Marine algal flora of São Miguel Island, Azores

Abstract Background The macroalgal flora of the Island of São Miguel (eastern group of the Azores Archipelago) has attracted the interest of many researchers in the past, the first publications going back to the nineteenth century. Initial studies were mainly taxonomic, resulting in the publication of a checklist of the Azorean benthic marine algae. Later, the establishment of the University of the Azores on the Island permitted the logistic conditions to develop both temporal studies and long-term research and this resulted in a significant increase on research directed at the benthic marine algae and littoral communities of the Island and consequent publications. Prior to the present paper, the known macroalgal flora of São Miguel Island comprised around 260 species. Despite this richness, a significant amount of the research was never made public, notably Masters and PhD theses encompassing information regarding presence data recorded at littoral and sublittoral levels down to a depth of approximately 40 m around the Island and the many collections made, which resulted in vouchers deposited in the AZB Herbarium Ruy Telles Palhinha and the LSM- Molecular Systematics Laboratory at the Faculty of Sciences and Technology of the University of the Azores. The present publication lists the macroalgal taxonomic records, together with information on their ecology and occurrence around São Miguel Island, improving the knowledge of the Azorean macroalgal flora at local and regional scales. New information A total of 12,781 specimens (including some identified only to genus) belonging to 431 taxa of macroalgae are registered, comprising 284 Rhodophyta, 59 Chlorophyta and 88 Ochrophyta (Phaeophyceae). Of these, 323 were identified to species level (212 Rhodophyta, 48 Chlorophyta and 63 Ochrophyta), of which 61 are new records for the Island (42 Rhodophyta, 9 Chlorophyta and 10 Ochrophyta), one an Azorean endemic (Predaea feldmannii subsp. azorica Gabriel), five are Macaronesian endemisms (the red algae Botryocladia macaronesica Afonso-Carrillo, Sobrino, Tittley & Neto, Laurencia viridis Gil-Rodríguez & Haroun, Millerella tinerfensis (Seoane-Camba) S.M.Boo & J.M.Rico, Phyllophora gelidioides P.Crouan & H.Crouan ex Karsakoff and the green alga Codium elisabethiae O.C.Schmidt), 19 are introduced species (15 Rhodophyta, two Chlorophyta and two Ochrophyta) and 32 are of uncertain status (21 Rhodophyta, five Chlorophyta and six Ochrophyta).


Introduction
Research on the marine algae from the Azores started in the mid-nineteenth century (1838) when Guthnick and the two Hochstetters, father and son, visited the Archipelago . Since then, many other researchers and naturalists have visited the Archipelago, resulting in several publications on the marine algal flora of this region (see summary in , Neto 1997. Most initial studies were taxonomic, focusing on the production of species lists. Almost a century later, the German botanist Otto Christian Schmidt visited several islands, including São Miguel and initiated a more comprehensive ecological approach describing species associations and their spatial organisation (Schmidt 1931). Ever since the first half of the last century, several studies have focused more widely on intertidal and shallow subtidal communities providing information on the vertical distribution of macroalgae and invertebrates and their trophic relations (see Neto 1992, Neto 2000, Neto 2001 for a review on this subject). Taxonomic investigations have continued and the first checklist of the Azorean benthic marine algae published by  brought together the existing published information, provided distributional records within the Archipelago and reported 307 species, indicating a moderately rich flora given its isolated mid-Atlantic position. A revision of this first checklist was made by Parente (2010), increasing the number of algae species to 327, but without providing their distributional information on the Archipelago. Later, Rosas-Alquicira et al. (2011) published a catalogue of non-fossil geniculate coralline red algae (Corallinales, Rhodophyta) of the Macaronesia, in which they made both a critical review of species and infraspecific taxa, as well as an assessment of species diversity in the region. Research by local teams was also dedicated to the Azorean littoral communities and biota conservation (see, for example, Abecasis et al. 2015, Amorim et al. 2015, Chainho et al. 2015. Taxonomic, ecological and biotechnological investigations have continued generating knowledge on the Azorean macroalgae flora, its biotechnological potential and also on the structure and functioning of littoral communities (see revisions on . Recently, several additional studies have been published with important information on the Azorean algae biodiversity, biogeography, conservation, ecology and taxonomy (see, for example, Bruno de Sousa et al. 2019, Cacabelos et al. 2020, Freitas et al. 2019, Kellaris et al. 2019, Parente et al. 2020, Patarra et al. 2017, Patarra et al. 2019, Faria et al. 2020a, Faria et al. 2020b, Vieira et al. 2020).
The paper by Freitas et al. (2019) increased the number of macroalgae species occurring in the Azores to 405 and reported that, amongst the mid-Atlantic archipelagos, the Azores is second in species richness after the Canary Islands, with 689 species, followed by Madeira (396), Cabo Verde (333) and Selvagens (295 species). For some species, the Azores Archipelago forms a boundary in their distribution. Codium effusum (Rafinesque) Delle Chiaje, for example, is as its western distribution limit in the Archipelago (Leon-Cisneros et al. 2012), whereas for Dudresnaya crassa M.Howe, a western Atlantic warmwater species, the Azores extends its known distributional range to the east. Some northern species such as the red alga Schizymenia dubyi (Chauvin ex Duby) J.Agardh and Lomentaria orcadensis (Harvey) Collins come close to their southern limit of distribution in the Azores, while some southern warm-water species, such as green alga Anadyomene stellata (Wulfen) C.Agardh and the red alga Sebdenia rodrigueziana (Feldmann) Codomier ex Athanasiadis, reach their Atlantic northern limit of distribution on the Islands , Leon-Cisneros et al. 2012. Some species, relatively common in the region a few years ago, have become uncommon or even very rare, for example, Scytosiphon lomentaria (Lyngbye) Link, Schimmelmannia schousboei (J.Agardh) J.Agardh. In contrast, there has been an increase in unexpected macroalgae in the Azores, with the arrival and establishment of several non-native species (see Cardigos et al. 2006, Micael et al. 2014, Vaz-Pinto et al. 2014, Cacabelos et al. 2020. Within the spread of the Archipelago, there are no marked differences between floras of individual Islands or Island groups and, biogeographically, the Azores algal flora reveals itself to have a mixed nature, with species shared with Macaronesia, North Africa, the Mediterranean Sea, Atlantic Europe and America (Tittley and Neto 1995, Tittley and Neto 2006, Tittley 2003, Wallenstein et al. 2009b). This nature of the Azorean marine algal flora was reinforced by the work of Freitas et al. (2019), who, using an extensive analysis encompassing data on coastal fishes, brachyurans, polychaetes, gastropods echinoderms and macroalgae, suggested that the Azores should be a biogeographical entity on its own and proposed a re-definition of the Lusitanian biogeographical province, in which they included four ecoregions: the South European Atlantic Shelf, the Saharan Upwelling area, the Azores ecoregion and a new ecoregion they named Webbnesia, which comprises the archipelagos of Madeira, Selvagens and the Canary Islands.
Not all the Azorean Islands have received the same attention regarding the studies on macroalgae. Furthermore, many species may have been overlooked due to their small size, opportunistic nature or ephemeral life span.
To overcome this and gain a better and up-to-date knowledge of the Archipelago's macroalgae flora, an effort was made by resident teams to undertake a considerable amount of research over the past three decades on several Islands. The present paper is the last one of a series and presents physical, occurrence data and information gathered from macroalgal surveys undertaken on São Miguel Island between 1989 and 2019 mainly by the Island Aquatic Research Group of the Azorean Biodiversity Centre of the University of the Azores (Link: https://ce3c.ciencias.ulisboa.pt/sub-team/island-aquatic-ecology), the BIOISLE, Biodiversity and Islands Research Group of CIBIO-Açores at the University of the Azores (Link: https://cibio.up.pt/research-groups-1/details/bioisle) and the OKEANOS Centre of the University of the Azores (Link: http://www.okeanos.uac.pt). In these surveys, particular attention was given to the small filamentous and thin sheet-like forms that are often short-lived and fast-growing and usually very difficult to identify in the field, without the aid of a microscope and specialised literature in the laboratory.
This paper aims to provide a valuable marine biological tool to aid research on the systematics, diversity and conservation, biological monitoring, climate change, ecology and more applied studies, such as biotechnological applications, which will be of assistance to Study area description: The Azores Archipelago (38°43′49″N, 27°19′10″W, Fig. 1), isolated in the mid-Atlantic Ocean, comprises nine volcanic Islands and several islets spread over 500 km in a WNW-ESE direction, emerging from the Azores Plateau and located above an active triple junction between three of the world's largest tectonic plates (the North American Plate, the Eurasian Plate and the African Plate, Hildenbrand et al. 2014).
The Archipelago comprises nine volcanic Islands and several small Islets in three separate groups (eastern, central and western). The Azores, its location in the Atlantic and São Miguel Island highlighted in black (by Nuno V. Álvaro). Fig. 1), approximately 750 km² in size, is the largest and most volcanically-active Island (Gaspar et al. 2015). Located in the eastern group of the Archipelago (37°54'58''N, -25°51'52''W, Fig. 2), its formation followed a series of volcanic events, with different parts of the Island having different ages. The oldest portion (4 M years old) is the eastern side, Nordeste, where Pico da Vara (the highest mountain of the Island with 1103 m a.s.l.) is located. The Island was then progressively formed to the west: Povoação (2 M years); Furnas (750,000 years); Serra de Água de Pau (250,000 years). The Sete Cidades complex appeared 500,000 years ago and only later (50,000 years ago) was connected to Serra de Agua de Pau through the Serra Gorda and its succeeding line of peaks (Zbyszewski et al. 1958, Zbyszewski andFerreira 1959).

São Miguel (in black in
As in the other Azorean Islands, the climate is considerably influenced by the surrounding ocean and is characterised by regular rainfall, medium levels of relative humidity and persistent winds, mainly during the winter and autumn seasons (Morton et al. 1998). The tidal range is small (< 2 m) and the coastal extension is restricted, with deep waters occurring within a few kilometres offshore (Hidrográfico 1981). Most sea-shores are subject to swell and surge most of the year and few are sheltered, except for some bays and harbours. Extremely heavy seas occur during winter .
São Miguel has the longest coastline in the archipelago, about 155 km, corresponding to 25.3% of the whole Azorean coastline. The coastal topology, resulting from the effect of the maritime agitation, responsible for the predominance of erosive morphologies, is mainly composed of high, steep cliffs with a variety of stack, arch and gully formations and is mostly difficult to access by land. Most of the cliffs and coastal slopes are less than 50 m a.s.l. (Borges 2003) and fall directly into the sea. The coastline is mainly composed of irregular compact, bedrock platforms, alternating with boulder and cobble locations. On some shores, boulders entrap coarse sand and gravel and there are a few sandy beaches (Wallenstein et al. 2009b). Intertidal communities of São Miguel Island, as on the other islands of the Archipelago, are primarily dominated by macroalgae, which mainly exhibit a mosaic and/or zoned distribution pattern and have a predominance of algal turfs that cover the rocks as a carpet (Wallenstein et al. 2009), best seen when rocks are uncovered at low tide. There is a very distinct horizontal pattern of species distribution, with three major zones commonly found on bedrock and boulder shores (Neto 2000, Wallenstein et al. 2009b. The uppermost intertidal level is dominated by littorinids (Fig. 3), while the mid-level zone is usually characterised by a fringe of chthamalid barnacles (Fig. 4), in which sometimes algae and limpets can occur (Fig. 5), followed by a lower area, in which either algal turf (generally monospecific and usually composed of Caulacanthus ustulatus (Turner) Kützing) dominates (Fig. 6) or patches of the brown alga Fucus spiralis Linnaeus and the red agarophyte Gelidium microdon Kützing (Fig. 7) grow interspaced with barnacles and algal turf. The lowest intertidal zone, representing the transition to the sublittoral envrironment, is either dominated by algal turf (generally multispecific and commonly dominated by coralline algae, Fig. 8) or by various species of frondose algae growing in bands (e.g. the brown alga Gongolaria abies-marina (S.G.Gmelin) Kuntze, Fig. 9) or forming patches amongst and over turf species (e.g. the agarophyte Pterocladiella capillacea (S.G.Gmelin) Santelices & Hommersand and the calcareous Ellisolandia elongata (J.Ellis & Solander) K.R.Hind & G.W.Saunders, Fig. 10). The brown alga Colpomenia sinuosa (Mertens ex Roth) Derbès & Solier is very common at this level, growing epiphytically on several other algae. Seasonally, the red algae Porphyra/ Neopyropia and/or Nemalion elminthoides (Velley) Batters can be seen growing in patches at the mid-intertidal level. In some locations, the brown crust Nemoderma tingitanum Schousboe ex Bornet can be common at this shore level , Wallenstein et al. 2009b. In spring and summer, considerable amounts of the introduced red alga Asparagopsis armata Harvey can be seen at the lower intertidal level, normally as an epiphyte on other algae (Neto, personal observation).
At cobble locations, the zonation pattern of macroalgae species is not clear (Costa 1994). The many microhabitats and substrate instability tend to mask and attenuate the limits of the biological zones. Nevertheless, in locations where cobbles are large and their size enlarges towards the sea (e.g. Fenais da Luz, north shore), the profile is steeper and usually the mid-intertidal level is dominated by the green macroalgae Ulva linza Linnaeus,  U. clathrata (Roth) C.Agardh and U. rigida C.Agardh; the lower level is characterised by the presence of algal turf, mainly composed of Jania crassa J. V. Lamouroux and Corallina officinalis Linnaeus, with epiphytic Rhodophyta, such as Asparagopsis armata Harvey, phase Falkenbergia rufolanosa (Harvey) F.Schmitz, Centroceras clavulatum (C.Agardh) Montagne, Ceramium ciliatum (J.Ellis) Ducluzeau, C. deslongchampsii Chauvin ex Duby and Polysiphonia atlantica Kapraun & J.N.Norris. In locations with small cobbles (e.g. Caloura, south coast) or where there is a mixture of large and small cobbles (e.g. Povoação, south coast), the mid-intertidal level is usually characterised by fast growing algae, such as Cyanobacteria and the green algae Ulva spp., whereas the lowest level is also dominated by algal turf, but here mainly composed of C. officinalis and C. clavulatum (Caloura) or by C. clavulatum, Chondracanthus acicularis (Roth) Fredericq, Jania sp. and Lophosiphonia sp. (Povoação).     The brown alga Gongolaria abies-marina growing in bands at the low shore level (by the Island Aquatic Ecology Subgroup of cE3c-ABG). Important habitats at the shore level in bedrock locations are rock pools (Fig. 11). Differing in shape and size, they recreate a shallow subtidal habitat which may contain a rich diversity of marine algae and other marine organisms , Wallenstein et al. 2010).
The macroalgae diversity varies according to the pool location on the shore. Pools in the upper shore region are dominated by green algae, whilst those lower on the shore are dominated by red and brown algae. Similarly, faunal diversity in rock pools is greater at lower intertidal levels.

Design description:
The sampling referred to in this study was performed across littoral and sublittoral levels down to approximately 40 m. Each sampling location was visited several times and, on each occasion, a careful and extensive survey was undertaken to provide a good coverage of the area. Both presence recording and physical collections were made by walking over the intertidal shores during low tides or by SCUBA diving in the subtidal. The specimens collected were taken to the laboratory for identification and preservation and the resulting vouchers were deposited at the AZB Herbarium Ruy Telles Palhinha and the LSM -Molecular Systematics Laboratory at the Faculty of Sciences and Technology of the University of the Azores.
Funding: This study was mainly financially supported by the following projects/scientific expeditions: • Portuguese Regional Funds, through DRCT -Regional Directorate for Science and Technology, within several projects, 2019 and 2020 and SRMCT /DRAM -Regional Secretariat for the Sea, Science and Technology, Regional Directorate for Sea Affairs; Sampling description: Sampling involved species presence recording and/or specimen collecting at each sampling location. Species recording data were gathered by registering all species present in the sampled locations (Fig. 14). Destructive samples were obtained by scraping and/or manually collecting one or two specimens of every species found (Fig.  15). Intertidal collections were made during low tide by walking over the shores. Subtidal collections were made by SCUBA diving.
Quality control: Each specimen collected was identified by trained taxonomists and involved morphological and anatomical observations of whole specimens by eye and/or of histological preparations under the microscope to determine the main diagnostic features of each species as described in literature.
Step description: At the laboratory, specimen sorting and macroalgae identification followed standard procedures. A combination of morphological and anatomical characters and reproductive structures was used for species identification. For small and simple thalli, this required the observation of the entire thallus with the naked eye and/or using dissecting and compound microscopes. For larger and more complex algae, investigation of the thallus anatomy required histological preparations (longitudinal and transverse sections) or squashed preparations of mucilaginous thalli, sometimes after staining, to observe vegetative and reproductive structures and other diagnostic features.
A reference collection was made for all collected specimens by assigning them a herbarium code number and depositing them at the AZB Herbarium Ruy Telles Palhinha and the LSM -Molecular Systematics Laboratory, University of Azores. Depending on the species and on planned further research, different methods of preservation were used, namely (i) wet collections using 5% buffered formaldehyde seawater and then replacing it by the fixing agent Kew (Bridsen and Forman 1999); (ii) dried collections, either by pressing the algae (most species) as described by Gayral and Cosson (1986)

Additional information
This paper accommodates the 12,781 specimens of macroalgae recorded from São Miguel Island in 431 taxa comprising 323 confirmed species (Tables 2, 3) and 108 taxa identified only to genus level. The confirmed species (Table 3)   Many species were only sporadically recorded, but 10 were widely recorded around the Island and occurred quite abundantly in some locations, namely: the Rhodophyta Table 3.
Summary of the macroalgal flora of the Island of São Miguel (N spec-number of specimens; N taxa-taxa; N spp-number of species) with information on the species origin and status (Introdintroduced; Uncrt-uncertain origin; Azo end-Azores endemism; Mac end-Macaronesia endemism; New rec-new record). This paper increases the total of macroalgae species previously listed for São Miguel Island by 63 (44 Rhodophyta, 9 Chlorophyta and 10 Ochrophyta). When compared with the other Azorean Islands (Table 4), São Miguel has the highest number of species in all phylla, which reflects the greater amount of research undertaken on this Island on a more regular basis, involving both temporal and long-term studies. In general and in keeping with other warm-water areas of the North Atlantic Ocean, the Azorean macroalgae flora has a larger proportion of red seaweeds.
A relatively high number of non-native species has been recorded on São Miguel Island (see Tables 2, 3), similarly to what has been reported for Santa Maria (Neto et al. 2020d), Terceira , Graciosa (Neto et al. 2020c), Pico ) and Flores and Corvo (Neto et al. 2021a). Research over the past 15 years (Cardigos et al. 2006, Micael et al. 2014, Vaz-Pinto et al. 2014, Chainho et al. 2015, Cacabelos et al. 2020) has indicated that the arrival of non-native species is increasing in the Azores, which is very likely to be related to the geostrategic position of the Archipelago as a "crossroad" in the distribution of marine algae in the North Atlantic, with documented exchanges with European, Mediterranean and American coasts. This may favour the arrival of new species via maritime traffic, both commercial and recreational (hull fouling, ballast waters), which may be a high risk to local marine ecosystems, as non-indigenous species can become invasive, resulting in impacts on ecosystem services and biodiversity (e.g. Katsanevakis et al. 2014).
The discovery of the new macroalgae records on São Miguel Island (present study) and on the other Azorean Islands , Neto et al. 2020c, Neto et al. 2020d, Neto et al. 2021a, demonstrates the need for continuing taxonomic and floristic studies in this region of the Atlantic Ocean. The biogeographically-variable nature of the Table 4.
new records found confirms the overall mixed nature of the marine algal flora of the Azores with elements shared with Macaronesia, the Mediterranean Sea, Atlantic Europe and the subtropical and tropical Atlantic America.

Taxonomic mismatch
A mismatch regarding the GBIF backbone taxonomy of some of the macroalgae species names was identified as detailed in Suppl. material 1.