Effects of fragmentation and seawater submergence on photochemical efficiency and growth in the clonal invader Carpobrotus edulis

Accepted Manuscript Title: Effects of fragmentation and seawater submergence on photochemical efficiency and growth in the clonal invader Carpobrotus edulis Author: Sergio R. Roiloa Rub´ en Retuerto PII: S0367-2530(16)30147-5 DOI: http://dx.doi.org/doi:10.1016/j.flora.2016.10.002 Reference: FLORA 51022 To appear in: Received date: 11-8-2016 Revised date: 9-10-2016 Accepted date: 11-10-2016 Please cite this article as: Roiloa, Sergio R., Retuerto, Rub´ en, Effects of fragmentation and seawater submergence on photochemical efficiency and growth in the clonal invader Carpobrotus edulis.Flora http://dx.doi.org/10.1016/j.flora.2016.10.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Effects of fragmentation and seawater submergence on photochemical efficiency and growth in the clonal invader Carpobrotus edulis Sergio R. Roiloa1and Rubén Retuerto2 1BioCost Group, Department de Animal Biology, Plant Biology and Ecology, Faculty of Sciences, Universidade da Coruña, 15071 A Coruña, Spain. 2Unit of Ecology, Faculty of Biology, University of Santiago de Compostela, 15781 Santiago de Compostela, Spain. Author for correspondence: Sergio R Roiloa, Email: [email protected] Phone: (+34) 981 16 70 00 ext. 2159 Fax: (+34) 981 16 70 65 2 Highlights • Clonal propagation has been suggested as important traits for plant invasions • Carbohydrates stored in the stolon can be mobilized to buffer stress conditions • We simulated fragmentation and sea-water submergence in a clonal invader • Stolons, as a source of carbohydrates, allows the expansion of clonal invaders Abstract Clonal plants are frequently affected by process of disturbance as fragmentation. The capacity of these fragments to survive and grow after disturbance has important implications for the expansion of clonal plants, and could have special consequences for the colonization of new environments by invasive clonal species. Stolon internodes of clonal plants represent important reserve organs. These storage structures can play a crucial role in the survival and re-growth of clonal plants after an event of disturbance. In this study we simulated physical disturbance by fragmentation of clones of the stoloniferous invader Carpobrotus edulis into ramets with short and long stolon lengths, and a subsequent event of seawater submergence and de-submergence. Ramets with long stolons showed a significantly higher total biomass than ramets with short stolons, supporting the idea that stolon length is related with the amount of reserves stored and with the benefit reported in terms of growth. Our results showed that the benefit of having long stolons was also important for clonal fragments that suffered a process of seawater submergence. Our study suggests that the use of stolon as a source of resources can represent a suitable mechanism for colonization of coastal sand dunes by the aggressive invader C. edulis. 3 Key-words: chlorophyll fluorescence, clonal growth, fragmentation, disturbance, plant invasions, seawater submergence, spectral reflectance, stolon length, storage organ. 1. Introduction Clonal propagation allows the production of a number of ramets that can remain physically connected through stolons or rhizomes, forming large structures that may occupy considerable areas (Cain, 1997; Hutchings et al., 2004; Klimes et al., 1997; Oborny and Price and Marshall, 1999). These large clonal structures may frequently be affected by processes of disturbance that tear them up into fragments of different size (Barrat-Segretain and Bornette, 2000; Latzel and Klimesŏvá, 2009; Stuefer and Huber, 1999). The capacity of these fragments to survive and re-grow after disturbance has important implications for the expansion of clonal plants, and could play a crucial role in the colonization of new environments by invasive clonal species (Dong et al., 2010, 2012; Konlechner et al., 2016; Lin et al., 2012; Song et al., 2013a;). In particular, in rocky coasts, clonal structures as rhizomes or stolons can be very often fragmented by intense wave action (Maun, 1994), and long-distance transported by sea waves, representing an important dispersion strategy after tempests (Aptekar and Marcel, 2000; de la Peña et al., 2011; Harris and Davy, 1986a, 1986b; Huiskes, 1979; Konlechner and Hilton, 2009). Specifically, in coastal sand dunes species, it has been showed that both seeds and clonal structures play an important role in the colonization of new environments (Harris and Davy, 1986a, 1986b). Even more, plant establishment on coastal sand dune can be more successful from clonal 4 fragments than from seeds, because of the large amount of stored reserves in clonal organs (Maun, 2009). Seawater submergence can be a common situation for plants inhabiting rocky coast and foredune habitats. Water submergence usually produces a photosynthetic collapse, due to critical reduction of light and gas exchange, with the resulting energy crisis for the plant. Generally, after the energy crisis the plant will suffer a depletion of carbohydrates that cannot be fully restocked by the acceleration of glycolysis or the induction of fermentative metabolism (Bailey-Serres and Voesenek, 2008; Colmer and Voesenek, 2009; Sairam et al., 2008;). Damages caused by submergence in the plant can continue after de-submergence due to post-anoxic injury (Sarkar et al., 2006). In addition, seawater can induce salt injure on chloroplasts, affecting electron transport, and consequently reducing photosynthetic efficiency (Gao et al., 2015; Larcher, 1995). Saltwater increase the concentration of Na+ and Clin plant tissues, altering ionic ratios in plants and producing ion toxicity (Barrett-Lennard and Shabala, 2013; Grattan and Grieve, 1998; Rhoades et al., 1999). As a result, saltwater can have a negative impact on growth, compromising plant development and survival (Barrett-Lennard and Shabala, 2013; Im et al., 2014) Stolon connections allow clonal plants to be physiologically integrated (i.e. to shareresources between connected modules). During the last decades, many studies have been oriented to explore the benefits of this integration for the colonization of a wide variety of environments by clonal plants (e.g. Alpert and Mooney, 1986; Alpert, 1999; Hartnett and Bazzaz, 1983; Roiloa et al., 2014c; Roiloa and Retuerto, 2006; Slade and Hutchings, 1987). However, the potential benefits for clonal plants of the reserves storage in the stolons have been less explored (but see; Dong et al., 2012, 2011, 2010; Isogimi et al., 2014; Lin et al., 2012; Stuefer and Huber, 1999; You et al., 5 2014). By storing resources, stolons can play a decisive role in the survival and regrowth of clonal plants after disturbance events. Resources stored in the stolon can be mobilized helping to buffer stress conditions (Goulas et al., 2001; Stuefer and Huber, 1999; Suzuki and Stuefer, 1999), and therefore can represent an additional benefit from clonal attributes. Carpobrotus edulis L. is a stoloniferous invader that inhabits coastal habitats wheretheir clonal clumps may experience natural disturbances, as tempests, with fragmentation of the clonal system in pieces of different size, These may be wash down by the sea and later returned to land. In this study, we simulate physical disturbance in C.edulis by fragmenting their clones into ramets with short and long stolon lengths, and a subsequent event of seawater submergence and de-submergence. The objective of this study is to determine the role of stolons as storage organs in maintaining photochemical activity and growth of C. edulis clones after fragmentation and seawater submergence. Recent studies have been conducted to determine the importance of clonal integration (i.e. resource sharing between connected modules of the clone via stolons) in the expansion of C. edulis (Roiloa et al., 2014a, 2014b, 2013, 2010). However, the role of clonal structures as storage organs and their potential contribution to the expansion of this invader has not yet been investigated. Specifically, we aim to respond to the following questions: (1) does stolon length affect photochemical activity and growth of C. edulis after physical fragmentation? Because stolons can act as reserve organs (Goulas et al., 2001; Stuefer and Huber, 1999; Suzuki and Stuefer, 1999), and the stolon length may be positively correlated with the amount of reserves stored (Dong et al., 2010), we expect that increased stolon length will positively affect the photochemical activity and growth of the fragmented ramets. (2) Are growth and photochemistry of longer ramets less 6 affected by a stressful event of seawater submergence? Because reserve storage could be critical to mitigate the carbohydrate crisis due to submergence (Striker, 2012), we predict that the benefits of increased stolon length, in terms of growth and photochemical activity, will be greater for ramets subjected to seawater submergence. 2. Material and methods 2.1. Study species Carpobrotus edulis (L.) N.E. Br. is a mat-forming succulent plant native to the Cape (South Africa), and currently an aggressive invader in all the Mediterranean climate areas around the world, where it colonizes rocky coast and coastal sand dunes (D’Antonio, 1993; D’Antonio and Mahall, 1991; Traveset et al., 2008; Vilà et al., 2008). C. edulis propagates clonally by the production of stolons, showing a radial growth with a structure of nodes and internodes that allows an effective colonization of the surrounding area (Wisura and Glen, 1993). 2.2. Experimental design Forty similar size un-rooted ramets of C. edulis were collected in a dune system in Quiaios (Portugal) (40°13'N, 8°53'W). Each ramet was obtained by excising the fourth unit from the apex of a maternal clump. Normally, the fourth unit from the apex represents in C. edulis a well-developed ramet. This protocol allowed to standardize the age, size and developmental stage of the plant material used in the experiment. Ramets were collected from a total of 10 maternal clumps (4 ramets from each) separated from each other by at least 50 m. Because plant material was collected over a relatively large area, we assume that each clump represents a different genotype. The experimental design included two crossed factors: ‘stolon 7 length’ (short, long) and ‘seawater submergence’ (submerged, non submerged) (see Fig. 1). To test the effect of stolon length as a storage organ we prepared ramets with short (2.11 ± 0.09 cm, mean ± SE) and long (9.06 ± 0.18 cm, mean ± SE) stolons by cutting the appropriated length with scissors. We did not observe any negative effect of cutting the stolon (as sudden death or diseases). Long and short stolons significantly differed in length (F1,38 = 1095.752, P < 0.001), but not in initial fresh biomass of the ramets (F1,38 = 3.041, P = 0.089). To test the effect of seawater submergence on plant performance, half of the ramets in each ‘stolon length’ treatment were seawater submerged during 48h in a unique cycle, and the other half were not. Submerged ramets (leaves + stolon) where immersed in a 20cm-depth tray with seawater, whereas non-submerged plants remained in a tray without water. We used seawater collected from the Atlantic Ocean, in the proximities where C. edulis inhabit (ca. 34 g salts/L water; Na 11 g/L water). After this, all the ramets were planted individually in 5L plastic pots filled with sand from dunes where C. edulis inhabits. These treatments imitate natural conditions, where clones of C. edulis inhabiting rocky coasts are fragmented into pieces of different size by the action of storm waves, transported along the shore and stranded later on the beaches and foredunes. Although the period of seawater submergence could be quite variable, submergence during 48h is a realistic scenario for C. edulis, where plant fragments are abandoned again in the beach after a relatively short period of time. Ramets from each of the original maternal clumps were equally represented in each combination of treatments. Each treatment was replicated 10 times. The experiment was carried out in a greenhouse at the University of Santiago de Compostela (Spain) during 3 months, from March 27 until harvest on June 29. All the ramets were randomly arranged in the 8 greenhouse. Plants grew under a natural day/night light cycle and were watered regularly to prevent water stress. 2.3. Growth and physiological measurements Growth: At the end of the experiment, ramets were harvested individually, divided into shoots (leaves and stolons) and roots, oven-dried at 60 °C to constant weight and weighed to the nearest 0.0001 g (Mettler AJ100, Greifensee, Switzerland). Total mass was calculated for each ramet as the sum of shoot and root dry mass. In addition, the proportional biomass allocated to roots was also determined as the ratio root/total mass (root mass ratio, RMR). Chlorophyll fluorescence: Chlorophyll fluorescence parameters were measured every 30 days from the start of the experiment in all the ramets by the saturation pulse method (Schreiber et al. 1998), using a portable fluorometer (MINI-PAM photosynthesis yield analyser; Walz GmbH, Effeltrich, Germany). The maximum (Fv/Fm) and actual (ΦPSII) quantum yield of photosystem II (PSII) were determined as follows: Fv/Fm was calculated as (Fm - F0) / Fm (Bolhàr-Nordenkampf et al., 1989), where F0 and Fm are, respectively, the minimal and maximal fluorescence yield of a dark-adapted sample, when all PSII reaction centres are fully open (i.e. all primary acceptors oxidized). The Fv/Fm ratio provides information on the efficiency of excitation energy capture by open PSII reaction centres (Butler and Kitajima, 1975) and is correlated with the amount of carbon gained per unit of light absorbed (BolhàrNordenkampf and Öquist, 1993). On the other hand, ΦPSII was calculated as (F′m - Ft) / F′m (Genty et al., 1989), where F′m is the maximal fluorescence yield reached in a pulse of saturating light with an illuminated sample, and Ft is the fluorescence yield of the leaf at a given photosynthetic photon flux density. This parameter was measured 15 representing an important mechanism for dispersion and colonization of new coastal environments (Harris and Davy 1986a, 1986b). In this sense, the use of stolons as storage organs could be playing an important role in the expansion of clonal invaders (Dong et al., 2012, 2011, 2010; Konlechner et al., 2016; Lin et al., 2012). Attributes associate to clonal propagation have been recently suggested as important traits for plant invasions (Liu et al., 2006; Song et al., 2013b). Most of the studies have been focused on the benefits of physiological integration (Song et al., 2013b; Wang et al,. 2008; Yu et al., 2009) for clonal invaders, and recent studies explicitly conducted with C. edulis have reported benefits of clonal integration at physiological and morphological level (Roiloa et al., 2016, 2014a, 2014b, 2013, 2010). However, this is the first research exploring the benefit of clonal structures as storage organs for the invader C. edulis. Our study suggests that the use of stolons as a source of resources can represent a suitable mechanism for colonization of new coastal dunes habitats by the aggressive invader C. edulis. In this regard, our results indicate that C. edulis inhabiting rocky coast could be considered as a source of propagules, with potential to be transported along shore by waves and colonize new coastal habitats. This information is particularly interesting for managing plant invasions. Acknowledgements We thank to A.R. Blanco for assistance in the greenhouse. Financial support for this study was provided by the Spanish Ministry of Economy and Competitiveness (projects Ref. CGL2013-44519-R, awarded to S.R.R. and Ref. CGL2013-48885-C22-R, awarded to R.R.). These projects were co-financed by the European Regional Development Fund (ERDF). This is a contribution from the Alien Species Network 16 (Ref. R2014/036 – Xunta de Galicia, Autonomous Government of Galicia). 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Fv/Fm ΦPSII CHL PRI Between-subject effects df F P df F P df F P df F P Stolon 1 0.892 0.351 1 0.182 0.672 1 2.257 0.142 1 2.711 0.108 Submergence 1 4.738 0.036 1 2.016 0.164 1 10.544 0.003 1 1.245 0.272 Stolon x submergence 1 0.818 0.372 1 0.540 0.467 1 0.335 0.566 1 0.924 0.343 Error 36 36 36 36 Within-subject effects Time 2 11.541 <0.001 2 26.845 <0.001 2 108.741 <0.001 2 17.334 <0.001 Stolon x time 2 1.571 0.215 2 0.432 0.651 2 0.016 0.542 2 2.462 0.092 Submergence x time 2 1.720 0.186 2 0.066 0.936 2 0.097 0.031 2 0.919 0.404 Stolon x submergence x time 2 0.555 0.576 2 0.267 0.766 2 0.014 0.588 2 0.884 0.417 Error 72 72 72 72