First evidence of root morphological and architectural variations in young Posidonia oceanica plants colonizing different substrate typologies
Elena Balestria,*, Davide de Battistia, Flavia Vallerinia and Claudio Lardiccia a Department of Biology, University of Pisa, Pisa, Via Derna 1, 56126, Italy
*Corresponding author. Department of Biology, University of Pisa, Pisa 1, 56126, Italy Tel: + 390502211442. Fax: +39050 2211410
Abstract
Root morphology and root system architecture of young Posidonia oceanica plants established on two contrasting substrate types, sand and rock, were examined to provide insights into the strategy of adaptation of seagrasses to their environment. After germination, seedlings were planted on sandy patches and on rock within the same area, and survived plants were collected five years later for measurements of the size of the entire root complex and analysis of individual morphological and architectural root traits. Collected plants exhibited up to nine highly intermingled root systems and approx. 2.5 m of total root length. Maximum horizontal extension, total biomass and total length of roots were not significantly affected by substrate. However, on sand roots grew vertically reaching up to 13 cm, while on rock they extended more horizontally and did not penetrate deeper than 5-7 cm leading to the formation of a shallow, densely packed root complex. On rock, the number and the length of second-order laterals on an individual root system were reduced and the topological index higher than on sand (0.8 vs. 0.7) reflecting a more simple (herringbone) branching pattern. Again, root diameter was greater than on sand. The results suggest that P. oceanica can adjust root traits early during plant development according to substrate typology to maximize anchorage and substrate exploration efficiency. This plasticity enables the species to establish and persist also on rocky bottoms which generally prevent establishment of the majority of seagrasses.
Key-words: adaptive strategy; Mediterranean Sea; root plasticity; seedling recruitment; seagrass meadows; substrate
1. Introduction
Seagrasses are a dominant component of communities in shallow marine habitats where they form extensive root/rhizome systems (Hemminga, 1998) which grow both vertically and horizontally on a variety of soft substrates like sand and mud. However, a number of species can also occur on hard substrates such as reefs and rocks, and few species grow predominantly on rocks (Table 1). Yet, very little is known about the shape of the entire root system and its structure or root system architecture (Lynch, 1995), especially at the early phases of plant development which are the most critical in the life seagrass history (Duarte et al., 1998; Kuo and den Hartog, 2006; Kiswara et al., 2009; Hovey et al., 2011; Statton et al., 2014). This information is needed to better understand the adaptive mechanisms that enable some species to establish and colonize environments differing in substrate typology, in particular sandy and rocky substrates. Indeed, the establishment of new seagrass clones may be largely dependent on the ability of seedlings to produce rapidly an extensive root system and penetrate deep into the substrate so to cope with frequent physical disturbances and scarce availability of nutrients (Duarte and Sand-Jensen, 1996; Reed et al., 1998; Balestri and Bertini, 2003; Orth et al., 2006; Statton et al., 2013, Statton et al., 2014). The typology of substrate is expected to be important in determining the growth and spatial distribution of roots. There is evidence from terrestrial habitats that soft substrates of low cohesive strength may facilitate root penetration and lateral root expansion, even though predispose young plants to uprooting and complete dislodgment by physical disturbances (Dupuy et al., 2005). On the other hand, compacted or hard substrates such as rocks may obstacle root penetration due to structural constraints, making root growth quite problematic. In particular, the mechanical resistance to growing roots may constrain post-embryonic root development and the spatial configuration of roots, restricting root penetration and reducing root elongation rates in many species (Dexter, 1988; Materechera et al., 1991; Masle, 2002; Vocanson et al., 2006; Konôpka et al., 2009; Bécel et al., 2012; Tracy et al., 2012). These alterations are deleterious to plant growth because they limit the volume of soil
exploited and reduce uprooting resistance (Ehlers et al., 1983; Dexter, 1988; Goodman and Ennos, 1999; Masle, 2002; Pagès, 2002; Grzesiaka et al., 2013). However, some species have shown morphological features and compensatory mechanisms, including greater elongation of laterals and thickening of the roots, which enable them to continue growth on hard substrates (Zwieniecki and Newton, 1995; Pott et al., 2012).
Members of the genus Phyllospadix, the only seagrasses inhabiting predominantly rocky substrates, are known to have morphological and anatomical root adaptations, such as extremely short roots covered with fine short root-hairs to penetrate minute irregularities of the substrate surface, that enable seedlings to firmly attach to rocks (Cox et al., 1992, Reed et al., 1998). However, accurate, quantitative information on seagrass root system architecture is presently restricted to a limited number of species that usually grow on soft substrates (two temperate species, Posidonia australis Hok. f and P. sinuosa Cambridge et Kuo, and six tropical species Cymodocea rotundata Ehrenb. et Hempr. ex Aschers., Cymodocea serrulata (R. Br.) Aschers. et Magnus, Halodule uninervis (Forssk.) Aschers., Syringodium isoetifolium (Aschers.) Dandy, Enhalus acoroides (L. f.) Royle) and Thalassia hemprichii (Ehrenb.) Aschers. (Kiswara et al., 2009; Hovey et al., 2012; Statton et al., 2013, Statton et al., 2014). These plants have been shown to posses a relatively simple, conservative (herringbone) root system architecture (i.e. a main root axis with many first-order laterals roots but few higher-order laterals), commonly observed in slow-growing plant species from nutrient poor environments (Fitter, 1991). In P. australis seedlings, textural sediment proprieties have been found to substantially alter the development of roots (Statton et al., 2013, Statton et al., 2014). Sediments containing a higher percentage of fine-textured particles “lubricate” roots allowing them to penetrate deeper the sediment, whereas coarse sandy sediments producing an interlocking matrix impede root growth (Statton et al., 2013). However, how seagrass species that commonly inhabit soft substrates manage the overall form of their root system when they established on hard rocky substrates remains to be elucidated.
Table 1. approx. here
Posidonia oceanica, L. Delile, the endemic seagass to the Mediterranean, is an extremely long-lived and slow-growing species that forms large clones spreading over up to 14 kilometers (Arnaud-Haond et al., 2012) and a dense network of roots and rhizomes that can extend several meters deep into soft substrates (Molinier and Picard, 1952; Duarte et al., 1998). It is one of the few species capable of colonizing sandy bottoms and hard substrates such rocks and artificial rubble mounds without forming a high matte (Di Carlo et al. 2007; Montefalcone et al., 2014). Seedlings has been rarely observed in nature, but there is growing evidence that recruitment via seedlings plays an important role in the processes of colonization of new areas and recolonization after disturbances (Balestri and Cinelli, 2003; Diaz-Almela et al., 2006; Balestri and Lardicci, 2008). Recent studies have documented the presence of germinated seeds of P. oceanica on a variety of soft substrates and rocks (Balestri et al., 1998a; Piazzi et al., 1999; Balestri and Lardicci, 2008; Domínguez et al., 2012; Alagna et al., 2013). Available data on seedlings established on sand and rock have not provided evidence for substantial effects of substrate type on their performance at least within the first two years (Balestri et al., 1998a; Piazzi et al., 1999; Balestri and Lardicci, 2008; Domínguez et al., 2012; Alagna et al., 2013). This could suggest that in this species root growth is programmed to cope with the substrates which germinating seeds initially encounter. However, information on the root system of seedlings is limited to variables such as root number, length and biomass of roots (Balestri et al., 1998a,b; Balestri and Bertini, 2003; Belzunce et al., 2008; Balestri et al., 2009; Alagna et al., 2013), which alone cannot provide a complete understanding of the influence of substrate on root development and architecture.
Knowledge of how root morphology and architecture of individual plants change early with the type of rooting substrate may provide valuable insights into the strategies of adaptation of seagrasses to their environment. It may also provide new elements that could be useful in restoring meadows. Due to the decline of P. oceanica populations in many areas (Marba` et al., 2005) and
consequent loss of important ecosystem functions and services they provide, the conservation and restoration of meadows is a critical need for coastal management (Hemminga and Duarte, 2000; Duarte, 2002; Vassallo et al., 2013; Duarte et al., 2013). Planting seedlings is likely to be an effective and sustainable technique for restoring damaged meadows, and a better knowledge of root system proprieties and substrate conditions conducive to great or faster root development in young seagrasses is essential to improve restoration success (Balestri et al., 1998a; Reed et al., 1998; Terrados et al., 2003; Balestri and Bertini, 2003; Balestri and Lardicci, 2006; Orth et al. 2006; Dominiguez et al., 2012; Statton et al.., 2013; Statton et al., 2013 ).
The aim of this study was to examine how root morphology and architecture of the root system of young P. oceanica plants change with the typology of substrate. Germinated seeds were transplanted in the field within the same area but in contrasting microhabitat typologies (sandy patches and rock crevices), and successfully established plants were harvested five years later for measurements of the whole root complex and analyses of individual morphological and architectural traits. The “whole root complex” refers here to the entire belowground parts of an individual plant (clone) that consisted of a variable number of adventitious roots with their lateral roots, while the “individual root system” refers as a single adventitious root with its laterals produced on a single ramet. We hypothesized that (i) young plants have a simple (herringbone) root system on the basis of Fitter's topological prediction for slow-growing species from nutrient poor environments (Fitter, 1991) and (ii) root morphology and root branching pattern traits are modified by rocky substrate but whole root system size remained unchanged due to compensatory responses.
2. Materials and methods
Naturally detached fruits of Posidonia oceanica were collected in June 2004 soon after their deposition on a beach near Livorno (Calambrone, 43°37' N, Ligurian Sea, Italy) and transported to the laboratory where seeds were manually extracted from fruits. Previous observations indicated that these fruits were produced on a P. oceanica meadow located in front of the beach that covers a large coastal area between 3 and 25 m deep (Balestri et al., 2006). Mature seeds were germinated in floating plastic boxes maintained in an aquaculture tank equipped according to the protocol described in Balestri and Lardicci (2012). One month after germination (July 2004), seedlings (200) of uniform size were transplanted into a shallow and relatively sheltered coastal oligotrophic site near to the upper limit of the parent meadow (Antignano, Livorno, 43° 30' N). The site is characterized by a rocky bottom (beach rock) and interspersed patches of medium-fine calcareous sand. The occurrence of germinated seeds in the past (Piazzi et al., 1999) suggested that this site was suitable for seedling growth. Seedlings were individually planted, at least 50 cm apart each other, within the same area (150 m2) at about 1.5 m depth. One half of seedlings (100) were planted
on sandy patches and the remaining half (100) were placed into crevices on beach rock colonized by algal turfs. At the time of planting, seedlings consisted of a single shoot with 3-5 leaves, one primary root and two adventitious root primordia. In P. oceanica, the primary root differentiates from the radicle of seed already present in the seed embryo and gives rise to an unbranched root axis that generally ceases growth 3-4 weeks after emergence (Balestri et al., 1998b; Balestri et al., 2008; Belzunce et al., 2008). Adventitious roots differentiate from rhizome nodes on ramets in coordination with the development of the rhizome system, and lateral roots originate from the branching of the main root axis of adventitious roots (Balestri et al., 1998b; Belzunce et al., 2008). No artificial structure was used to anchor seedlings to the substrate to avoid any possible interferance on root growth.
Five years after planting (July 2009), five surviving plants per substrate type were randomly chosen. At this time, plants exhibited one emerging lateral rhizome branch and hence initiated clone development. All root systems on each plant were excavated using small hand tools with great care to preserve the integrity of the entire root complex. Before transporting plants to the laboratory for root measurements, the maximum width (i.e., maximum horizontal spread) and maximum rooting depth of the root complex and the length of the rhizome of each plant were recorded in situ to avoid possible deformation. Measurements were made to the nearest millimetre with a ruler.
In the laboratory collected plants were gently rinsed with fresh seawater to remove fine sediment particles and a digital photo of each plant was made. All root systems on each plant were dissected from the rhizome, carefully separated each from others, counted and tagged. Each individual root system was decomposed in the main root and lateral roots which were then cut into root segments (1-4 cm in length) according to changes in growth direction and insertion on the main root axis to facilitate root measurements. Root segments were spread on a squared paper and scanned. Images were analysed using ImageJ 1-42 software (http://rsbweb.nih.gov/ij/) after resizing the images to 300 dpi/inch black-and-white resolution. Image data were used for determination of total root length of the entire root complex of each plant.
Subsequently, two individual root systems produced on different ramets on each plant were chosen for topological analysis. This analysis was based on the root branching pattern, particularly the number of external links (i.e., terminal root sections between the meristem and the nearest branching point) also referred to as magnitude, the longest path (largest number of links between an external link and the base link) also referred as altitude, and total pathlength defined as the sum of all paths for each external link. The topological index (TI) was calculated as the ratio of the natural logarithm of the altitude and the natural logarithm of the magnitude. Although not necessarily the best index to describe topology (Berntson, 1994, 1995), it is the most commonly used index in the literature on root topology (Taub and Goldberg, 1996; Arrendondo and Johnson, 1999). A TI close to 0 is typical of dichotomous branching pattern while a TI close to 1 indicates a monopodial or
herringbone pattern (Fig. 1; Fitter, 1991, 1996). The number, total lengths of first- and higher-order lateral roots and the total length of each individual root system (main root plus laterals) were also calculated. Additionally, the maximum diameter of the main root and first-order laterals on each of the two systems was measured with a stereo microscope. Finally, all the roots on a root system were dried at 70°C for 48 h and weighed to determine their biomass, and the root biomasses of all the root systems produced by each plant were summed to obtain the biomass of the whole root complex of each plant.
Figure 1. approx. here
2.3. Statistical analysis
Treatment (sand or rock) effects on the different variables used to characterise the whole root complex (rooting depth, horizontal spread, number of individual root systems, total root length and root biomass) were analysed by one-way analysis of variance (ANOVA). Architectural and morphological characteristics (number and length of laterals, total root length, topological index and diameters of the main root axis and first-order laterals) of individual root systems were analysed by two factor nested ANOVA (mixed model) with substrate as fixed factor, individual plant as random factor nested in substrate and individual root system nested in plant and substrate treatment. This approach allowed us to test the null hypotheses that there were (i) no difference in the mean values between the two substrate types and (ii) no difference in means between all possible plants in any of the two substrate type. When significant differences were found in ANOVAs, Tukey test was performed to identify differences between treatments. Prior to ANOVAs, all data were analysed for normality using normal probability plots of residuals. Since the number and the length of secondary-order branches and root diameter data were not normal, they were log transformed to
correct for deviations from normality before analysis. The assumption of homoscedasticity was checked with Cochran C test (Underwood, 1997). This test indicated that for rooting depth data variances were not homogenous, and standard transformation procedures were not effective in restoring homoscedasticity. However, ANOVA is likely to be generally robust to moderate violations deviations from the assumption of homogeneity of variances particular in the case of balanced (equal n) designs (Underwood, 1997), so the analysis was continued using original rooting depth data setting the F-test alpha to 0.01. Two variables, number and length of third-order laterals, were not individually analyzed with ANOVA for the effect of substrate type because there was not enough number of plants (three plants) with third-order laterals for statistical comparison. All statistical analyses were performed with StatSoft (2007).
3. Results
Five-year old Posidonia oceanica plants had a relatively short plagiotropic rhizome axis, mean rhizome length 3.1 cm ± 0.3 (standard error, SE) on sand and 3 (± 0.2) on rock, with one living shoot and one emerging lateral rhizome branch. They also exhibited a variable number (4-9) of highly intermingled root systems along the plagiotropic rhizome (Table 2). On average, there were 2.3 root systems every 1 cm of rhizome produced. The network formed by these systems reached a maximum depth of about 12 cm and a maximum horizontal extension of about 11 cm (Table 2).
Table 2. approx. here
Branching intensity on root systems was relatively low and branching orders did not exceed 3. However, only three of the selected plants had third-order laterals, and amount of these roots was negligible (less than 2 roots, 1-2 mm long). .Both root penetration and distribution of roots within the substrate matrix were significantly influenced by substrate type (Table 2). On sand, roots grew
typically vertically within the substrate and the maximum rooting depth of the entire root system was about 12 cm (Fig. 2a). In contrast, on rock roots initiated elongation in vertical direction but later they changed direction and grew more horizontally within about 6 cm of substrate (Fig. 2b). As a result, the maximum rooting depth of plants on sand resulted about two fold greater than that of plants on rock (Table 2).
No significant differences were detected for the number of individual root systems, total root biomass and total length of the entire root complex between the two substrates (Table 2). The maximum horizontal spread of the root complex of plants on rock tended to be greater than that of plants on sand, but ANOVA did not detected significant differences between the two substrates for this variable (Table 2).
Figure 2. approx. here
Significant differences between the two substrate types were found for some root morphological and architecture traits. Specifically, the number and the length of second-order laterals on an individual root system were significant lower for plants on rock than on sand (Fig. 3; Table 3).
Table 3. approx. here
Instead, the maximum diameters of the main root axis and first-order laterals of plants on rock were greater than those on sand (Fig. 4; Table 3). Again, the topological index of the root system on rock was significantly higher than that on sand (Fig. 5, Table 3). The root system of plants established on rock had a branching pattern close to herringbone (mean topological index, 0.88 ± 0.02) while that of plants on sand tended to a more branched or dichotomous pattern (mean topological index 0.78 ± 0.02; Fig. 5). Significant differences were also observed among plants for the number and the length
of second-order laterals and the total root length of the individual root system (Figs. 3 and 5; Table 3).
Figure 3. approx. here
Figure 4. approx. here
Figure 5. approx. here
4. Discussion
Results of the study show that five years after establishment Posidonia oceanica plants had produced up to 9 individual root systems highly intermingled with each other, each consisting of a main axis with relatively many first-order laterals and few higher-order laterals. On average, the total length reached by roots was rather surprising (up to 2.5 m) if compared with the poor root development reported for adult rhizomes of this species (Lepoint et al., 2004; Balestri and Lardicci, 2006). This might reflect the importance to form early a significant root presence for anchorage and resource capture. The relative root length to the horizontal rhizome axis (approx. 0.7 m of root length per cm of rhizome) was comparable to that of two closely related temperate species, P. australis and P. sinuosa (approx. 0.5 m of root length per cm of rhizome) which, however, grow faster than P. oceanica (Hovey et al., 2011; Hovey et al., 2012). The type of rooting substrate substantially affected root distribution and it also caused the roots to increase in diameter. In particular, rooting depth was reduced by 50% on rock because roots were unable to physically penetrate deep into the substrate matrix. The mechanical forces imposed by rock on roots induced most of them to change the trajectory of growth and expand horizontally possibly to increase the chance to access fissures or to encounter areas of less resistance to penetration: This explains the
development of a shallower and more densely packed root structure on rock comparatively with sand. The capacity of roots to alter growth direction revealed a flexibility that the might be related to large proportion of cortical tissue in roots (Belzunce et al., 2008). Instead, the increased diameter of roots might be interpreted as a higher investment of resources in strengthening of roots in response to mechanical resistance. Both the forms of response by roots to rocky substrate are consistent with the most common root responses to compacted sediments or rock recorded in some terrestrial plants (Materechera et al., 1991, 1992; Clark et al., 2003; Fageria et al., 2006; Tracy et al., 2012). Our results also revealed differences among plants for some architectural traits (number and length of second-order laterals and total root length of individual root systems) independently of substrate type. This suggests the existence of genotypic variability in these traits, a phenomenon reported in some terrestrial plants (Ludlow and Muchow, 1990; Manschadi et al., 2008).
Results of topological analysis revealed that the root system of young plants had relatively simple architecture with topological indices close to those previously reported for adult seagrasses that commonly occur in sandy substrates (0.7 to 0.8; Kiswara et al., 2009; Hovey et al., 2012), confirming our hypothesis. Moreover, they indicated that the topological pattern may be partially modified by the nature of the rooting environment. Indeed, plants established on rock had a root system topology conforming nearly to the herringbone pattern indicating an explorative strategy of root system architectural development, while those grown on sand had a more complex topology tending to a more dichotomous or intermediate pattern.
It is well known that the topology of the root system plays an important role in resource uptake and anchorage efficiency. Cost–benefit analyses for terrestrial plants suggested that a more herringbone pattern may be favorable to slow-growing species or in habitats where resources are scarce or difficult to obtain as it minimizes intra-plant root competition, but is more expensive to construct and maintain than a dichotomous one (Fitter, 1991). Instead, a more dichotomous pattern may be more suitable for fast-growing species or in rich-nutrient habitats as it causes more inter-root competition, but is less expensive to construct (Fitter, 1991; Fitter and Stickland, 1991;
Berntson and Woodward, 1992; Taub and Goldberg, 1996). Moreover, this typology is more efficient and resistant to uprooting than a herringbone one on soft substrates of low cohesive strength due to the increased rooting depth and higher number of laterals (Stokes et al., 1995). In seagrasses, a more dichotomous pattern has been previously reported for two temperate species, P. sinuosa and P. australis, and this pattern has been related to hydrodynamic exposure in the wave-exposed sandy habitats where these species were found (Hovey et al., 2009). As P. oceanica usually lives in poor-nutrient and exposed habitats, the development of a more dichotomous pattern and deeper root system (up to 12 cm) observed here on sand might be therefore interpreted as an adaptive strategy to increase plant anchorage efficiency on non-cohesive substrates so to reduce the risk of uprooting by hydrodynamic forces. On the other hand, rocks may offer a stable platform for root attachment. Indeed, the results indicated that roots of P. oceanica tended to be mostly concentrated in the upper layer of substrate and this physical segregation generated root system overlap. A recent study has shown reduced growth rate in adult shoots of P. oceanica at rocky sites compared with sandy sites, and this reduction has been related to possible restricted root penetration and nutrient deficiency (Di Maida et al., 2013). Here, total length and total biomass of all root systems produced by plants were conserved on rock, confirming the hypothesis that restricted root penetration was compensated at least during the first five years of plant development. This therefore seems to support the hypothesis that root growth is programmed to cope with the substrates which germinating seeds initially encounter. However, the construction of a more herringbone pattern could have a high cost for plants and further long term observations are needed to assess if this cost effectively penalize shoot growth.
5. Conclusion
This study is the first to describe how the root system of seagrasses interacts with two different substrate typologies, rock and sand, during the early phases of clone formation. The results show
that rocky substrate reduces root penetration, induces poorer root architecture and makes roots thicker without affecting the total size and biomass of the root systems of young Posidonia oceanica plants. Overall, these findings indicate that this species has the capacity to adjust root traits early during plant development according to the nature of substrate so to ensure efficiency in anchorage and exploration and exploitation of resources. This adaptive propriety may contribute to the ability of the species to establish on different environments, particularly rocky bottoms which typically prevent establishment of the majority of seagrasses.
Understanding how root growth and distribution are influenced by substrate type may assist in planning more efficient future restoration actions. Practices such exogenous application of hormones and addition of nutrients to sediments have been recently proposed to increase the rooting success of transplanted seedlings and cuttings (Balestri et al., 1998a; Balestri and Bertini, 2003; Balestri and Lardicci, 2006, Hovey et al., 2012; Statton et al., 2013; Statton et al., 2014.). However, knowledge of the potential interactive effects of such practices and substrate typology on seagrass root architecture is still limited (Hovey et al., 2012; Statton et al., 2013; Statton et al., 2014). This information may be important for determining the best practice (in terms of optimal composition and concentration of hormones or nutrients) to be applied under different substrate conditions in order to maximize root architecture efficiency and rooting depth. For example, treatments that stimulate lateral root elongation and rooting depth would be preferred for restoration actions at sandy sites to reduce the risk of uprooting. Other practices aimed at ameliorating sediment characteristics in terms of sediment texture, or even transplanting seedlings in containers with an optimal sediment composition at the restoration site, could be effective alternatives to expensive hormone/nutrient treatments because they increase lateral spread and rooting depth of the root system (Statton et al., 2013). On the other hand, the creation of cracks with hand tools close to seedlings and transplants would be useful at rocky sites or on dead mat to increase root explorative capacity.
Acknowledgements
The work described here was in part funded by Solvay Chimica Italia Spa (Rosignano Solvay, Italy), and in part by the University of Pisa, Italy (Lardicci C: 308-60% 2011). The founding sources had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.
References
Alagna, A., Tomás Vega, F., Terlizzi, A., Badalamenti, F., 2013. Influence of microhabitat on seedling survival and growth of the mediterranean seagrass Posidonia oceanica (l.) Delile. Estuarine Coastal Shelf Science 119, 119-125.
Arnaud-Haond, S., Duarte, C.M., Díaz-Almela, E., Marbà, N., Sintes, T., Serrão, E.A., 2012. Implication of extreme life span in clonal organisms: millenary clones in meadows of the threatened seagrass Posidonia oceanica. PlosONE 7(2) e30454. doi:10.1371/journal.pone.0030454.
Arredondo, J. T., Johnson, D.A., 1999. Root architecture and biomass allocation of three range grasses in response to non uniform supply of nutrients and shoot defoliation. New Phytologists 143, 373–385.
Balestri, E, Bertini, S., 2003. Growth and development of Posidonia oceanica seedlings treated with plant growth regulators: possible implications for meadow restoration. Aquatic Botany 76, 291-297
Balestri, E., Cinelli, F., 2003. Sexual reproductive success in Posidonia oceanica. Aquatic Botany 75, 21-32.
Balestri, E., Lardicci, C., 2006. Stimulation of root formation in Posidonia oceanica cuttings by application of auxins (NAA and IBA). Marine Biology 149, 393−400.
Balestri, E., Lardicci, C., 2008. First evidence of a massive recruitment event in Posidonia oceanica: Spatial variation in first-year seedling abundance on a heterogeneous substrate. Estuarine Coastal Shelf Science 76, 634-641.
Balestri, E., Lardicci, C., 2012. Nursery-propagated plants from seed: a novel tool to improve the effectiveness and sustainability of seagrass restoration. Journal of Applied Ecology 49, 1426-1435.
Balestri, E., Piazzi, L., Cinelli, F., 1998a. Survival and growth of transplanted and natural seedlings of Posidonia oceanica (L.) Delile in a damaged coastal area. Journal of Experimental Marine Biology and Ecology 228, 209–225.
Balestri, E., Piazzi, L., Cinelli, F., 1998b. In vitro germination and seedling development of Posidonia oceanica. Aquatic Botany 60, 83-93.
Balestri, E., Vallerini, F., Lardicci, C., 2006. A qualitative and quantitative assessment of the reproductive litter from Posidonia oceanica accumulated on a sand beach following a storm. Estuarine Coastal Shelf Science 66, 30-34.
Balestri, E., Luccarini, G., Lardicci, C., 2008. Abnormal embryo development in the seagrass Posidoina oceanica. Aquatic Botany 89, 71-75.
Balestri, E., Gobert, S., Lepoint, G., Lardicci, C., 2009. Seed nutrient content and nutritional status of Posidonia oceanica seedlings in the northwestern Mediterranean Sea. Marine Ecology Progress Series 388, 99–109.
Bécel, C., Vercambre, G,, Pagès, L., 2012. Soil penetration resistance, a suitable soil property to account for variations in root elongation and branching. Plant Soil 353, 169–180.
Belzunce, M., Navarro, R.M., Rapoport, H.F., 2008. Posidonia oceanica seedling root structure and development. Aquatic Botany 88, 203-210.
Berntson, G.M., 1994. Topological scaling and plant root system architecture: developmental and functional hierarchies. New Phtyologists 135, 621-634.
Berntson, G.M., 1995. The characterization of topology: comparison of four topological indices for rooted binary trees. Journal of Theoretical Biology 177, 271-281.
Berntson, G.M., Woodward, F.I., 1992. The root system architecture and development of Senecio vulgaris in elevated carbon dioxide and drought. Functional Ecology 6, 324-333.
Clark, L.J., Whalley, W.R., Barraclough, P.B., 2003. How do roots penetrate strong soil? Plant and Soil 255, 93-104.
Cox, P.A., Tomlinson, P.B,. Nieznanski, K., 1992. Hydrophilous pollination and reproductive morphology in the seagrass Phyllospadix scouleri (Zosteraceae). P1ant Systematics and Evolution 180, 65-75..
Dexter, A.R., 1988. Advances in characterization of soil structure. Soil and Tillage Research 11, 199-239.
Di Carlo, G.., Badalamenti, F., Terlizzi, A.., 2007. Recruitment of Posidonia oceanica on rubble mounds: substratum effects on biomass partitioning and leaf morphology. Aquatic Botany 87, 97-103.
Di Maida, G., Tomasello, A., Sciandra, M., Pirrotta, M., Milazzo, M., Calvo, S., 2013. Effect of different substrata on rhizome growth, leaf biometry and shoot density of Posidonia oceanica. Marine Environmental Research 87, 96-102.
Diaz-Almela, E., Marbà, N., Álvarez, E., Balestri, E., Ruiz-Fernández, J., Duarte, C., 2006. Patterns of seagrass (Posidonia oceanica) flowering in the western Mediterranean. Marine Biology 148, 723–742.
Domínguez, M., Celdran, D., Munoz-Vera, A., Infantes, E., Martinez-Banos, P., Marín, A., Terrados, J., 2012. Experimental evaluation of the restoration capacity of a fish-farm impacted area with Posidonia oceanica (L.) Delile seedlings. Restoration Ecology 20, 180-187.
Duarte, C.M., Sand-Jensen, K., 1996. Nutrient constraints on establishment from seed and on vegetative expansion of the Mediterranean seagrass Cymodocea nodosa. Aquatic Botany 54, 279-286.
Duarte, C.M., Losada, I.J., Hendriks, I.E., Mazarrasa, I., Marbá, M., 2013. The role of coastal plant communities for climate change mitigation and adaptation. Nature Climate Change 3, 961-968. Duarte, C.M., Merino, M., Agawin, N.S.R, Uri, J., Fortes, M.D., Gallegos, M.E., Marbá, N.,
Hemminga, M.A., 1998. Root production and belowground seagrass biomass. Marine Ecology Progress Series 171, 97-108.
Dupuy, L., Fourcaud, T., Stokes, A., 2005. A numerical investigation into the influence of soil type and root architecture on tree anchorage. Plant and Soil 278, 119–134.
Ehlers, W., Köpke, U., Hesse, F., Bőhrm, W., 1983. Penetration resistance and root growth of oats in tilled and untilled loess soil. Soil Tillage Research 3, 261-275.
Fageria, N.K., Balingar, V.C., Clark, R.B., 2006. Physiology of crop production. The Haworth Press Inc., New York, London, Oxford, pp. 23-60.
Fitter, A.H., 1991. The ecological significance of root system architecture: an economic approach. In: Atkinson, D. (Ed.), Plant root growth: an ecological perspective. Blackwell Scientific Publishers, London, pp. 229−243.
Fitter, A.H., 1996. Characteristics and functions of root system. In: Waisel, Y., Eshel, A., Kafkafi, U. (Eds.), Plant roots, the hidden half. Marcel Dekker, New York, pp. 1–20.
Fitter, A.H., Stickland, T.R., 1991. Architectural analysis of plant root systems. 2. Influence of nutrient supply on architecture in contrasting plant species. New Phytologists 118, 383−389. Fitter, A.H., Stickland, T.R., Harvey, M.L., Wilson, G.W., 1991. Architectural analysis of plant root
systems. 1. Architectural correlates of exploitation efficiency. New Phytologists 118, 375−382. Goodman, A.M., Ennos, A.R., 1999. The effects of soil bulk density on the morphology and
Green, E.P, Short, F., 2003. World Atlas of Seagrasses. UIMEP World Conservation Monitoring Centre. University of California Press, Berkeley, USA.
Grzesiaka, S., Grzesiaka, M.T., Huraa, T., Marcińskaa, I., Rzepka, A., 2013. Changes in root system structure, leaf water potential and gas exchange of maize and triticale seedlings affected by soil compaction. Environmental and Experimental Botany 88, 2–10.
Hemminga, M.A., 1998. The root/rhizome system of seagrasses: an asset and a burden. Journal of Sea Research 39, 183–196.
Hemminga, M., Duarte, C.M., 2000. Seagrass ecology. Cambridge University Press, Cambridge. Hovey, R.K., Cambridge, M.L., Kendrick, G.A., 2011. Direct measurements of root growth and
productivity in the seagrasses Posidonia australis and P. sinuosa. Limnology Oceanography 56, 394–402.
Hovey, R.K., Cambridge, M.L., Kendrick, G.A., 2012. Season and sediment nutrient additions affect root architecture in the temperate seagrasses Posidonia australis and P. sinuosa. Marine Ecology Progress Series 446, 23–30.
Lepoint, G., Vangeluwe, D., Eisinger, M., Paster, M., Vann Treeck, P., Bouquegneau, J:M:, Gobert, S., 2004. Nitrogen dynamics in Posidonia oceanica cuttings: Implications for transplantation experiments. Marine Pollution Bulletin 48, 465–470.
Kiswara, W., Behnke, N., van Avesaath, P., Huiskes, A.H.L., Erftemeijer, P.L.A., Bouma, T.J., 2009. Root architecture of six tropical seagrass species, growing in three contrasting habitats in Indonesian waters. Aquatic Botany 90, 235−245.
Kuo, J., den Hartog C., 2006. Seagrass morphology, anatomy, and ultrastructure. In: Larkum, A.W.D., Orth, R.J., Duarte, C.M. (Eds.), Seagrasses: Biology, Ecology and Conservation. Springer, Dordrecht, The Netherlands, pp. 51–87.
Konôpka, B., Pages, L., Doussan, C., 2009. Soil compaction modifies morphological characteristics of seminal maize roots. Plant Soil Environment 55, 1–10.
Ludlow, M.M, Muchow, R.C., 1990. A critical evaluation of traits for improving crop yields in water-limited environments. Advances in Agronomy 43, 107–153.
Lynch, J., 1995. Root architecture and plant productivity. Plant Physiology 109, 7-13.
Manschadi, A.M., Hammer, G.L., Christopher, J.T., DeVoil, P., 2008. Genotypic variation in seedling root architectural traits and implications for drought adaptation in wheat (Triticum aestivum L.). Plant and Soil 303, 115-129.
Marbá, N., Duarte, C.M., Diaz-Almela, E., Terrados, J., Álvarez, E., Martínez, R., Santiago, R., Gacia, E., Grau, A.M., 2005. Direct evidence of imbalanced seagrass (Posidonia oceanica) shoot population dynamics in the Spanish Mediterranean. Estuaries 28, 53–62.
Masle, J., 2002. Root impedance and plant performance- physiology, genetic determinants. In: Waisel, Y., Eshel, A., Kafkafi, U. (Eds.), Plant Roots, The Hidden Half. Marcel Dekker, Inc. Publ, NewYork, pp. 807-819.
Materechera, S.A., Dexter, A.R., Alston, A.M., 1991. Penetration of very strong soils by seedling roots of different plant species. Plant and Soil 135, 31-41.
Materechera, S.A., Alston, A.M., Kirby, J.M., Dexter, A.R., 1992. Influence of root diameter on the penetration of seminal roots into a compacted subsoil. Plant and Soil 144, 297–303.
Molinier, R., Picard, J., 1952. Recherches sur les herbiers de phanerogames marines du littoral mediterraneen français. Ann. l'Institut. Oceanogr. Paris 27 (3), 157-234.
Montefalcone, M., Vassallo, P., Gatti a, G., Parravicini, V., Paoli, C., Morri, C., Nike Bianchi, C., 2014. The exergy of a phase shift: Ecosystem functioning loss in seagrass meadows of the
Mediterranean Sea. Estuarine, Coastal and Shelf Science
http://dx.doi.org/10.1016/j.ecss.2014.12.001.
Orth, R.J., Harwell, M.C., Inglis, G.J., 2006. Ecology of seagrass seed and dispersal strategies. In: Larkum, A.W.D., Orth, R.J., Duarte, C.M. (Eds.), Seagrasses: Biology, Ecology and Conservation. Springer, Dordrecht, The Netherlands, pp. 111–133.
Pagès, L., 2002. Modeling root system architecture In: Waisel, Y., Eshel, A., Kafkafi, U. (Eds.), Plant roots: the hidden half. Marcel Dekker Pub., pp. 359-382.
Piazzi, L., Acunto, S., Cinelli, F., 1999. In situ survival and development of Posidonia oceanica (L.) Delile seedlings. Aquatic Botany 63, 103–112.
Poot, P., Hopper, S.D., van Diggelen, J.M.H., 2012. Exploring rock fissures: does a specialized root morphology explain endemism on granite outcrops? Annals of Botany 110, 291–300.
Reed, D.C., Holbrook, S.J., Solomon, E., Anghera, M., 1998. Studies on germination and root development in the surfgrass Phyllospadix torreyi: implications for habitat restoration. Aquatic Botany 62, 71–80.
StatSoft, Inc, 2007. STATISTICA (Data Analysis Software System), Version 8.0. www.statsoft.com.
Statton, J., Cambridge, M.L:, Dixon, K.W., Kendrick G.A., 2013. Aquaculture of Posidonia australis seedlings for seagrass restoration programs: Effect of sediment type and organic enrichment on growth. Restoration Ecology 21, 250-259.
Statton, J., Kendrick G.A., Dixon, K.W., Cambridge, M.L, 2014. Inorganic nutrient supplements constrain restoration potential of seedlings of the seagrass, Posidonia australis. Restoration Ecology 22, 196-203.
Stokes, A., Atger, C., Bengough, A.G., Fourcaud, T., Sidle, R.C., 1995. Responses of young trees to wind and shading: effects on root architecture. Journal of Experimental Botany 46, 1139–1146. Taub, D.R., Goldberg, D., 1996. Root system topology of plants from habitats differing in soil
resource availability. Functional Ecology 10, 258–264.
Terrados, J., Marin, A., Celdrian, D., 2003. Use of Posidonia oceanica seedlings from beach-cast fruits for seagrass planting. Botanica Marina 56, 182-195.
Tracy, S.R., Black, C.R., Roberts, J.A., Sturrock, C., Mairhofer, S., Craigon, J., Mooney, S.J., 2012. Quantifying the impact of soil compaction on root system architecture in tomato (Solanum lycopersicum) by X-ray micro-computed tomography. Annals of Botany 110, 511-519.
Underwood, A.J., 1997. Experiments in ecology: their ecological design and interpretation using analysis of variance. Cambridge University Press, Cambridge
Vassallo, P., Paoli, C., Rovere, A., Montefalcone, M., Morri, C., Bianchi, C.N., 2013. The value of the seagrass Posidonia oceanica: A natural capital assessment. Marine Pollution Bulletin 75, 157-167.
Vocanson, A., Roger-Estrade, J., Boizard, H., Jeuffroy, M.H., 2006. Effects of soil structure on pea (Pisum sativum L.) root development according to sowing date and cultivar. Plant Soil 281, 121–135.
Zwieniecki, M.A., Newton, M., 1995. Roots growing in rock fissures: Their morphological adaptation. Plant and Soil 172, 181-187.
Table 1. Seagrass species found on soft and hard substrates and species reported to occur
predominantly on hard substrates, and substrate characteristics. Sources: Green and Short, 2003; Kuo and den Hartog 2006; Di Carlo et al., 2007; The IUCN “Red List of Threatened Species”. www.iucnredlist.org/.
Species Substrate characteristics
Substrate typology
Soft and hard substrates Cymodoceaceae
Amphibolis griffithiis Gravel, clay, sand covered rocks
Halodule wrightii Sand, coralligenous sand, rock
Thalassodendron ciliatum Coarse sand with dead coral, rock, reefs
Thalassodendron pachyrhizum Sand, sand covered limestone, reefs Hydrocharitaceae
Halophila engelmanni Sand, gravel, rock
Halophila stipulacea Mud, sand, coralligenous sand, rock
Thalassia hemprichii Mud, sand, coarse sand mixed with
coral and shell debris, reefs Posidoniaceae
Posidonia oceanica Sand, dead matte, rock, rubble mounds
Zosteraceae
Zostera marina Mud, sand, coarse sand; silt; stones
Hard substrates Zosteraceae
Phyilospadix iwatensis Rock
Phyltospadix japonicus Rock
Phyllospadix scouleri Rock
Phyllospadix serrulatus Rock
Phyllospadix torreyi Rock
Table 2
Posidonia oceanica. Morphological characteristics and size of the entire root complex of five-year old plants grown on sand and rock, and results of one-way ANOVA for substrate type effect. Data are means ± SE. (n = 5)
Substrate type ANOVA statistics
Variable Sand Rock MS F p
Number of adventitious roots 7.4 ± 0.68 7 ± 1.05 0.400 0.103 0.757 Total root length (cm) 247.15 ± 34.83 175.08 ± 48.39 12985 0.881 0.375 Maximum horizontal spread (cm) 5.33 ± 0.67 7.33 ±1.05 10.211 1.844 0.211 Maximum rooting depth (cm) 11.16 ± 0.26 6.20 ± 0.73 61.618 21.777 0.001 Total root mass (g DW) 0.44 ± 0.04 0.46 ± 0.06 0.001 0.101 0.757
Table 3
Posidonia oceanica. Results of nested ANOVA for substrate type and plant effects on root architectural traits, topological index and root diameters of individual root systems from five-year old plants.
Number of first-order laterals Length of first-order laterals Number of second-order laterals
Source df MS F p MS F p MS F p
Between substrates 1 0.05 0.001 0.972 54.260 0.248 0.629 5.611 26.364 0.0006 Among plants 8 34.525 0.896 0.553 350.54 1.605 0.238 1.201 5.643 0.009 (substrate)
Within samples 10 38.55 218.44 0.213
Length of second-order lateralsTotal root length Diameter of the main root axis
Source df MS F p MS F p MS F p
Between substrates 1 7.428 21.535 0.0012 1590.3 3.638 0.086 0.286 21.283 0.009 Among plants 8 2.064 5.984 0.007 1069.4 2.446 0.009 0.040 2.998 0.053 (substrate)
Within plants 10 0.345 437.11 0.013
Diameter of first-order laterals Topological index
Source df MS F p MS F p
Between substrates 1 0.022 6.376 0.030 0.046 10.153 0.009 Among plants 8 0.044 1.290 0.346 0.011 2.351 0.103 (substrate)
Fig. 1. Example of the two possible extremes in branching patterns (herringbone vs. dichotomous
branching) of plant root systems (Fitter, 1991). .Magnitude (M) refers to the number of exterior links (those ending with a meristem); Altitude (A) refers to the centrifugal branching order
Fig. 2. Posidonia oceanica. Five-year old plants from seed grown on sand (left panels) and rock
(right panels). Entire root complex of plants (A,B) and a representative individual root system (C,D) excised from the root complex of plants (2-column fitting image)
Fig. 3. Posidonia oceanica. Morphological characteristics of two individual root systems excised
from the root complex of different five-year old plants (n = 5) grown on sand (left panel) and rock (right panel): mean number of (A;B) and second-order lateral roots (C,D), mean length of first-(E,F) and second-order laterals (G,H) and total root length (I,J). Error bars represent SE (2-column
Fig. 4. Posidonia oceanica. Mean diameter of the main root axis (A,B) and first-order laterals (C,D)
of two individual root systems excised from the root complex of different five-year old plants (n = 5) grown on sand (left panel) and rock (right panel). Error bars represent SE (2-column fitting
Fig. 5. Posidonia oceanica Topological characteristics of two individual root systems excised from
the root complex of different five-year old plants (n = 5) grown on sand (left panel) and rock (right panel): altitude (A,B), magnitude (C,D), pathlength (E,F) and topological index (G,H). Error bars represent SE (2-column fitting image)
