0.6601 Rain exclusion:shrub density 1 11 5.4255 0.0399
- 111 -
Tab 3.3 Starch results, considering rain exclusion, shrub density and time and the interactions between these variables. Degrees of freedom (DF), F value and P value are reported. In bold are indicated the significant outcomes (p < 0.05; *).
PARAMETERS numDF denDF F-value p-value Intercept 1 28 111.49587 <.0001 Rain exclusion 1 28 0.49415 0.4879 Shrub density 1 28 0.01018 0.9204 time 1 28 104.71884 <.0001* Rain exclusion:shrub density 1 28 2.29333 0.1411 Rain exclusion:time 1 28 1.77972 0.1929 Shrub density:time 1 28 0.08367 0.7745 Rain exclusion:shrub density:time 1 28 0.22099 0.6419
- 112 -
Tab 3.4 Soluble NSC results, considering rain exclusion, shrub density and time and the interactions between these variables. Degrees of freedom (DF), F value and P value are reported. In bold are indicated the significant outcomes (p < 0.05; *).
PARAMETERS numDF denDF F-value p-value Intercept 1 28 713.4362 <.0001 Rain exclusion 1 28 0.2746 0.6044 Shrub density 1 28 5.4173 0.0274* time 1 28 2.8483 0.1026 Rain exclusion:shrub density 1 28 0.1245 0.7269 Rain exclusion:time 1 28 0.0086 0.9266 Shrub density:time 1 28 1.5526 0.2231 Rain exclusion:shrub density:time 1 28 0.2144 0.6469
- 113 -
Tab 3.5 Linear Model for soluble NSC results, considering rain exclusion, shrub density and time and the interactions between these variables. Degrees of freedom (DF), F value and P value are reported. In bold are indicated the significant outcomes (p < 0.05; *).
PARAMETERS numDF denDF F-value p-value Intercept 1 7 1319.6406 <.0001 Rain exclusion 1 7 0.6080 0.4611 Shrub density 1 7 1.8683 0.2139 Stem length 08 1 7 0.7597 0.4123 Nsc 06 1 7 2.2619 0.1763 Rain exclusion:shrub density 1 7 1.1768 0.3139 Rain exclusion:stem length 08 1 7 5.3282 0.0543* Shrub density:stem length 08 1 7 10.5973 0.0140* Rain exclusion:shrub density:stem length 08 1 7 4.9083 0.0623
- 114 -
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CHAPTER 4
Hydraulic recovery from xylem embolism in excised branches of twelve woody species: relationships with parenchyma cells and non-structural carbohydrates
Patrizia Trifilò1, Natasa Kiorapostolou2, Francesco Petruzzellis3, Stefano Vitti3,4, Giai Petit2, Maria A. Lo Gullo1, Andrea Nardini3 and Valentino Casolo4
1. Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università di Messina, Viale Ferdinando Stagno d’Alcontres 31, 98166 Messina, Italy 2. Dipartimento Territorio e Sistemi Agro-Forestali, Università di Padova, Viale dell’Università 16, 35020 Legnaro (PD), Italy 3. Dipartimento di Scienze della Vita, Università di Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy 4. Dipartimento di Scienze AgroAlimentari, Ambientali e Animali, Università di Udine, via delle Scienze 91, 33100 Udine, Italy
Corresponding Author: [email protected]
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Abstract Embolism repair ability has been documented in numerous species. Although the actual mechanism driving this phenomenon is still debated, experimental findings suggest that non-structural carbohydrates (NSC) stored in wood parenchyma would provide the osmotic forces to drive the refilling of embolised conduits. We selected 12 broadleaved species differing in vulnerability to xylem embolism (P50) and amount of wood parenchyma in order to check direct evidence about the possible link(s) between parenchyma cells abundance, NSC availability and species-specific capacity to reverse xylem embolism. Branches were dehydrated until ~50% loss of hydraulic conductivity was recorded (PLC ⁓50%). Hydraulic recovery (DPLC) and NSC content was, then, assessed after 1 h of rehydration. Species showed a different ability to recover their hydraulic conductivity from PLC⁓50%. Removing the bark in the species showing hydraulic recovery inhibited the embolism reversal. Strong correlations between the DPLC and: a) the amount of parenchyma cells (mainly driven by the pith area), b) the consumption of soluble NSC have been recorded. Our results support the hypothesis that refilling of embolised vessels is mediated by the mobilization of soluble NSC and it is mainly recorded in species with a higher percentage of parenchyma cells that may be important in the hydraulic recovery mechanism as a source of carbohydrates and/or as a source of water.
Keywords: Hydraulic failure; Embolism repair; Non-structural carbohydrates; Parenchyma cells; P50
- 121 -
Introduction Terrestrial plants face the contrasting needs of maintaining a relatively stable water content, while losing large amounts of water vapour to the atmosphere when stomata are open to allow CO2 diffusion into the leaf (Raschke, 1976). In vascular homoiohydric plants, this delicate balance is made possible by continuous absorption of water from the soil, that is then transported to the foliage through the xylem conduits while under negative pressure (Nardini et al., 2018; Venturas et al., 2017). Intense or prolonged drought events pose a serious threat to the survival of plants, including crops and forest trees (Lesk et al., 2016). Drought and heat waves reduce soil water availability and increase atmospheric evaporative demand, putting plants at risk of desiccation. Under these conditions, timely stomatal closure is a fundamental mechanism allowing plants to maintain their hydration status and resist drought stress (Martin-StPaul et al., 2017). However, under prolonged drought, plants can continue to lose significant amounts of water via residual cuticular transpiration (Schuster et al., 2017), leading to progressive dehydration and decreasing xylem pressure. When xylem pressure drops below the species-specific critical thresholds, an air-phase can be aspirated into water-filled xylem conduits through pit membrane pores, leading to gas-filled and non-conductive elements (Mayr et al., 2014; Tyree and Sperry, 1989). When this so-called xylem embolism process spreads to a significant number of conduits in the plant body, leading to high loss of plant hydraulic conductance, the residual water transport capacity cannot assure adequate hydration of tissues and the plant can eventually die by terminal desiccation (Choat et al., 2018; Nardini et al., 2013; Tyree et al., 2002). This typically occurs when xylem hydraulic conductance is reduced by more than 80% in angiosperm species (Nardini et al., 2013; Urli et al., 2013). However, even when trees experiencing non-
- 122 - lethal drought, they can also undergo xylem embolism and suffer from reduced hydraulic efficiency, which potentially translates in decreased photosynthesis, reduced content of non-structural carbohydrates (NSC) and progressive decline of tree vigour and health due to carbon starvation (Peguero-Pina et al., 2018; Tomasella et al. 2019; Zhang et al., 2015). Although trees can recover full hydraulic functionality by producing new xylem conduits (Zhang et al., 2019), different studies have reported that some plants, under specific conditions, can refill embolised conduits with water (Beikircher and Mayr, 2017; Gleason et al., 2017; Love and Sperry, 2018; Savi et al., 2016; Trifilò et al., 2015; Yoshimura et al., 2016). Considering the importance of hydraulic recovery for the overall performance of forest trees and crops under future climate scenarios (Klein et al., 2018), and despite scepticism around the actual occurrence of this phenomenon in planta (Cochard and Delzon, 2013; Wheeler et al., 2013), the possible mechanisms allowing plants to refill embolized conduits have been subjected to intense scrutiny by several studies in recent years (Brodersen et al., 2018; Martorell et al., 2014; Nardini et al., 2018; Ooeda et al., 2017; Secchi and Zwieniecki, 2016; Trifilò et al., 2017). It has been shown that xylem embolism reversal is frequently accompanied by starch degradation and release of sugars in the conduits (Beikircher and Mayr, 2017; Salleo et al., 2009; Secchi and Zwieniecki, 2011; Secchi and Zwieniecki, 2012; Tomasella et al., 2017; Yoshimura et al., 2016; Wang et al., 2018), apparently ‘priming’ the xylem for refilling via an osmotic mechanism when plants are rehydrated and xylem tension is released (Brodersen and McElrone, 2013; Knipfer et al., 2016; Nardini et al., 2011). In accordance, enhanced expression of several genes involved in NSC metabolism and transport, as well as of aquaporins have been reported in plants undergoing hydraulic recovery (Chitarra et al., 2014; Perrone et al.,
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2012; Secchi et al., 2011), and metabolic inhibitors have been shown to block the refilling process (Secchi and Zwieniecki, 2016; Trifilò et al., 2014). Considering the proposed role of stored NSC in the hydraulic recovery process, it has been postulated that vessel-associated parenchyma and/or phloem play key roles in the xylem refilling mechanism (Salleo et al., 2004; Secchi et al., 2017). In fact, the amount of wood parenchyma correlates to the quantity of NSC stored in the stem (Morris et al., 2018; Plavcovà et al., 2016; Pratt and Jacobsen, 2017) and, hence, with the potential osmotic forces generated in refilling conduits after drought relief (Nardini et al., 2018). Previous studies have also shown that post-drought hydraulic recovery is often vigorous and complete in species characterized by low wood density and high stem capacitance (Ogasa et al., 2013; Trifilò et al., 2015), while species with denser wood often do not recover at all their xylem functionality upon rehydration. Wood density and capacitance are inversely correlated (Savi et al., 2017) and generally associated with the percentage of wood volume occupied by parenchyma (Pratt et al., 2007; Pratt and Jacobsen, 2017; Scholz et al., 2007; Zheng and Martinez-Cabrera, 2013), suggesting a possible link between parenchyma abundance, NSC availability, and species- specific capacity to reverse xylem embolism upon rehydration. In the present study, we explicitly tested the hypothesis that species-specific recovery from embolism is correlated with parenchyma abundance, as a proxy for the amount of NSC available to generate the osmotic forces during the refilling process.
Material and Methods Experiments were performed during summer 2017 on twelve adult angiosperm species (Table 4.1), growing in the campus of University of Messina, Italy (38o15’36’’N; 15o35’53’’E; 51 m a.s.l.). Species were
- 124 - selected on the basis of a preliminary screening, considering the xylem parenchyma fraction (Fig. 4.1) and the species-specific vulnerability to xylem embolism (see subsection 4.2.3). Measurements of branches anatomical traits Three branches per species, including those used for hydraulic measurements, were collected from different individuals to estimate the amount of wood parenchyma. For each sample, transverse and tangential micro-sections (25 µm thick) were cut with a rotary microtome (RM 2245, Leica Biosystems, Nussloch, Germany). Sections were stained with a solution of safranin and Astra Blue (1% and 0.5% in distilled water respectively), and fixed on glass slides with Eukitt (BiOptica, Milano, Italy). Images were acquired at 100x magnification, using a D-sight slide scanner (Menarini Group, Florence, Italy) and analysed with ImageJ version 2.0.0. The area of axial parenchyma (AP), including both apotracheal and paratracheal cells, and of radial parenchyma (RP), were measured. For each image, an area of approximately 1 mm2 was selected and the areas occupied by the parenchyma cells were manually outlined. AP and RP areas were converted in percentage values by dividing the recorded areas for the corresponding analysed area. Since the axial and radial parenchyma cells orientate perpendicularly to the transverse and tangential section, respectively, the percentage of xylem parenchyma cells in a unit of wood volume (RAP) was estimated as: RAP = AP + RP. Additional anatomical measurements were performed on branch cross sections using ROXAS v3.0.139 (Arx and Dietz 2005; von Arx and Carrer 2014). Analysis was performed on a wedge of known angle (α) centred at the pith for each image. A first manual editing of the images to outline the contour of the pith and of each ring was carried out. For each outlined sector in the wedge, ROXAS automatically measured the area (A), the number of xylem conduits (Nc) and
- 125 - the mean conduit area (MCA). Then, traits’ data (Yˈ) of pith area (Apith), xylem ring area (RA), total xylem area (XA) and total vessel number were up-scaled to the whole cross section as Y=Yˈ/α× 360. Vessel density (Vd) has been estimated as: (Nc/XA). Measurements of maximum vessel length In order to avoid possible excision artefacts during hydraulic measurements (Venturas et al., 2017), maximum vessel length (MVL) was estimated for each species using an air injection method. At least three branches (more than 1 m long) were collected for each species from different individuals, enclosed in a plastic bag and transported to the laboratory within 10 min. The apical end of the samples was cut and connected to a pressure chamber equipped with a precision pressure test gauge (A4A, Ashcroft Inc., Stratford,CT, USA), while the basal end was immersed in a water-filled tray. A pressure of 60 kPa was applied and the basal portion of the stem was observed with a magnifying lens. Progressive 2 cm cuts were made at the basal end, until a stream of air bubbles emerged from the basal cut section, indicating the presence of at least one vessel cut open at both ends. This length was recorded as an estimate of MVL (Table 4.1).
Vulnerability to xylem embolism Species-specific vulnerability to xylem embolism was assessed in terms of the xylem pressure values inducing 50% loss of xylem hydraulic conductivity (P50). P50 values were obtained by published data for all species (see references in Table 4.1), except in M. alba. For this species, a vulnerability curve was measured using the bench dehydration technique on samples of similar age and dimensions of that used for estimating the ability to recover from xylem embolism. Summarizing, P50 values have been obtained by vulnerability curves performed by the bench dehydration
- 126 - method for all species except for A. unedo and P. latifolia. For these species,
P50 values have been obtained by vulnerability curves performed by the air- injection method.
Estimating dehydration time required to reach 50% loss of hydraulic conductivity The target 50% loss of hydraulic conductivity was set for experiments aimed at verifying species-specific ability to recover from xylem embolism upon rehydration (see below). Preliminary experiments were performed to get information about species-specific dehydration time intervals necessary to reach the desired loss of conductivity. Samples longer at least 2 times the species-specific MVL were detached from plants, put in large plastic bags and transported to the laboratory within about 5 min. In the laboratory, branches were exposed to air, and the percentage loss of hydraulic conductivity (PLC) was measured on stem samples detached at different time intervals (see below for details on hydraulic measurements) until PLC ~ 50% was recorded.
Estimating species-specific capacity for embolism recovery Branches at least 2 times longer than the species-specific MVL were cut in air (Torres-Ruiz et al., 2015), enclosed in a plastic bag and transported to the laboratory where they were bench-dehydrated for the species-specific time required to reach a PLC of ~50% (see subsection 4.2.4). Then, the basal end of samples was put into a water-filled tray, and 10 cm cuts were progressively made under water to progressively relax xylem tension and reconnect the xylem network with water, until obtaining sub-samples shorter than the MVL (relaxed samples, R). This experimental procedure was aimed at avoiding spurious embolism in terminal shoot samples during dehydration
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(Wheeler et al., 2013). Xylem tension relaxation was checked in at least two branches per species, collected from different samples, by measuring the water potential of leaves previously wrapped in cling film and aluminium foil (at least 1 h before the measurement) to assure equilibration with xylem pressure (Yx). Water potential was measured using a pressure bomb in: a) samples collected when PLC ~50% was reached (native samples, N); b) samples at the end of the cutting procedure (relaxed samples, R); c) R samples maintained with their basal end immersed in water for 10 min after the cutting procedure (R10min); d) R samples after 1 h rehydration (R1h). The initial PLC was measured in R samples (PLCR), and the eventual hydraulic recovery was measured in R1h samples (PLCR1h). Xylem recover ability was calculated as ΔPLC = PLCR – PLCR1h. All hydraulic measurements were performed on 10-15 cm long branch segments, using a hydraulic apparatus. Samples were perfused with a solution based on a commercial mineral water containing several ions and with [K+] adjusted to a value of 15 mM by adding KCl in order to simulate native xylem sap content (Trifilò et al., 2014). The solution was initially perfused at a pressure (P) of 8 kPa and, when the flow (F) became stable, the initial xylem-specific stem hydraulic conductivity (Ks) was calculated as
(F/P) × (L/Ax), where L is the sample length and Ax is the xylem cross sectional area. Samples were then flushed at P = 0.2 MPa for 20 min to remove embolism and flow was re-measured at P = 8 kPa to obtain Kmax. The
PLC was calculated as 1 - (Ks / Kmax) × 100.
Impact of phloem girdling on embolism recovery According to previous studies (Trifilò et al., 2014; Venturas et al., 2015), stem girdling inhibits the embolism repair process. Hence, the effect of girdling on hydraulic recovery was tested in 6 out of 12 species. Branches 2-
- 128 - fold longer than species-specific MVL were cut in air, and processed as described in the subsection 4.2.5. However, 1 h before starting the hydraulic measurements, the samples were girdled for their entire length at 15 cm intervals, by removing about 5 mm wide bark rings, avoiding (when possible) areas when leaves were attached. The exposed wood was immediately covered with a thin layer of silicone grease to prevent desiccation (Trifilò et al., 2014). Girdled samples were then measured to estimate their PLC at the end of the relaxation procedure and after 1 h rehydration (see subsection 4.2.5). Estimating non-structural carbohydrates Non-structural carbohydrates (NSC) were measured on the same samples used for hydraulic measurements. Just before hydraulic measurements, a 5 cm stem segment was collected from at least 5 samples per treatment, and microwaved at 700 W for 3 min to stop enzymatic activities. Samples were then oven-dried at 70 °C for 24 h and ground to fine powder (particle size < 0.15 mm). Then, 15 ± 1 mg of material were transferred in a 1.5 ml Eppendorf test vial. The super-natant was collected to measure the soluble
NSC fraction (NSCsol) by means of the anthrone-sulfuric acid assay. A glucose standard curve was used to compare the colorimetric response of the samples, whose absorbance was read at 620 nm, and the NSC content was expressed as mg [glucose] g-1 [DW]. The insoluble fraction was used for starch measurement. Starch digestion was performed overnight at 55 °C, using 100 U of α-Amylase (EC 3.2.1.1, Sigma-Aldrich,St. Louis, MO, USA) and 25 U of Amylogucosidase (EC 3.2.1.3, Sigma-Aldrich, St. Louis, MO, USA) per sample. To prevent further degradation, the samples were boiled for 3 min. For analyses, 2 µl of final supernatant were transferred in a plate reader (1420 Multilabel Counter Victor3, Perkin Elmer), with 300 µl final + volume of essay buffer (Tris-HCl with MgCl2 5 mM, NADP 125 µM, and
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MgATP 1 mM) and evaluated by measuring the florescence of NADPH (329 and 460 nm for excitation and emission wavelength, respectively). Known amounts of Amylose were also processed and analysed to obtain a calibration curve. The final concentration of starch in the sample was then expressed as mg [starch] g-1 [DW].
To quantify eventual variations in NSCsol and in starch content during hydraulic recovery following the rehydration procedure, DNSCsol and
DStarch were also estimated. In detail, DNSCsol was calculated as: (NSCsol, R
- NSCsol, R1h) and DStarch was calculated as: (StarchR - StarchR1h) where
NSCsol, R and StarchR were the NSCsol and the starch content, respectively, as recorded in relaxed samples while NSCsol, R1h and StarchR1h were the NSCsol and the starch content as recorded in in R samples after 1 h rehydration.
Statistical analysis All statistical analyses were performed with the software R (v. 3.3.3, R Core Team, 2017). Differences in PLC between treatments were tested for each species separately, using Welch’s t-test (for species with only 2 treatments) and one-way-Welch’s ANOVA followed by Games-Howell post hoc comparisons (for species with more than 2 treatments) to account for non- homogenous variances. Differences were considered as highly significant for P < 0.05, and marginally significant for P < 0.1. Spearman's rho (ρ) correlation coefficient among anatomical and hydraulic parameters and NSC of all studied species was calculated. For statistically significant correlations (P < 0.05), a regression model was calculated.
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Results Vulnerability to xylem embolism and anatomical traits Across our set of studied species, large variability in vulnerability to xylem embolism estimated in terms of P50 was present (from -0.9 MPa in M. alba to -6.5 MPa in P. latifolia, Table 1). The total amount of wood parenchyma (RAP) ranged from about 14% of wood volume in P. lentiscus and N. oleander to about 29% in M. alba (Fig. 4.1, Table 4.1). Considerable variation was also observed in terms of maximum vessel length, that ranged between 0.2 m in C. siliquastrum to 0.9 m in E. camaldulensis and Q. ilex (Table 4.1). Ray parenchyma (RP) was more abundant than axial parenchyma (AP) in all the study species (Table S4.1). Pith area (Apith) ranged widely between species, from about 6% of stem cross-sectional area in P. lentiscus and Q. ilex, to almost 40% in C. siliqua (Table S4.1, Fig. 4.3). Large differences were also observed across species in terms of vessel density, Vd (from 73 conduits mm-2 in C. siliqua to 420 conduits mm-2 in A. unedo and P. latifolia), mean conduit area, MCA (from 200 µm2 in P. latifolia to 1400 µm2 in M. alba) and number of conduits, Nc (from 500 in M. alba to 3300 in A. unedo) (Table S4.1).
Embolism recovery in response to rehydration in intact and girdled samples
On the basis of species-specific P50 and preliminary experiments, branches of the different species were dehydrated to xylem water potential (Yx) values between about -1 MPa (C. siliquastrum and M. alba) and -6 MPa (P. latifolia) (Table 4.2), to induce PLC values between 40 and 60% (Fig. 4.2). PLC values were measured in samples where xylem tension had been relaxed by progressive cutting samples underwater (Fig. 4.2, R samples). This
- 131 - procedure induced a significant increase in Yx that was already observed at the end of the cutting sequence, when values between -0.4 MPa (C. siliqua, N. oleander and Q. ilex) and -1.1 MPa (A. unedo and O. europaea) were recorded. After R samples had been rehydrated for 10 min (Table 2, R10min),
Yx further increased and ranged between -0.2 and -0.45 MPa. After 1 h of rehydration (R1h), Yx was between -0.02 and -0.1 MPa in all the study species (data not shown). The girdling procedure applied to 6 out of 12 species did not affected the pattern of xylem water potential changes. Girdled branches were dehydrated to statistically similar Yx values (Table 2, NG), and after the fast relaxation procedure (i.e. RG samples) their Yx was not statistically different from that of non-girdled branches. Similarly, RG samples had Yx values similar to R sample after both 10 min and 1 h rehydration. When branches of different species experiencing PLC values of 40-60% (Fig. 4.2, R samples) were rehydrated for 1 h after reconnecting the xylem system to the water source through sequential cuttings based on species- specific MVL, different patterns of hydraulic recovery were observed. In seven out of 12 species PLC significantly decreased within 1 h (Fig. 4.2,
R1h), reaching values between 10 and 20%, while in the remaining five species non-significant or only marginally significant decreases of PLC were recorded. It can be noted that, among the four species showing no rise in PLC (i.e. embolism reversal), three had RAP values < 15% (i.e. Ec, No, Pl), while all species showing a significant or a marginally significant PLC recovery had RAP values > 15% (Table 4.1). Among the species where a significant hydraulic recovery was observed, six were treated with a girdling procedure. In all these species, removing the bark resulted in the suppression of the PLC recovery pattern, that remained close to values recorded in R samples even after 1 h rehydration (Fig. 4.2).
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NSC and starch consumption: relationships with the embolism reversal ability The different studied species also widely differed in terms of starch and soluble carbohydrates (NSCsol). In particular, in R samples, starch content -1 ranged between about 3 (Q. ilex) and 190 mg g (C. siliqua), while NSCsol ranged between 50 (P. latifolia) and 150 mg g-1 (P. lentiscus) (Table S4.2,
Fig. 4.3). Both starch and NSCsol were different between R and R1h samples, but because of the large variability of data, these differences turned out to be non-significant. Nonetheless, NSCsol decreased in the species showing a significant decrease of PLC following rehydration. In fact, a positive correlation was recorded between the decrease in PLC following re- hydration (DPLC) and DNSCsol (Fig. 4.3a). Correlations between pith area and xylem hydraulic traits
Both P50 and DPLC were correlated with the percentage pith area, with species showing the largest volume of wood occupied by pith being more vulnerable to xylem embolism and showing the largest PLC recovery upon rehydration (Fig. 4.3b, c).
Discussion Our results show that excised branches of twelve woody species have different capacities to recover from embolism after 1 h rehydration, despite similar starting PLC levels and similarly high (close to 0) water potential reached at the end of the rehydration period. We show that this species- specific capacity to reverse embolism is correlated with the amount of parenchyma cells, especially of the pith area, and apparently depends on the consumption of soluble NSC. In accordance with previous studies (Trifilò et al., 2014; Venturas et al., 2015), detached branches experiencing substantial embolism level recovered
- 133 - their hydraulic functionality following short-term rehydration. It has been suggested that cut branches put in contact with water can refill xylem conduits via purely physical effects related to capillarity, that would not occur in intact plants (Knipfer et al., 2017). However, it can be noted that capillarity may partially explain the ascent of sap within a vessel. In accordance, it cannot occur in longer and wider vessels. The species-specific occurrence of embolism reversal detected in our experiments, despite similar water potential values reached at the end of the rehydration, calls for a role of biological processes in this phenomenon. In addition, it is unlikely that our PLC data were affected to a significant extent by a ‘cutting artefact’ as described by Wheeler et al. (2013). This is because the samples were processed taking into careful account species-specific maximum vessel length, and were also subjected to a xylem tension relaxation procedure before performing hydraulic measurements. This procedure resulted in xylem water potential values between -0.2 and -0.5 MPa in all the species at the time of cutting, i.e. a range of values recommended by Torres-Ruiz et al. (2015) as optimal for final excision of samples to be used in hydraulic measurements. The biological nature of the embolism refilling process detected in our experiments is also confirmed by the effects of experimental girdling on the recovery process. In fact, selective removal of the bark from branches resulted in similar patterns of water potential decline and recovery during dehydration and rehydration, and similar PLC levels at the end of dehydration. However, girdled samples did not recover the PLC values after 1 h rehydration, in accordance with previous observations in excised branches (Trifilò et al., 2014; Venturas et al., 2015) or intact plants (Christman et al., 2012). In a recent study, Salmon et al. (2018) have shown that detached branches of Betula pendula can refill embolized conduits even
- 134 - under moderate residual negative stem water potential, but the recovery process disappeared when living cells were killed with a heat treatment. Schmitz et al. (2012) reported that inhibition of NSC production by bark and wood photosynthesis resulted in the impairment of embolism repair processes in different mangrove species. All these effects would be consistent with the proposed role of bark, phloem and living cells in general as sources of energy, solutes and water necessary to provide the driving forces and the water volumes to refill embolized xylem conduits (Nardini et al., 2018). Our results suggest that embolism reversal was more effective in species with higher amount of parenchyma cells in their stems, and especially with higher pith area. Taking into account the potential impact of wood parenchyma on wood density, our data are in line with previous studies suggesting that embolism reversal is more likely to be observed in species with lower wood density, which are often those more vulnerable to xylem embolism formation under drought (Nardini et al., 2013; Santini et al., 2016). Although we could not detect a correlation between amount of wood parenchyma and quantity of stored NSC, such a relationship has been previously reported in different assemblages of species (Plavcovà et al., 2016; Pratt and Jacobsen, 2017). Moreover, the correlations recorded between the magnitude of PLC recovery and the consumption of soluble
NSC and between the DPLC and Apith strongly suggest that abundant parenchyma cells might be important in the hydraulic recovery mechanism as a source of carbohydrates, or as a source of water due to the generally high capacitance of woods with high parenchyma fractions (Pratt et al., 2007; Secchi et al., 2017; Trifilò et al., 2015). Pith cells have been recognized acting as water and carbon storage reservoirs especially in succulent species (Goldstein et al., 1984; Hearn, 2009).
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Nevertheless, starch grains have been found also in the pith of some deciduous species and in palms (Essiamah and Eschrich, 1985; Pei-Lang et al., 2006; Piispanen and Saranpää, 2001). Therefore, it could be speculated that an abundant percentage of pith cells and RP, contributing to water and/or carbon storage, are both involved in xylem embolism reversal, as our results suggest. This hypothesis, however, needs to be further investigated in planta. The correlation observed across species between DPLC recovery and the change in DNSCsol recorded during the recovery process is consistent with previous studies suggesting a role for carbohydrates in the embolism reversal process (Savi et al., 2016; Yoshimura et al., 2016; Wang et al., 2018). Progressive depletion of starch and accumulation of soluble sugars in xylem sap has been reported for poplar stems undergoing drought stress (Secchi and Zwieniecki, 2012). It has been suggested that this process primes the xylem for embolism repair via generation of localized osmotic pressures once plant water potential rises again to water potential close to zero following drought relief (Secchi and Zwieniecki, 2016). In our study species, only those undergoing a decline of NSCsol during the rehydration phase finally showed a significant recovery of PLC, supporting the view that during the dehydration phase soluble sugars were released and then used for the refilling process. However, the fate of these sugars after hydraulic recovery is not clear, and our data do not provide evidence for their inter-conversion in starch, or consumption by respiratory metabolism. The conditions experienced by detached branches dehydrated and rehydrated in the laboratory are obviously different from those of an intact plant. Hence, our results do not necessarily provide evidence that embolism reversal does occur in planta. One of the most obvious differences between detached branches and intact plants is related to the conditions during the rehydration phase. In fact, detached branches put in contact with water might eventually
- 136 - rehydrate to water potential values close to zero, a condition that might favor the spontaneous dissolution of the gas phase in embolized conduits (Venturas et al., 2017), or would strongly favor active refilling of conduits avoiding drawdown of refilling water by still functioning conduits under tension. A condition of ‘zero water potential’ is generally not possible for intact plants, unless stem or root pressure are generated (Gleason et al., 2017). In fact, even when at field capacity, soils have slightly negative water potential due to the presence of solutes, and in any case perfect equilibration of plant water potential with soil water potential is rare (Donovan et al., 2001). As a consequence, most woody plants rehydrated by natural precipitation following a prolonged drought are likely to experience some residual negative pressure in their functioning xylem conduits, possibly making impossible embolism reversal. Previous studies have suggested that embolism reversal is possible even under conditions of residual negative water potential (Nardini et al., 2008; Stiller et al., 2005), and this has been explained by anatomically based hydraulic isolation of refilling conduits from still functioning ones (Ooeda et al., 2017). In conclusion, while the eventual occurrence of embolism refilling under tension in the study species needs to be further investigated, our data reports experimental results supporting the hypothesis that refilling capacity of branches is species-specific and related to the amount of parenchyma and NSC.
Author contributions AN, PT and VC conceived and designed the experiment. PT, NK, VC and SV performed the measurements. FP performed the statistical analysis. AN and PT wrote the manuscript, with contributions by all Authors. All authors read and approved.
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Acknowledgments We are very grateful to Dipartimento Regionale Azienda Foreste Demaniali, Messina, Sicily, Italy, for kindly providing plant material.
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Urli, M., Porté, A.J., Cochard, H., Guengant, Y., Burlett, R., Delzon, S., 2013 Xylem embolism threshold for catastrophic hydraulic failure in angiosperm trees. Tree Physiol. 33, 672-683. Venturas, M.D., MacKinnon, E.D., Jacobsen, A.L., Pratt, R.B., 2015. Excising stem samples underwater at native tension does not induce xylem cavitation. Plant Cell Environ. 38, 1060-1068. Venturas, M.D., Sperry, J.S., Hacke, U.G., 2017. Plant xylem hydraulics: what we understand, current research, and future challenges. J. Int. Plant Biol. 59, 356-389. von Arx, G., Carrer, M., 2014. ROXAS – a new tool to build centuries-long tracheid-lumen chronologies in conifers. Dendrochron. 32, 290-293. Wang, A.Y., Han, S.J., Zhang, J.H., Wang, M., Yin, X.H., Fang, L.D., Yang, D., Hao, G.Y., 2018. The interaction between nonstructural carbohydrate reserves and xylem hydraulics in Korean pine trees across an altitudinal gradient. Tree Physiol. 38, 1792-1804. Wheeler, J.K., Huggett, B.A., Tofte, A.N., Rockwell, F.E., Holbrook, N.M., 2013. Cutting xylem under tension or supersaturated with gas can generate PLC and the appearance of rapid recovery from embolism. Plant Cell Environ. 36, 1938-1949. Yoshimura, K., Saiki, S.T., Yazaki, K., Ogasa, M.Y., Shirai, M., Nakano, T., Yoshimura, J., Ishida, A., 2016. The dynamics of carbon stored in xylem sapwood to drought-induced hydraulic stress in mature trees. Sci. Rep. 6, 24513. Zhang, T., Cao, Y., Chen, Y., Liu, G., 2015. Non-structural carbohydrate dynamics in Robinia pseudoacacia saplings under three levels of continuous drought stress. Trees 29,1837-1849. Zhang, Y., Xu, J., Su, W., Zhao, X., Xu, X., 2019. Spring precipitation effects on formation of first row of earlywood vessels in Quercus
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variabilis at Qinling Mountain (China). Trees, doi:10.1007/s00468-018- 1792-y Zheng, J., Martínez-Cabrera, H.I., 2013- Wood anatomical correlates with theoretical conductivity and wood density across China: evolutionary evidence of the functional differentiation of axial and radial parenchyma. Ann. Bot. 112, 927-935.
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Figure legends Figure 4.1: Images at 100x magnification of transverse sections of: (a) Arbutus unedo, (b) Ceratonia siliqua, (c) Cercis siliquastrum, (d) Eycalyptus camaldulensis, (e) Laurus nobilis, (f) Morus alba, (g) Myrtus communis, (h) Nerium oleander, (i) Olea europaea, (j) Phillyrea latifolia, (k) Pistacia lentiscus, (l) Quercus ilex. Scale bars at 100 µm are shown. Figure 4.2: Mean values ± SEM of the percentage loss of hydraulic conductivity (PLC) as recorded in relaxed samples (R), R samples after 1 h rehydration (R1h), in girdled samples (RG) and in RG samples after 1 h rehydration (RG1h). Different letters indicate significant differences from Tukey’s pairwise comparisons or t-test (n ≥ 6). Figure 4.3: Relationships between: a) changes in the soluble non-structural carbohydrate content during hydraulic recovery following the rehydration
(DNSCsol) and the xylem recovery ability (DPLC); b) recovery ability (DPLC) and pith area (Apith) and c) pith area (Apith) and xylem pressure values inducing 50% loss of xylem hydraulic conductivity (P50). Species abbreviations are reported in Table 1. Regression lines (dotted lines) and related b coefficients values are reported. Significance level: *P<0.05,**P<0.01.
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Table 4.1: Names of species, family, abbreviations (Abbr), xylem pressure values inducing ⁓50% loss of xylem hydraulic conductivity (P50), mean values ± SEM of the maximum vessel length (MVL, n=3) and of the percentage of wood parenchyma cells (RAP, n=3).
Species Family Abbr P50 MVL RAP (MPa) (m) (%) Arbutus unedo L. Ericaceae Au -3.1 0.24 ± 0.01 19.2 ± 1.0 (Martinez-Vilalta et al. 2002) Ceratonia siliqua L. Fabaceae Cs -2.6 0.36 ± 0.005 25.6 ± 4.0 (Trifilò et al. 2015) Cercis siliquastrum L. Fabaceae Csq -1.8 0.20 ± 0.01 17.4 ± 2.2 (Nardini et al. 2003) Eucalyptus Myrtaceae Ec -4.6 0.90 ± 0.005 14.8 ± 0.2 camaldulensis Dehnh (Trifilò et al. 2015) Laurus nobilis L. Lauraceae Ln -2.5 0.42 ± 0.01 17.1 ± 0.5 (Trifilò et al. 2015) Morus alba L. Moaraceae Ma -0.9 0.32 ± 0.01 28.6 ± 1.9
Myrtus communis L. Myrtaceae Mc -3.1 0.31 ± 0.02 23.0 ± 1.6 (Trifilò et al. 2015) Nerium oleander L. Apocynaceae No -1.5 0.30 ± 0.02 14.0 ± 1.3 (Trifilò et al. 2015) Olea europaea L. Oleaceae Oe -2.1 0.66 ± 0.005 16.8 ± 0.8 (Trifilò et al. 2015) Phillyrea latifolia L. Oleaceae Phi -6.5 0.26 ± 0.03 20.0 ± 1.7 (Martinez-Vilalta et al. 2002) Pistacia lentiscus L. Anacardacieae Pl -4.1 0.32 ± 0.03 13.7 ± 1.5 (Trifilò et al. 2015) Quercus ilex L. Fagaceae Qi -3.3 0.91 ± 0.01 28 ± 4.0 (Trifilò et al. 2015)
Table 4.2: Mean values ± SD (at least n=2) of the xylem water potential measured in: intact and girdled samples collected when a percentage loss of hydraulic conductivity of about 50% was reached (N and NG, respectively), intact and girdled samples at the end of the cuttings procedure (R and RG, respectively) as well as in R and RG samples maintained with their basal end immersed in water for 10 min after the cutting procedure (R10min and RG, 10min, respectively). Different letters indicate significant differences for Tukey’s pairwise comparisons.
Abbr N NG R RG R10min RG, 10min Au -3.90 ± 0.23a -4.05 ± 0.03a -1.13 ± 0.21b -1.07 ± 0.14b -0.35 ± 0.03c -0.27 ± 0.02c Cs -0.96 ± 0.03a -1.13 ± 0.17a - 0.45 ± 0.05bc -0.60 ± 0.17b -0.20 ± 0.00c -0.22 ± 0.01c Csq -2.302 ± 0.02a -2.42 ± 0.08a -0.39 ± 0.16b -0.4 ± 0.05b -0.20 ± 0.09b -0.20 ± 0.01b Ec -2.27 ± 0.38a / -0.65 ± 0.05b / -0.33 ± 0.06b / Ln -2.75 ± 0.15a / -0.83 ± 0.07b / -0.19 ± 0.09c / Ma -1.05 ± 0.05a -1.18 ± 0.19a -0.5 ± 0.00b -0.5 ± 0.01b -0.18 ± 0.00b -0.19 ± 0.00b Mc -3.40 ± 0.29a / -1.07 ± 0.37b / -0.43 ± 0.09b / No -1.08 ± 0.19a / -0.42 ± 0.02b / -0.25 ± 0.05b / Oe -2.57 ± 0.25a -2.71 ± 0.11a -1.10a ± 0.2bc -1.15 ± 0.25b -0.45 ± 0.05c -0.45 ± 0.04c Phi <-5.27a / -1.34 ± 0.10b / -0.43 ± 0.04c / Pl -4.30 ± 0.24a / -1.37 ± 0.24b / -0.47 ± 0.12c / Qi -3.08 ± 0.12a -3.35 ± 0.05a -0.40 ± 0.07b -0.45 ± 0.05b -0.25 ± 0.05b -0.25 ± 0.05b
Figure 4.1
Figure 4.2
Figure 4.3
CONCLUSIONS
Global change is now occurring. Probably it will continue for the foreseeable future and is likely to intensify in many aspects. It is an emerging reality that will increasingly impact the political process, on regional strategic planning, and on the daily lives of resource managers; learning to live with global change, to avoid the worst hazards and capitalize on opportunities as they arise, requires creative and innovative strategies (Walker and Steffen 1997). The consequences of taking no action now, may not be felt until the middle of the next century, but when these consequences do occur, they could be serious and very difficult to cope with.
The overall aim of this research was therefore to investigate the effects of climate changes, analyzing the effects of changing climatic factors, to plant ecosystems, in the view to better understand global changes in harsh environments, investigating how global changes can influence plant distribution, linked to the different degrees of tolerance and their traits (Pellegrini 2016). This work confirmed the central role of global changes in determining environmental conditions, leading to determining plant distribution (Chapter 1 and 3).
Coastal areas are highly endangered habitats due to their susceptibility to global changes (Defeo et al. 2009) and biological invasion (Campos et al. 2004b; Giulio et al. 2020), often leading to serious ecological problems to the entire ecosystem (Vilà et al. 2010; Simberloff et al. 2013).
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Findings suggest that main soil properties and plant functional traits are linked to the plant invasion across the sea-saltmarsh gradient in lagoon barrier islands. Soil conductivity tends to block both the abundance and specific richness of alien species, favoring the presence of native species; nitrogen and organic carbon in soil were linked to plant abundance. Backdune is the most sensitive habitat to invasion, while foredune and saltmarsh were mostly unaffected by plants. In general, results highlight that in coastal systems some plant communities are much more prone to receive invasion than others, representing real conservation priorities.
Alpine ecosystems and arctic shrubs are extremely sensitive to temperature variation (Sturm et al. 2001; Dial et al. 2007; Blok et al. 2011; Myers-Smith et al. 2015). The anticipated climatic warming has been hypothesized to strongly affect alpine heaths, by extending plant life (Shi et al. 2014), altering nutrient content and/or modifying plant abundance (Kaarlejärvi et al. 2012), and conducting to a common reduction if biodiversity (Ratajczak et al. 2012; Boscutti et al. 2018). Plant–plant interactions represent one of the major selective forces driving population and community dynamics (Callaway and Walker 1997). In general, the obtained results showed an effect of the reduction of precipitation for the selected parameters, with a starch content five times higher values in June, if compared with September. Soluble NSC content shows higher values in LD stands, and this occurs because, in the summer period, plants need more nutrients and intra-specific competition is higher in HD stands. The only parameter that did not shows a significant difference is the annual shoot density.
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The shrub density showed important modifications of the morphological features of the annual shoots. In HD stands, it was found that the shoots length is relevantly higher than in LD stands, suggesting that under normal conditions the growth is faster. It has therefore been verified that drought has a concrete effect on the HD populations of Vaccinium myrtillus by modifying other morphological features.
Across both world and local scales competition between alien species and native species in harsh environments plant communities, in the view of global changes, is clearly of crucial relevance in understanding community changes and to refine strategies for alien species management and containment. However, there is a need for long term monitoring within harsh environments. In chapter 1 annual monitoring of vegetation, as well as sediment characteristics, would reveal whether or not cyclical changes occur. The comparison between fixed points over time is crucial to understand how communities have changed, evaluating how such changes are associated with prevailing abiotic factors. This study would also avoid criticisms about the use of the space-time substitution method (spatial and temporal variation are equivalent) (Pickett 1989; Blois et al. 2013).
This research has supply important insights about the factors affecting the success of alien species in harsh environments and their relative impacts on native plant diversity. However, some key questions are present. Firstly, in chapter 1 our findings suggest that main soil properties and plant functional traits are related to the plant invasion across the shore-saltmarsh gradient in barrier islands. The initial hypotheses were supported by our results, showing that soil conductivity, influenced by rainfall regime, curb both abundance
- 157 - and specific richness of alien species, favoring the presence of native species. This highlights the role of assessing the interactions between alien species and environmental variables when we evaluate their impact on native communities. Secondly, in chapter 3, it has been verified that rain exclusion has a moderate effect on the alpine shrubs by modifying their morphological features.
The results demonstrate the importance of studying a wide range of variables to understand plant responses to environmental global changes. Moreover, methods of sampling and analyses have to be improved. The analysis methodology itself requires reevaluation in further research, to verify if the applied rating system is extensive enough to be able to correctly assess the relevance of the components and the quality of the data. The limited-time available prevented an extensive evaluation of the applicability of the evaluation method.
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APPENDIX
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Study of plant traits, soil features and plant invasion in sandy beach of Grado and Marano lagoon (Northern Adriatic Sea)
Stefano Vitti1,2, Francesco Boscutti2, Elisa Pellegrini2, Valentino Casolo2
1Department of Life Sciences, University of Trieste, Via Weiss 2, 34128 Trieste, Italy;
2Department of Agricultural and Environmental Sciences, Plant Biology Unit, University of Udine, Via delle Scienze 91, 33100 Udine, Italy
113° Congresso della Società Botanica Italiana
V INTERNATIONAL PLANT SCIENCE CONFERENCE (IPSC) Fisciano (SA) 12 - 15 September 2018
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Study of plant traits, soil features and plant invasion in sandy beach of Grado and Marano lagoon (Northern Adriatic Sea)
Stefano Vitti1,2, Francesco Boscutti2, Elisa Pellegrini2, Valentino Casolo2
1Department of Life Sciences, University of Trieste, Via Weiss 2, 34128 Trieste, Italy;
2Department of Agricultural and Environmental Sciences, Plant Biology Unit, University of Udine, Via delle Scienze 91, 33100 Udine, Italy
Biotic invasions are altering the world's natural communities and their ecological characters at an unprecedented rate. If we fail to implement effective strategies to curb the most damaging impacts of invaders, we risk impoverishing and homogenizing the ecosystems on which we rely to sustain ecosystem services given by biodiversity with irreplaceable natural services (1). As also other extreme environments, sandy beaches are undergoing severe changes due to global changes. Climate changes are drastically shifting the most important ecological drivers affecting their plant communities (e.g., sand salinity, sand deposition, wind, marine aerosol, and water table). These modifications can trigger rapid changes in species composition and abundance of a naturally highly dynamic ecosystem, often favoring invasive alien plants. This study aims to evaluate which are the ecological drivers that cause alien species plant invasion, analyzing plant
- 161 - response from cellular to the community level, through a laboratory-field approach, measuring plant traits, and evaluating the species composition of the whole plant community. The study sites are barrier islands of the Marano and Grado lagoon (45°40’40’’ N 13°03’50’’ E to 45°46’30’’ N 13°27’20’’ E). It is located in the north part of the Adriatic Sea (2). The lagoon is morphologically classified as a leaky lagoon (3), with strong tidal influence. The experimental design includes a gradient study (from water edge to dune’s backside) of alien species on native species occurring in diverse plant communities evaluating plant traits and species composition in relation to the most important soil features (e.g. soil salinity, grain size). Plant traits can be divided into physiological (e. g. Non-Structural Carbohydrates, osmolites, secondary metabolites) and morphological (e. g. Specific Leaf Area, biomass, height, number, and, dimension of leaves) while plant communities can be evaluated with taxonomical and functional diversity and plant – plant interactions. Particular attention is given to Ambrosia psilostachya DC., Cyperus esculentus L., and Sporobolus pumilus (Roth) P.M. Peterson & Saarela (4, 5). We expect to clarify the role of soil proprieties on biological invasion, focusing on salt tolerance strategies, which might be a key driver for future scenarios of rainfall reduction and the consequent increase of soil sand content
1) R.N. Mack, D. Simberloff, W.M. Lonsdale, et al. (2000) Ecological Applications, 10, 689-710 2) F. Boscutti, I. Marcorin, M. Sigura, et al. (2015) Estuarine, Coastal and Shelf Science, 164, 183-193 3) B. Kjerfe (1994) Coastal Lagoon Processes. In: B. Kjerfve (ed.) Elsevier Oceanography Series. Elsevier, New York, NY, pp. 1-8 4) G. Galasso et al (2018) An updated checklist of the vascular flora alien to Italy, Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology 5) S. Pignatti (2017) Flora d’Italia, 1-3. Edagricole - New Business Media
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Seagrass - waterbirds interactions in a lagoon ecosystem of the Northern Adriatic Sea
Stefano Vitti1,2, Francesco Boscutti2, Valentino Casolo2, Stefano Sponza3
1Department of Life Sciences, University of Trieste, Via Weiss 2, 34128 Trieste, Italy;
2Department of Agricultural and Environmental Sciences, Plant Biology Unit, University of Udine, Via delle Scienze 91, 33100 Udine, Italy
3Department of Mathematics and Geoscience, University of Trieste, Trieste, Italy
114° Congresso della Società Botanica Italiana VI INTERNATIONAL PLANT SCIENCE CONFERENCE (IPSC) PADOVA (PD) 4 - 7 September 2019
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Seagrass - waterbirds interactions in a lagoon ecosystem of the Northern Adriatic Sea
Stefano Vitti1,2, Francesco Boscutti2, Valentino Casolo2, Stefano Sponza3
1Department of Life Science, University of Trieste, Via Weiss 2, 34128 Trieste, Italy;
2Department of Agricultural and Environmental Sciences, Plant Biology Unit, University of Udine, Via delle Scienze 91, 33100 Udine, Italy;
3Department of Mathematics and Geoscience, University of Trieste, Trieste, Italy
The main goal of this work is to understand the interactions between primary producers (plants) and consumers (herbivore birds) for the conservation of ecosystems such as lagoons, in the view of plant-herbivore interactions. We studied the relationships between the abundance of the herbivore bird Eurasian Wigeon (Anas penelope) and the distribution of three seagrass species (Cymodocea nodosa, Zoostera marina, and Nanozostera noltei) occurring in the Marano and Grado lagoon. This is a large water body located in the north part of the Adriatic Sea, in the Friuli Venezia Giulia region, Italy (1) and it is morphologically classified as a leaky lagoon (2). Twelve bird monitoring areas were monthly surveyed during three years and seagrass distribution data were collected. The overall number of individuals of A.
- 165 - penelope was related to seagrass meadow extension and species cover by using a multiscale approach in four circle buffers (with a radius of 500 m, 750 m, 1,000 m, and 1,250 m). Among the considered scales, the 500 m radius and 1,250 m radius showed similar statistical scores, having lower performances for all the considered statistical parameters. The 750 m radius scale had the best performances. The total number of Eurasian wigeon individuals was related to the area occupied by seagrass meadows and the mean percentage cover of C. nodosa and N. noltei. In particular, the number of observed individuals of wigeons increased where there was a larger area occupied by seagrasses meadows. Results showed also that when C. nodosa mean percentage cover increased the number of wigeons decreased, while if N. noltei mean percentage cover increased wigeon number increased. Finally, Z. marina showed a not statistically relevant influence in all the tested scales. Our findings confirmed that, in lagoon ecosystems, wigeon wintering populations shows co-occurrence on the abundance of seagrass meadows.
1) F. Boscutti, I. Marcorin, M. Sigura, et al. (2015) Estuarine, Coastal and Shelf Science, 164, 183-193 2) B. Kjerfe (1994) Coastal Lagoon Processes. In: B. Kjerfve (ed.) Elsevier Oceanography Series. Elsevier, New York, NY, pp. 1-8
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