Distribution of testate amoebae in bryophyte communities in São Miguel Island (Azores Archipelago)

Abstract Background Testate amoebae are a polyphyletic group of protists living preferentially in soils, freshwaters and wetlands. These Protozoa have a worldwide distribution, but their presence and diversity in the Azores (a remote oceanic archipelago) is poorly known, with only twelve taxa recorded so far. The published information reflects occasional collections from sporadic field visits from naturalists to São Miguel Island, mainly in the nineteenth century. To overcome this limitation, a standardised survey was carried out on the Island, sampling different types of habitats from several localities to provide the distribution and information on species ecology of testate amoebae. New information In this study, 43 species of testate amoebae were recorded (within a total of 499 occurrences), belonging to two orders of Protista (26 Arcellinida and 17 Euglyphida). The most frequently occurring testate amoebae were Euglypha strigosa, Trinema lineare, Euglypha rotunda, Assulina muscorum and Cyclopyxis eurystoma. The most diverse genus was Euglypha (six species). A total of 38 species are new records for the Azores Archipelago. These data help to improve knowledge of the geographical distribution of testate amoebae in the northern hemisphere and their diversity in the Azores Archipelago.


Introduction
Testate amoebae are a polyphyletic group of small size [ranging from 7 to 500 µm long (Clarke 2003)] protists, enclosed within a xenosomic or idiosomic shell or test, made from proteinaceous, calcareous and/or siliceous material (Mitchell et al. 2008), with one or two oral apertures. They have a worldwide distribution, occurring in aquatic and terrestrial systems (Smith et al. 2007). In aquatic environments, they play an important role, especially in material cycling and energy flow (Glime 2017), while in terrestrial habitats, they play a crucial role in carbon and nitrogen cycling (Puppe et al. 2015). Due to their importance and sensitivity to environmental changes in both systems, they have been frequently used as bioindicators of environmental quality or stress or ecosystem resilience , Nguyen-Viet et al. 2007, Zapata et al. 2008). In addition, their shells are usually well preserved in sedimentary records and remain nearly unchanged over time.
As the species composition of these protists depends on environmental conditions, they are frequently used to reconstruct the past climate and environment (Ellison 1995, Mitchell et al. 2001, Mitchell et al. 2008. The increasing use of testate amoebae in palaeoecological studies in the last decades demands the knowledge of modern assemblages for comparative analysis (Mitchell et al. 2001, Charman 2001, Booth 2002, Swindles et al. 2015 and for the establishment of their functional traits , Amesbury et al. 2016, Marcisz et al. 2020. Despite their great importance, current knowledge of testate amoebae in the Azores Archipelago is limited when compared to other groups (e.g. Borges et al. 2010, Raposeiro et al. 2012) and previous studies are fragmented and unsystematic. Interest in Azorean testate amoebae started almost two centuries ago with the work of Ehrenberg (1854), which reported three protist species Difflugia azorica, Difflugia oligodon and Trinema enchelys, found in soil collected on São Miguel Island. Difflugia azorica was described by Ehrenberg (1871) as an endemic species, although the diagnosis can be applied to many species of the genus and may correspond to a variety of Difflugia pyriformis. Leidy (1879), in his work "Fresh-water rhizopods of North America", quotes the The main objective of this data paper is to provide a record of the diversity and detailed distribution of testate amoebae in São Miguel Island (Azores Archipelago, Portugal).

Study area description:
The Azores is an oceanic archipelago located in the middle of the North Atlantic, about 1500 and 2100 km off the coast of Portugal (Europe) and North America, respectively (Fig. 1).
Native forests cover less than 10% of the total area, mostly at elevations > 800 m a.s.l. (Borges et al. 2010, DDRF 2014, being a priority habitat in the Natura 2000 network (Guimarães and Olmeda 2008). Dominant tree species of this endemic forest are   (Cruz et al. 2007), located especially at elevations > 400 m a.s.l. (DDRF 2014). These Japanese cedar forests are very dense, limiting the development of ferns and some mosses. The bryophyte communities present under the canopy of this forest is dominated by Leucobryum juniperoideum (Brid.) Müll. Hal., Marchantia paleacea Bertol., Trichocolea tomentella (Ehrh.) Dumort, Thuidium tamariscinum (Hedw.) Schimp. and Hypnum cupressiforme Hedw. Peatlands, mainly located in depressions in high elevation areas and cover an area of 3000 ha (Mendes and Dias 2017), are characterised by the strong development of different species of Sphagnum and other bryophytes (Dias and Mendes 2007). Apart from their ecological importance, peatlands are, together with lakes (e.g. , Vázquez-Loureiro et al. 2019, the best paleoecological archives available in the Azores. Due to the existence of active volcanoes, São Miguel Island is particularly rich in hydrothermal vent fields (Gaspar et al. 2015). Biological communities of wetlands located close to these hydrothermal sites are influenced by higher temperatures and CO -rich mineral waters. In this specific habitat, plant communities consist mainly of vascular plants, such as Juncus effusus L. and Equisetum telmateia Ehrh. Hydrothermal carbonisation of different wetland biomass wastes allows for the development of a rich community of bryophytes characterised by species that tolerate extreme conditions (Elmarsdóttir et al. 2015), such as lawn communities of Sphagnum spp, Calliergon sp. and Polytrichum sp. According to (Porteiro 2000), São Miguel Island has 33 lakes, located at a range of between 260 m (Azul and Verde) and 830 m in altitude (Éguas Norte

Native forest
Sampling description: Testate amoebae collections were taken at the beginning of the growing season, between spring (February -March) and summer 2020 (May -June). In each location, several types of vegetation were chosen for their homogeneity and Table 2.
Habitat characteristics and location of the sixteen studied localities in São Miguel abundance (e.g. forest, lowland shrub, shrubland, wetland, peatland, riparian communities ( Fig. 2)) and within these communities, the most abundant bryophytes were sampled. In each subsampling site, three homogeneous subplots (10 × 10 cm) were chosen, defining a total 138 sampling points (Table 2).
Step description: Testate amoebae were sorted by fragmenting, washing and stirring of 10 × 10 cm of a wet mass of moss material into 1 litre of distilled water and then sieved through a 300 μm mesh size to remove large moss particles. The samples were concentrated by sedimentation and stored in vials with 50% alcohol at 4°C. A small aliquot of each sample was stored as a reference collection.
A drop of each sample (three subplots) was mounted on a semi-permanent slide and all testate amoebae were identified at 200x and 400x magnification using a compound microscope Leica DM2500. All measurements were made on photomicrographs (Leica DFC495 camera) of at least than 10 specimens, using image analysis software (Leica Application Suite version 3.8.0).
In order to obtain Scanning Electronic Microscopy (SEM) images, an aliquot was dried on aluminium supports with a carbon film. They were metallised with Iridium (40 nm) in a BioRad Microscience ion plating system and examined in a Zeiss Ultra Plus field emission microscope, at 5 kV high electric voltage.
The identification of testate amoebae was based on Ogden and Hedley 1980, Todorov and Bankov 2019. The classification at higher ranks follows Adl et al. (2019). Identification of the bryophyte species follow Smith and Smith (2004). Accepted names and authorities for vascular plants and bryophytes were checked in http://www.theplantlist.org (June 2020). Different habitats sampled in this study: 1) native forest, 2) Cryptomeria forest, 3) lakeshore, 4) hydrothermal vent and 5) peatlands.
Comparison of species richness (S) amongst different habitats was tested using one-way analysis of variance (ANOVA). Tukey's honest significant difference (HSD) test was used as the multiple comparison post-hoc test when significant differences were identified in the ANOVA.

Geographic coverage
Description: The Azores is an oceanic archipelago located in the middle of the North Atlantic, about 1500 and 2100 km off the coast of Portugal (Europe) and North America, respectively (Fig. 1) (Figs 3, 4).

Discussion
There are very few inventories of testate amoebae in the Azores, therefore the overall species richness of testate amoebae is unknown. Here, we present the first systematic study that explored the distribution of testate amoebae in bryophyte communities mainly in forest habitats. Forty-three species are recorded for São Miguel Island, 38 of these being new records for the Azores Archipelago. However, we must take into account the numerous cryptic taxa that testate amoebae present and some taxonomic uncertainty (Roland et al. 2017). For example, amongst Arcellinid, the Nebela tincta-bohemica-collaris species complex is a problematic group having very similar species (Heal 1964, Kosakyan et al. 2013. It is possible that what we identify here as Nebela collaris s.l. may include several taxa and, for this reason, it appears as the most abundant species. Another genus with similar difficulties is Quadrulella (Kosakyan et al. 2016). Considering the complexity of these groups, more detailed taxonomic work and more morphometric studies, combined with genetic approaches, such as the molecular barcoding effort, are needed to characterise this species complex.
The three most representative families in terms of species richness, Centropyxidae, Euglyphidae and Trinematidae, are, in general, the most commonly registered in other oceanic archipelagos (Smith 1986, Smith 1992, Beyens et al. 1990, Mieczan and Adamczuk 2015, Golemansky 2016 and in other parts of the world (Beyens et al. 1986, Bobrov et al. 1999, Mazei and Belyakova 2011, Acosta-Mercado et al. 2012, Šatkauskienė 2014. Considering the species richness for different testate amoebae genera in the distinct habitats studied, Euglypha and Trinema were the most frequent, followed by Corythion and Centropyxis (Fig. 5).
The 43 taxa recorded to the Azores is higher than what was reported to other oceanic archipelagos, such as the Canary Islands (10 species), Balearic Islands (15) and Island of Annobón (30 species). Testate amoebae assessment in the Balearic and Canary Islands was focused on mosses under Pinus forests of drier characteristics (Gracia 1965a, Gracia 1965b, while on the Island of Annobón (Equatorial Guinea), the work was performed on forest epiphyte mosses (Gracia 1963). However, these numbers cannot be used to draw conclusions about testate amoebae species richness in each archipelago since sampling efforts, habitats and approaches were different. In this context, it is essential to increase the sampling effort on other archipelagos, as well as to survey multiple habitats in order to find a greater diversity of testate amoebae.
The testate amoebae assemblages in São Miguel Island were composed mainly by genus with a cosmopolitan distribution which are also known from other oceanic islands. For example, in the comparable Annobón tropical rainforest, situated much further south (Gracia 1963), the diversity of testate amoebae is very similar (share 20 species). The cosmopolitan character of testate amoebae assemblages is also found in the mainland counterparts: the tropical mountain rainforest in Ecuador presented taxa geographically widespread with only nine species (6.7%) being considered tropical (Krashevska 2009). However, these biogeographic conclusions may be biased, on the one hand because of the number of cryptic species that exist and, on the other hand, because of the lack of habitat diversity surveyed. The information regarding protist communities cames from Sphagnum moss on peatlands (Glime 2017, Lamentowicz andMitchell 2005). Only a few studies were made in other types of bryophytes assemblages, mainly in northern areas including Devon Island (Beyens et al. 1990), Greenland (Beyens et al. 1986), Russia (Mazei and Belyakova 2011) or in sub-Antarctic areas (Smith 1992), such as Adelaide Island (Smith 1986), South Shetland Islands (Golemansky 2016), King George Island (Mieczan and Adamczuk 2015). The biogeographical situation of the Azores Archipelago between the Nearctic and the Palearctic offers unique possibilities to study the distribution of these organisms and their ability to colonise islands. This is the case of Argynnia caudata present in the Azores and with a tropical/subtropical distribution.
Moss biotopes are very abundant in the Azores, where extant bryoflora comprises about 430 species of mosses and hepatics (Sjögren 2003). The subtropical forest from the Azores is more or less constantly humid and warm and supports a very rich assemblage of moss species, including a higher proportion of endemic species (Sjögren 2001). The highest species richness of testate amoebae (n = 17, 40 species) occurred in native forest habitats and corresponds to epiphytic bryophytes that grow abundantly on the bark of living trees/shrubs. This alliance Echinodion prolixi Sjn. 93 is established in part of the native forest-phytocoenoses at high altitudes (600 m), dominated in the tree-layer of Laurus, Erica , Juniperus and Ilex (Sjögren 2003). In these epiphytic bryophytes, belonging to genera Frullania or Scapania, the most frequent species associated with these mosses were Corythion dubium, Centropyxis aerophila and Euglypha rotunda. Although, native forest communities are highly degraded in São Miguel, where the best-preserved area corresponds to the high eastern part of the Island (Fig. 1, Loc.: 14, 15 and 16) and in many places have been replaced by lowland shrub and peatlands (Fig. 2, habitats 1 and 5). In fact, peatland habitats can be considered in the Azores as an extension of these wet mountain forests, where the more abundant mosses are Polytrichum juniperinum Hedw. and Sphagnum spp. Despite that, they only share 42.5% of testate amoebae species, especially from genus Euglypha and a lower species diversity was observed (n = 2, 17 species). However, these results must be regarded with caution, because of the low number of replicates collected and analysed from peatlands.
Most of the natural vegetation of the Island has been replaced by pastures and Cryptomeria forests (Fig. 2, habitat 2). These Cryptomeria forests are the third most sampled habitat (n = 12, 31 species). Despite being conifer monocultures, the ecotone areas maintain a similar diversity of testate amoebae, when compared to native forest and it is easy to find native bryophyte communities, such as the case of Breutelia azorica (Mitt.) Cardot. The most common species of bryophyte Leucobryum juniperoideum grows in very dense, glaucous green, swollen cushions or hummocks. Some hummocks in woodland can be massive and colonised by other bryophytes and vascular plants. This eosinophilic moss which grows mainly at the base of Cryptomeria trunks, due to its dense growth structure, constitutes a favourable habitat for a high diversity of testate amoebae. In fact, this moss Breutelia azorica, presents the most diverse assemblages of testate amoebae within the Cryptomeria forest. Species from Nebela collaris complex (60%), Euglypha strigosa, Assulina muscorum and Trinema lineare dominated in cushion moss Leucobryum. The testate amoebae communities that are richest in species are those that develop on pleurocarpic mosses and Jungermannialian hepatics in forest habitats (Figs 5, 6).
The most frequent genera shared on these terrestrial and semi-terrestrial habitats are Nebela and Euglypha, which are less represented in the aquatic systems. According to Lansac-Tôha et al. (2007), these genera possess fragile shells, which limit their occurrence in more dynamic environments, especially lakes and hydrothermal vents. It is possible that these are eurytopic species or that they are more abundant because they have greater access to their food resources in these terrestrial habitats.
Bryophytes assemblages on lake shores presented a high diversity of testate amoebae (n = 13, 33 species). Acarcarpic mosses, like Fissidens or Campylopus in more open areas near lakes, maintain less developed testate amoebae communities (Fig. 6). However, the genera Sphagnum and Rhytidiadelphus squarrosus, especially in more waterlogged areas, maintain rich testate communities (n = 13, 33 species). Several works consider aquatic environments and sediments to be the preferred habitat for these organisms, yet they end up functioning as a data collector for the surrounding ecosystems and many species are actually terrestrial (Glime 2017). In order to better understand this issue, it is important to further study species' autoecology and habitat preferences.
On hydrothermal vents, lower diversity of testate amoebae was observed (n =3, 14 species). These vents develop an abundant bryophyte extension (Fig. 2), characterised by species that tolerate extreme conditions, such as Sphagnum spp, Calliergon sp. and Polytrichum juniperinum. The most frequent species are Quadrulella symmetrica and Trinema lineare. One possible explanation to this fact could be explained by their shell shape and composition, which are stiffer and more resistant, allowing their presence and permanence in this extreme habitat.

Final remarks
Here, we presented the first study that explored the distribution of testate amoebae of different habitats from the Azores Archipelago, mainly in São Miguel Island. Moreover, this work indicates that there are typical species on the different sampled habitats. This a matter of concern on islands, where large areas of native forest have been replaced by exotic forest and changes in land uses, driven by human activities, will affect population dynamics. In order to better understand the complexity of these habitats, population dynamics and species specificity need to be carried out. Larger datasets located in different islands and habitats are required to better understand how these communities respond to environment changes. Additionally, molecular barcoding is a useful tool, not only for species identification, but also for studying evolutionary and ecological processes. The results of this study provide indications that testate amoebae assemblages are habitat specific and therefore constitute a promising group for paleoenvironmental reconstruction of Azorean ecosystems. Future studies in drier ecosystems, coastal areas and hydrothermal zones may reveal and offer us a greater diversity of these organisms.