Introduction

A vast majority of terrestrial vascular plants form mutualistic root symbioses with a specialized group of soil fungi. These ancient associations (Redecker et al. 2000), called mycorrhizae (Smith and Read 2008), probably played a crucial role in the terrestrialization of vascular plants and their subsequent radiation on dry land (Selosse and Le Tacon 1998; Brundrett 2002). They represent one of the most intriguing plant adaptations for nutrient uptake, significantly affecting nutrient cycling in soils (Read and Perez-Moreno 2003; Read et al. 2004). Mycorrhizae vary in anatomy, morphology, ecophysiology and distribution (Read 1991; Brundrett 2004), but are functionally uniform, with a few exceptions, in that carbon flows from the photoautotrophic plant to the heterotrophic fungus and that water and mineral nutrients flow in the opposite direction. This requires the formation of specific exchange interfaces by the mycobiont inside the host root (Peterson and Massicotte 2004). It has been estimated that mycorrhizae are present in some 86 % of angiosperm species (Brundrett 2009), including freshwater plants (Søndergaard and Laegaard 1977; Beck-Nielsen and Madsen 2001; Sudová et al. 2011; Kohout et al. 2012) and plants from salt marshes (Radhika and Rodrigues 2007; Welsh et al. 2010; Eberl 2011) and mangroves (Sengupta and Chaudhuri 2002; Kothamasi et al. 2006). On the other hand, certain plant guilds do not require mycorrhizal symbioses for nutrient uptake (e.g. carnivorous plants, floating hydrophytes, parasites with haustoria, plants with cluster roots, etc.). Most of them belong to numerous families within the Brassicales, Caryophyllales, Lamiales, Poales and Santanales and also Alismatales, which comprise many hydrophytes including the only marine vascular plants—seagrasses (Brundrett 2009).

Besides mycorrhizal fungi, terrestrial plants live in symbioses with a wide spectrum of root endophytes that differ in their taxonomy, ecology and physiological significance for their hosts. In the Northern Hemisphere, their communities in temperate, boreal and subarctic plants are frequently dominated by dark septate endophytes (DSE), a group of miscellaneous soil fungi producing dematiaceous septate hyphae and melanized intracellular microsclerotia (Sieber 2002; Addy et al. 2005; Grünig et al. 2008). Additionally, some of them have been reported to form extra- and intraradical hyphal structures similar to those produced by mycorrhizal fungi for communication and nutrient exchange with their hosts, including dense parenchymatous hyphal nets on the root surface and intracellular hyphal loops (Vohník et al. 2003; Peterson and Massicotte 2004; Münzenberger et al. 2009; Vohník and Albrechtová 2011; Lukešová et al. 2015). Some authors even proved bi-directional nutrient flow between DSE and their host plants (Usuki and Narisawa 2007).

Seagrasses are a narrow ecological and taxonomic group of marine sessile macrophytes that evolved from terrestrial ancestors some 100 million years ago (Les et al. 1997). They comprise about 50–70 species (Hemminga and Duarte 2000; den Hartog and Kuo 2007) which have adapted to marine life by adopting several characteristic ecophysiological traits such as hydrophily and clonal growth. Seagrasses constitute less than 0.02 % of the angiosperm flora, yet they inhabit ca. 10 % of the coastal ocean area, represent ca. 1 % of the total marine plant biomass and are responsible for ca. 15 % of the total carbon storage in marine ecosystems (Hemminga and Duarte 2000). A recent estimate indicates that seagrasses can store up to twice as much carbon per square kilometre as temperate and tropical forests (Fourqurean et al. 2012). Seagrasses possess certain morphological, physiological and ecological traits which reduce the chance for symbioses with symbiotic fungi. Firstly, they take up nutrients through leaves (Khan and Belik 1995). Secondly, many species possess root hairs, and their roots develop extensive aerenchymatous systems in the cortex (Kuo and McComb 1989; Kuo and den Hartog 2007), i.e. in the tissue with the highest rates of arbuscular mycorrhizal (AM) colonization, the most common type of mycorrhiza, in terrestrial plants. Finally, many seagrasses live in muddy substrates low in available oxygen (Hemminga and Duarte 2000), which suppresses the development of extensive extraradical mycelia characteristic of many types of mycorrhiza (Nielsen et al. 1999). Congruently, the few studies focusing on seagrass root mycobionts (e.g. Cuomo et al. 1985; Nielsen et al. 1999; Panno et al. 2013; Torta et al. 2014) detected fungal hyphae or mycelia inside or on the surface of seagrass roots, but provided no clear evidence that seagrasses enter mycorrhizae or root endophytic associations similar to those occurring on dry land.

The genus Posidonia (Alismatales) is the evolutionary oldest genus of seagrasses, with the earliest fossil record from the Cretaceous (den Hartog 1970). It has a uniquely discontinuous distribution with eight of its nine species occurring in the Southern Hemisphere along the coast of Australia; the single Northern Hemisphere species Posidonia oceanica is endemic to the Mediterranean Sea (Green and Short 2003). Despite being a phanerogam, P. oceanica mostly relies on vegetative reproduction, which can be quite effective. Large climax meadows of the species dominate many sublittoral (depth of ca. 0–40 m) habitats along the Mediterranean coast, and the largest clones were estimated to spread over some 15 km while being hundreds to thousands of years old (Arnaud-Haond et al. 2012). Rhizomes, roots and senescent leaf sheaths of P. oceanica are exceptionally resistant to decay, which results in the formation of a characteristic peat-like sediment. This unique organic seabed layer (matte) can be several metres thick and thousands of years old (Hemminga and Duarte 2000). Vascular plants cannot effectively access such stocks of organically bound nutrients without the aid of symbiotic bacteria or fungi. However, the possibility that seagrasses in general and the matte-forming P. oceanica in particular host mycorrhizal or endophytic fungal symbionts has so far been examined only to a limited extent and with inconclusive results.

P. oceanica is sometimes accompanied by Cymodocea nodosa (Alismatales), a seagrass common in, but not restricted to, the Mediterranean Sea (den Hartog and Kuo 2007). This species usually occupies shallower waters and often grows in muddy substrates unsuitable for P. oceanica. Unlike P. oceanica, C. nodosa does not produce a matte, and its root system is characterized by vigorous production of root hairs (Kuo and McComb 1998). To our knowledge, possible root–fungus associations in C. nodosa have not yet been investigated.

In their pioneering work, Nielsen et al. (1999) did not find AM symbiosis in the seagrasses Thalassia testudinum and Zostera marina along the coasts of Denmark, Mexico and the USA. They nevertheless concluded that “it would be interesting to see data on possible AM colonization in seagrass species without root hairs from P-limited habitats, especially for fast growing species with hfaceigh nutrient demands” and encouraged other researchers “to investigate species like Cymodocea nodosa and Posidonia oceanica in the Mediterranean Sea”. Here, we report the results of our screening of these two Mediterranean seagrasses for mycorrhizal/root endophytic colonization.

Materials and methods

Root sampling

Root samples of P. oceanica (L.) Delile were collected using free and scuba diving between June and September 2012 at 11 localities in the NW Mediterranean Sea (Fig. 1, Table 1). We also sampled roots of the co-occurring seagrass C. nodosa (Ucria) Asch. where present (two localities; Table 1). The collection depths varied from 0.5 m in Antibes where leaves of P. oceanica emerged from the water at low tide to 31 m at Borak (Table 1). C. nodosa was found only at two localities (Finale Ligure and Sanary-sur-Mer), always occupying shallower habitats than P. oceanica. The two seagrasses never grew intermingled. P. oceanica always occurred on the rocky bottom or in coarse-grained sand whereas C. nodosa occurred in fine sand partly mixed with silt.

Fig. 1
figure 1

Location of the sampling sites within the NW Mediterranean Sea. Posidonia oceanica roots were sampled at 11 localities in Croatia (HR), Italy (IT), France (FR) and Spain (ES). The numbering of the localities follows Table 1. Bar = 500 km

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Table 1 A list of the localities sampled in this study
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At each locality, roots of five different individuals per species (at least 3 m apart) were carefully excavated from the substrate by bare hands, separated from the shoots and delivered to the surface. We tried to sample the whole diversity of substrates present at each locality, including sand, rocks and the matte. Terminal fine roots (ca. 1–2 mm in diam.) were then separated and inserted into 50-ml plastic beakers. Roots from all five individuals per locality were pooled to produce compound samples. The beakers with the roots were filled with seawater and stored in the dark in a portable refrigerator, and the seawater was later substituted by 30 % ethanol. The roots were then transported to the laboratory and stored in a refrigerator at 8 °C until processing.

Microscopic observations

Fungal colonization of the roots from each locality was investigated using an Olympus BX60 microscope equipped with DIC and an Olympus DP70 camera at magnifications of ×200, ×400 and ×1000. Scanning electron microscopy (SEM) was performed using a FEI Quanta 200 scanning electron microscope in the Olympus ESEM mode at low temperatures (−6 to −3 °C). We mainly screened hand-made semi-thin longitudinal (ca. 3–5 mm in length) and transversal root sections. For each locality, ten longitudinal sections from each of ten randomly chosen roots and five transversal sections from each of five randomly chosen roots were screened for any fungal structure. When needed, the sections were stained with 0.05 % trypan blue in lactoglycerol. For transmission electron microscopy, P. oceanica root segments were fixed in 5 % glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.2) and then in 2 % OsO4 in 0.1 M phosphate buffer. The samples were then dehydrated in increasing concentrations of ethanol, including a contrasting step with 1 % uranyl acetate. Infiltration was performed in increasing concentration series of propyleneoxide and Spurr Resin. Samples were embedded into Spurr Resin, and ultrathin sections (70 nm) were cut and contrasted with uranyl acetate and lead citrate. Photographs were taken with a digital TEM camera (Veleta, Olympus) using a JEOL 1011 microscope and modified for clarity in Paint.NET and Adobe Photoshop as needed.

Results

Roots of all sampled plants looked healthy, and the respective plants did not show any apparent signs of growth stress. There was no apparent extraradical mycelium in the form of rhizomorphs or sclerotia in the rhizosphere of the screened specimens. Seeking the identity of the respective mycobiont(s) forming the observed root–fungus association was beyond the scope of this work.

Light and scanning electron microscopy

The screened P. oceanica roots consisted of the stele surrounded by endodermis. The thick cortex frequently contained air lacunae. Its outer parts were formed by hypodermis with markedly thick-walled cells and rhizodermis without root hairs (Fig. 2a, b). Only one type of fungal colonization was found in the roots of P. oceanica, comprising characteristic melanized septate hyphae. Most of the fungal colonization occurred in the hypodermis, either intra- or intercellularly (Fig. 2c, d), and at the root surface (Fig. 2d, e). Rhizodermal cells were commonly (but not always) free of fungal colonization (Fig. 2c–e). The colonization in the hypodermis was formed either by intracellular microsclerotia (Figs. 2c, d and 3a) or narrow hyphae, occasionally reducing and expanding their diameter while exploring neighbouring longitudinally elongated hypodermal cells (Fig. 3a, b). On the root surface, the melanized hyphae produced sometimes extensive hyphal sheaths formed either by thick straight hyphae (Fig. 3c, d), parenchymatous labyrinthine nets of hyphae/hyphal sheaths (Fig. 3d–f) or a combination of both (Fig. 3d). Hyphae occurred in both living and dead cells (Fig. 3a, g), but it was unclear whether cell death resulted from the fungal colonization or whether the hyphae colonized already dead cells. This specific fungal colonization was regularly found in roots from all screened localities but varied in its extent. Infrequently, the whole hypodermis and outer parts of the root cortex were heavily colonized by melanized septate hyphae (Fig. 3g).

Fig. 2
figure 2

Anatomy of Posidonia oceanica roots possessing the dark septate endophytic colonization. a A transversal section through a typical P. oceanica root with the dark septate colonization. The stele (S) is surrounded by the endodermis (E). The multilayered cortex (C) contains several lacunae (L) and continues to the darkly brown hypodermis (H) formed by smaller cells with markedly thickened walls. The outer part of the root is the single-layer rhizodermis (R) with markedly inflated cells. Light microscopy with DIC, bar = 100 μm. b As in a but screened with scanning electron microscopy (SEM). Bar = 100 μm. c A transversal section through outer layers of a colonized root. The whole hypodermis is colonized by intracellular microsclerotia (arrows). The cortex and rhizodermis are free of any fungal colonization; an extraradical hyphal sheath is missing. SEM, bar = 50 μm. d Fungal intracellular colonization as in c but accompanied by a layered extraradical hyphal sheath (arrows). SEM, bar = 25 μm. e Intracellular fungal microsclerotia (arrowheads) accompanied by an extraradical hyphal sheath (arrows). Light microscopy with DIC, bar = 25 μm

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Fig. 3
figure 3

Anatomy and morphology of the dark septate colonization in Posidonia oceanica roots. a The root hypodermis colonized by lightly to heavily melanized septate hyphae (Hy), exploring hyphae (arrows) and intracellular elongated microsclerotia (M). Bar = 20 μm. b A longitudinal section through the hypodermis colonized by narrow dark septate hyphae (Hy). Note the transversal hypha repeatedly reducing and expanding its diameter while exploring the longitudinally elongated hypodermal cells (arrows). Bar = 20 μm. c An extraradical hyphal sheath (HS) formed by narrow dark septate hyphae. Bar = 20 μm. d An upright view through a layered hyphal sheath (HS) towards the rhizodermis. At the root surface, fungal hyphae produce a finger-like parenchymatous net (arrows) resembling structures formed by dark septate endophytic/mycorrhizal fungi in roots of terrestrial plants. Bar = 50 μm. e, f Details of the hyphal sheath (arrows). Bars = 20 μm. g The whole hypodermis and outer parts of the root cortex are heavily colonized by melanized hyphae. Bar = 20 μm. All pictures light microscopy with DIC

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In contrast to P. oceanica, C. nodosa roots lacked the thick-walled hypodermis (Fig. 4a, b), and its rhizodermis and root cortex were commonly free of any visible fungal colonization (Fig. 4c, d). Rhizodermal cells rarely contained single fungal hyphae, and single narrow hyphae were sometimes observed on the root surface. However, these significantly differed from those found in P. oceanica. All C. nodosa roots possessed numerous root hairs (Fig. 4e).

Fig. 4
figure 4

Anatomy and morphology of Cymodocea nodosa roots lacking any typical fungal colonization. a A transversal section through a typical C. nodosa root with no apparent fungal colonization. Similarly to P. oceanica, the stele (S) is surrounded by the endodermis (E), the multilayered cortex (C) contains several lacunae (L) and the outer part of the root is formed by the single-layer rhizodermis (R) which cells are however not inflated as in P. oceanica. The hypodermis with markedly thick-walled cells is missing. Light microscopy with DIC, bar = 100 μm. b As in a but visualized with SEM. Bar = 50 μm. c A detail of the rhizodermis (R) and the cortex (C) free of any apparent fungal colonization. SEM, bar = 50 μm. d As in c but visualized with light microscopy with DIC. Bar = 50 μm. e An SEM photograph of the C. nodosa root surface with numerous root hairs (arrows). Bar = 50 μm

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Transmission electron microscopy

Inflated rhizodermal cells of P. oceanica had approximately twice the diameter of hypodermal cells with markedly thickened outer cell walls. The cell walls of hypodermal cells were of a similar size as the rhizodermal outer cell walls, but much thicker than the inner rhizodermal cell walls (Fig. 5a). The spaces between neighbouring rhizodermal cells were usually occupied by prolonged, pointed, thick-walled, pentagonal to hexagonal cells which seemed to serve as entry points for fungal colonization (hence “entry cells”); the entering hyphae never seemed to penetrate directly through the thick-walled outer rhizodermal cell walls (Fig. 5b, c). In heavily colonized roots, the intraradical fungal colonization was mostly present throughout the hypodermis (Fig. 5b). However, in roots with low colonization intensity, intraradical fungal hyphae were often limited only to entry cells (Fig. 5c). When entering the root symplast or spreading from one cell to another, the hyphae first degraded the cell wall components and then proliferated through the resulting spaces (Fig. 5c, d). Occasionally, intraradical hyphae grew through intercellular spaces (Fig. 5e). It was not possible to unambiguously decide whether the intracellular hyphae were always surrounded by the host’s cytoplasmic membrane (Fig. 5e). Rather, it seemed that sometimes they were and sometimes they were not. Although most colonized and non-colonized hypodermal cells seemed to be alive, some microsclerotia and intracellular hyphae were evidently formed in already dead cells (also compare with Figs. 2e and 3b, g). We have not found any structures which would resemble arbuscules and vesicles typical of AM, fine intracellular hyphal coils typical of ericoid mycorrhiza or hyphal pelotons typical of orchid mycorrhiza.

Fig. 5
figure 5

Anatomy of P. oceanica roots with the fungal colonization visualized by transmission electron microscopy. a A transversal section through the rhizodermis and the hypodermis of a P. oceanica root. The inflated rhizodermal cells (RC) have thickened outer cell walls whereas their inner cell walls are very thin (arrowheads). The layers below the rhizodermis are formed by hypodermal cells (HC) with thickened cell walls. Each two rhizodermal cells is accompanied by a prolonged pointed cell which is the entry point for fungal colonization (hence “entry cells”, EC). Note several fungal hyphae occurring on the root surface in the sulci between the rhizodermal cells, right against the entry cells (arrows). Bar = 10 μm. b A transversal section through the upper layers of a colonized root. The fungal colonization comprises extraradical hyphal net/sheath (HN) which hyphae penetrate the spaces between the rhizodermal cells (arrow), and numerous intracellular hyphae formed first in the entry cells (EC) and then spreading to the hypodermal cells (HC). The inflated rhizodermal cells (RC) are usually free of fungal colonization. Bar = 10 μm. c A detail of an extraradical hypha (arrow) penetrating the space between two rhizodermal cells (RC) and consequently forming vigorous fungal colonization in an entry cell (EC). The remaining host cells are free of fungal colonization. Bar = 10 μm. d A rare example of a fungal colonization in the rhizodermis: an intracellular fungal hypha (IH) degrades components of the cell wall (CW) resulting in a cell wall lysis (CWL) while passing from one cell to another. Bar = 2 μm. e The typical colonization pattern in a thick-walled hypodermal cell possessing both primary cell wall (PCW) and secondary cell wall (SCW). Note several intracellular hyphae (IH) as well as one intercellular hypha (arrow). Bar = 2 μm

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Discussion

First reports on interactions between seagrasses and root-inhabiting fungi date back to the 1980s. With respect to the genus Posidonia, Kuo et al. (1981) provided microscopic evidence for an association between certain fungi and roots of two Australian species, Posidonia australis and Posidonia sinuosa. Similarly to our observations in P. oceanica, the fungal colonization was mostly restricted to the thick-walled hypodermis. The fungi often lysed cell walls and middle lamellae of colonized hypodermal cells, and although the authors speculated that the fungi might play a role in the nutrient uptake of the seagrasses, the reported association resembled rather a parasitic/pathogenic than a mutualistic interaction. More recently, García-Martínez et al. (2005) noticed some fungal hyphae growing on the root surface while examining microbial colonization of P. oceanica roots by electron microscopy, but did not report any intraradical fungal colonization. The mycoflora of P. oceanica leaves, rhizomes, roots and matte has been studied by Panno et al. (2013); however, the authors did not report any specific fungal colonization pattern in the screened tissues. Most recently, Torta et al. (2014) reported fungal colonization of P. oceanica roots at two localities in NW Sicily. The observed septate hyphae and microsclerotia, however, seemed to lack the dark pigmentation typical of DSE. Although it was evident that seagrass roots harbour fungal endophytes, the reported colonization seemed to be rather random, i.e. resulting from common non-specific contacts with marine fungi, as the authors did not report any typical fungal structures attributed to so far recognized root–fungus associations. In contrast, here, for the first time, we describe a dark septate association in the roots of a seagrass exhibiting all typical structures, i.e. extraradical and intraradical dark septate hyphae, dense melanized parenchymatous nets/hyphal sheaths on the root surface and melanized intracellular microsclerotia.

DSE associations typically lack fungal structures designed for nutrient transport and communication with the host plant (Peterson et al. 2008). On the other hand, certain DSE are able to form the Hartig net, i.e. the hyphal structure typical of ectomycorrhizal symbiosis (Danielson and Visser 1990; O’Dell et al. 1993; Fernando and Currah 1996; Münzenberger et al. 2009; Lukešová et al. 2015). Moreover, some authors hypothesized that the dense parenchymatous net on the root surface (also known as DSE labyrinthine tissue), detected here for the first time in a seagrass, may functionally parallel the Hartig net (O’Dell et al. 1993; Fernando and Currah 1996; Wurzburger and Bledsoe 2001; Vohník and Albrechtová 2011). Due to its frequency in some of the P. oceanica roots screened, the functioning of this fungal structure apparently deserves further investigation, as it might play a role in nutrient uptake from the matte or the mineral substrate. On the other hand, DSE microsclerotia, here commonly found in P. oceanica hypodermis, serve as storage and propagation organs of the mycobiont and most likely do not play any role in the host’s nutrient uptake (Yu et al. 2001). In endomycorrhizal symbioses, the intracellular hyphal phase is always separated from the cell cytoplasm by a plant-derived membrane (Peterson and Massicotte 2004). As was the case with other studies on DSE (Peterson et al. 2008), we were unable to unambiguously decide whether this was always true for the screened P. oceanica roots. Some intracellular hyphae evidently occurred in dead host cells, which is not typical of mycorrhizal symbioses (Peterson and Massicotte 2004); in some cases, however, DSE may cause host cell breakdown (Peterson et al. 2008), and host cell death is required for the proliferation of the mutualistic endophyte Piriformospora indica within host roots (Deshmukh et al. 2006).

Intriguingly, most of the intraradical colonization appeared within the hypodermis (cf. Kuo et al. 1981) whereas the rest of the root cortex usually remained free of fungal colonization. Hypodermis is commonly defined as a uni- or multiseriate layer of cells morphologically distinct from those of the neighbouring cortex formed just below the rhizodermis, and thus forming the outer layer of the root cortex (Peterson 1989). Its cell walls usually contain varying amounts of suberin, lignin, carbohydrates and structural cell wall proteins forming an important apoplastic barrier as well as a barrier against pathogenic microorganisms (Schreiber et al. 1999). Kuo et al. (1981) hypothesized that it may restrict the free movement of water and ions from sediments to the stele and the diffusion of root exudates into the rhizosphere in the two Australian Posidonia species. Given the apparent ability of mycobionts associated with P. oceanica to lyse even hypodermal cell walls (cf. Kuo et al. 1981), it is unclear why they do not progress to the cortex. On the other hand, frequent lysis of hypodermal cell walls may eliminate the hypodermal apoplastic barrier with as yet unknown consequences for P. oceanica.

The fungal association reported here was absent in the roots of the neighbouring seagrass C. nodosa, which points at its host/substrate specificity. Similarly, co-occurring terrestrial plants may host different spectra of root endophytes (Tejesvi et al. 2013). The reason for this specificity remains unknown. It may involve complex molecular mechanisms or simply reflect the absence of a root tissue suitable for colonization by mycobionts, as C. nodosa lacked the thick-walled hypodermis found in all P. oceanica individuals screened in the present study. Moreover, in contrast to P. oceanica, roots of C. nodosa (at least those sampled) produced abundant root hairs, which aid nutrient uptake in many terrestrial vascular plants (Gilroy and Jones 2000) and therefore reduce the need for symbiosis with fungi facilitating nutrient uptake in exchange for photosynthates. As root hairs are usually reduced to absent in mycorrhizal plants, the absence of root hairs in P. oceanica might also indicate the involvement of the root–fungus symbiosis in the acquisition of mineral nutrients. This, however, needs to be addressed in future studies.

Conclusions

Our survey of seagrass roots from 11 localities in the NW Mediterranean Sea resulted in the discovery of a specific fungal symbiosis in P. oceanica. Its anatomy and morphology are unique among seagrasses and displays a high degree of similarity to the ubiquitous DSE association occurring in the roots of most terrestrial vascular plants. Despite its ubiquity and abundance in healthy looking populations of P. oceanica, it remains unclear whether the mycobionts engage in nutrient capture and transport to the host (as suggested by the presence of parenchymatous labyrinthine hyphal nets/hyphal sheaths on the root surface and the absence of root hairs) and, more generally, whether it is of any benefit to the dominant Mediterranean seagrass.