Biodiversity of epiphytic macroalgae (Chlorophyta, Ochrophyta, and Rhodophyta) on leaves of Zostera marina in the northwestern Iberian Peninsula ; Biodiversidad de macroalgas epifitas (Chlorophyta, Ochrophyta y Rhodophyta) en hojas de Zostera marina en el noroeste de la península ibérica

The composition, abundance, and distribution of epiphytic macroalgae living in meadows of Zostera marina L. in the northwestern Iberian Peninsula are here analyzed. We identified 63 species: 40 red algae, 16 brown algae, and 7 green algae. Most of them are classified as filamentous or filiform functional forms, while Pneophyllum fragile Kütz. was the only encrusting species. In general, the surface covered by epiphytes on the leaves of Zostera marina was low and a 43% of species were only found in juvenile stages. Regarding their frequency, 10 species were collected in the majority of the areas, while others were rare. Most species were found both epiphytic and in other substrata of the meadows, but 9 were exclusively epiphytic. We detected 9 introduced species. Resumen. Se han analizado la composición, la abundancia y la distribución de macroalgas epifitas que viven en praderas de Zostera marina L. del noroeste de la península ibérica. Se han identificado 63 especies: 40 algas rojas, 16 pardas y 7 verdes. La mayoría pertenecen a los grupos funcionales filamentosos o filiformes, excepto Pneophyllum fragile Kütz., la única especie costrosa. En general, la cobertura de epifitos en las hojas de Zostera marina fue baja y un 43% de las especies solo se encontraron como estadios juveniles. En cuanto a su frecuencia, 10 de ellas se recolectaron en la mayoría de las áreas, mientras que el resto fueron más raras. La mayor parte de las especies se encontraron tanto epifitas como en otros substratos de las praderas, si bien 9 resultaron exclusivamente epifitas. Detectamos 9 especies introducidas. How to cite this article: García-Redondo V., Bárbara I. & Díaz-Tapia P. 2019. Biodiversity of epiphytic macroalgae (Chlorophyta, Ochrophyta, and Rhodophyta) on leaves of Zostera maritima in the northwestern Iberian Peninsula. Anales del Jardín Botánico de Madrid 76 (1): e078. https://doi.org/10.3989/ajbm.2502. Title in Spanish: Biodiversidad de macroalgas epifitas ( Chlorophyta, Ochrophyta y Rhodophyta) en hojas de Z ostera marina en el noroeste de la península ibérica. Received: 22‒II‒2018; accepted: 4‒I‒2019; published on-line: 28‒II‒2019; Associate Editor: A. Flores.


INTRODUCTION
The eelgrass-Zostera marina L., Zosteraceae Dumort.-meadows represent an important marine ecosystem in the northern temperate region (den Hartog 1970;Homziak & al. 1982;Short & Coles 2001). The protection against predation and the food availability of this habitat are the major traits that attract many organisms to be permanent or temporary residents of seagrass meadows (Hemminga & Duarte 2000). The leaves of Zostera marina also provide the substrate to which many benthic organisms attach, such as hydroids, fungi, protozoa, bryozoans or algae. Physical and chemical characteristics of leaf surface vary during growth, onset of reproduction and senescence, and these changes influence the recruitment and distribution of colonists (Michael & al. 2008). Epiphytic algae are the most abundant and diverse group of organisms on Zostera marina, which grow especially on its leaves (Borowitzka & al. 2006).
The diversity of epiphytic macroalgae on Zostera marina varies with the age of leaves, as they are deciduous and the oldest ones accumulate more epiphytes (Cullinane & al. 1985). Once they shed, they still play an important role as a substrate for algal epiphytes (Novaczek 1987). Epiphytic macroalgae increase biodiversity and total biomass of eelgrass meadows and are a food source for herbivores (Duarte 2002;Orth & al. 2006;Michael & al. 2008). A particular set of species grows exclusively as epiphytes on Zostera marina, such as Rhodophysema georgei Batters (Saunders & Bird 1989), while a larger number of species can be found epiphytic and on the adjacent substrate. Diversity, distribution, and abundance of seagrasses epiphytes are influenced by abiotic and biotic Zostera marina grows mainly in the subtidal and, consequently, most collections were carried out by scuba diving (fig. 2). Samplings were performed between April 2014 and April 2017, during spring and summer, which are the most favorable periods from a floristic point of view. In total, 36 eelgrass meadows were sampled and, in each one, five quadrats of 0.0625 m 2 were haphazardly distributed (Duarte & Kirkman 2001). In turn, the six longest leaves of Zostera marina were collected in each quadrat to study the macroalgal epiphytes (García-Redondo & al. 2017). Samples were preserved in 4% formalin seawater and kept in darkness at 4ºC. In total, 1,080 leaves of Zostera marina were studied.
We estimated the abundance of each epiphytic species on each studied leaf. As most species had very low values of covering-< 0.03%-, epiphytes were classified into abundance categories according to their percent of covering on the leaves of Zostera marina. We calculated the mean and the quartiles, and according to this, we considered five categories: i) mean greater than the minimum and lower than the first quartile; ii) mean greater than or equal to the first quartile and lower than the second quartile; iii) mean greater than or equal to the second quartile and lower than the third quartile; iv) mean greater than or equal to the third quartile and less than the fourth quartile; and v) mean greater than or equal to the fourth quartile and less than the maximum value.

Floristic catalogue
In total, 63 epiphytic macroalgae were found on leaves of Zostera marina in the northwestern Iberian Peninsula (Table 1). Diversity was higher than the recorded in other areas of the Iberian Atlantic where eelgrass meadows hosted up to 38 species (Cullinane & al. 1985;Novaczek 1987;Johnson & al. 2005). By contrast, it was higher in Gran Canaria, where up to 79 species were recorded in seagrasses (González 1976).
Red algae-40 species (figs. 3-4)-were much more abundant than brown algae-16 species (fig. 5)-and green algae-7 species ( fig. 6)-. The distribution of red, brown, and green algae is similar to the observed in eelgrass meadows from other Iberian regions, and it is proportional to the number of recorded species for these three groups in the northwestern Iberian Peninsula (Bárbara & al. 2005). However, the diversity of epiphytes was lower than the observed in other benthic habitats of the study region, such as maërl beds (Peña Freire 2010)       1, mean greater than the minimum and lower than the first quartile; 2, mean greater than or equal to the first quartile and lower than the second quartile; 3, mean greater than or equal to the second quartile and lower than the third quartile; 4, mean greater than or equal to the third quartile and less than the fourth quartile; 5, mean greater than or equal to the fourth quartile and less than the maximum value. Morphofunctional groups (     (Reyes & al. 1995;Reyes & Sansón 1996).
Two functional forms, filamentous-23 spp.-and filiform-23 spp.-, accounted most of the functional diversity of the epiphytic flora of Zostera marina. Foliose species, both thin and intermediate, were also common-seven and six species, respectively-, while the corticated filiform and crustose species were rare-three and one species, respectively-. Unicellular, corticated foliose and articulated calcareous functional groups included in García-Fernández & Bárbara (2016) were never observed among epiphytes. This pattern is the expected considering the short life span that the leaves of Zostera marina provide for epiphytes, as mentioned above. Consequently, species with high growth rates thrive better in this particular habitat, while perennial species cannot persist. Filiform, filamentous, and foliose are the functional forms characterized by having the highest growth rates (Littler & Littler 1984). Pneophyllum fragile Kütz. is the only crustose species that occur on the leaves. It is a thin calcareous coralline algae with only a few cell layers (Irvine & Chamberlain 1994), so it can grow quickly and is adapted to eelgrass habitat. It is also a common epiphyte on the seagrass Cymodocea nodosa, in which Pneophyllum fragile is one of the first epiphytic species colonizing the young leaves (Reyes & Sansón 1996). Probably, the pioneer character of this species also applies in Zostera marina. Mean covering of epiphytic macroalgae on the leaves of Zostera marina is low. Table 1 shows that the category 2-0.007-0.033%-is the most common, while category 5-10-73% cover-is rare. Some species were occasionally observed covering a high percentage of leaves, such as Pneophyllum fragile-up to 73%-, Colaconema daviesii (Dillwyn) Stegenga-up to Polysiphonia fibrillosa (Dillwyn) Spreng. are filiform red algae whose thallus can be up to 1 cm in length. The brown algae Ectocarpus fasciculatus Kütz. and Ectocarpus siliculosus are filamentous species that can be up to 2 cm in length.
The diversity of epiphytes greatly varied among sampling sites (Table 1). Ferrol-FER-had the highest diversity and Sálvora-SAL-the lowest, 36 and five species, respectively. Interestingly, the low diversity found in Sálvora contrasts with the highest covering observed in this study-73%, Pneophyllum fragile-. Some of the species here recorded were only found in one of the areas (Table 1), and, for example, Litosiphon laminariae (Lyngb.) Harv., Apoglossum ruscifolium (Turner) J.Agardh, Champia parvula (C.Agardh) Harv., and Cladophora albida (Nees) Kütz. were only observed in Ferrol. Thus, it is important to study several meadows in order to achieve a comprehensive view of the epiphytic flora at a regional scale.
The present work, focused on the epiphytic macroalgae, was developed in the framework of a broader study on the flora associated with Zostera marina. This allows us to establish comparisons among the flora associated to the different habitats within the meadows. Nine species have been found in Zostera marina exclusively as epiphytes on leaves: Cladosiphon zosterae (J.Agardh) Kylin, Ectocarpus fasciculatus, Ectocarpus siliculosus, Feldmannia globifera The high diversity of introduced species might be facilitated by the placement of most of the eelgrass meadows in sheltered areas, which are subjected to a high incidence of the most relevant vectors for introduction and spread of non-native seaweeds, i.e., harbors or aquaculture facilities (Williams & Smith 2007).
Some of the species here recorded are unusual or scarcely known in the northwestern Iberian Peninsula. Rhodophysema georgei ( fig. 7) had been only recorded before by Miranda (1934) and remained unnoticed up to now. The lack of information on this species is probably explained because it is an obligate epiphyte on Zostera marina and its flora has been scarcely studied in the Iberian Peninsula. Morphological characters of Rhodophysema georgei from the northwestern Iberian Peninsula agree with the descriptions for other regions (Saunders & Bird 1989;Saunders & McLachlan 1989). Other scarcely known species are Asperococcus scaber Kuck., which is here recorded for the second time in the Iberian Atlantic-first report in Bárbara & al. (2015)-and for the first time in the province of Pontevedra-Ría de Vigo-; and Gayliella mazoyerae T.O.Cho, Fredericq & Hommers.that is recorded for the first and second time in Pontevedra-A Toxa-and Galicia, respectively-first report in .
In sum, our study shows that the epiphytic flora on the leaves of Zostera marina is a diverse assemblage, considering the continuous environmental variations associated with this habitat. The composition of the flora was highly variable in frequency and abundance among the studied regions, as it depends on the interaction of several factors and processes that operate at different spatial and temporal scales (Borowitzka & al. 2006). The supply of propagules is a key process that influences the epiphytic assemblage (Borowitzka & al. 2006). They can come from the meadows or from adjacent communities, which explain the high number of species that were observed only as juvenile stages because leaves are an unsuitable substrate for mature stages.

Key to the NW Iberian Peninsula epiphytic macroalgae on Zostera marina
Identification of epiphytes is sometime challenging because most part of specimens were found as juvenile and immature stages. Thus, some of the key vegetative and reproductive characters needed for morphological identifications were not properly developed. In order to facilitate the identification of epiphytic macroalgae of the leaves of Zostera marina, we provide the following identification key that can be successfully used independently of the maturity of specimens. Pictures showing the main characters of these species are provided in figs. 3-7.