Biodiversity Data Journal 9: e63290 CO) doi: 10.3897/BDJ.9.e63290 open access Data Paper Distribution of testate amoebae in bryophyte communities in Sao Miguel Island (Azores Archipelago) Martin Souto Souto*, Vitor Goncalves*'§, Xabier Pontevedra-Pomball, Pedro M. Raposeiro*$ + CIBIO, Centro de Investigagao em Biodiversidade e Recurso Genéticos — Polo dos Agores, InBio, Laboratério Associado / Universidade dos Acores, Ponta Delgada, Portugal § Faculty of Sciences and Technology, University of the Azores, Ponta Delgada, Portugal | Dpto. Edafoloxia e Quimica Agricola, Fac. Bioloxia, Universidade de Santiago de Compostela, Santiago de Compostela, Spain Corresponding author: Pedro M. Raposeiro (pedro.mv.raposeiro@uac.pt) Academic editor: Yasen Mutafchiev Received: 18 Jan 2021 | Accepted: 18 Feb 2021 | Published: 17 Mar 2021 Citation: Souto MS, Gongalves V, Pontevedra-Pombal X, Raposeiro PM (2021) Distribution of testate amoebae in bryophyte communities in Sao Miguel Island (Azores Archipelago). Biodiversity Data Journal 9: e63290. https://doi.org/10.3897/BDJ.9.e63290 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 Sao 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. © Souto M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Souto M etal 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. Keywords biodiversity, community ecology, island, moss, Protozoa, substratum specificity Introduction Testate amoebae are a polyphyletic group of small size [ranging from 7 to 500 um 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 (Mitchell et al. 2003, 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 (Mitchell et al. 2014, 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, Pereira et al. 2014) 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 Sao 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 Distribution of testate amoebae in bryophyte communities in Sao Miguel ... 3 species again, without adding any comment and Difflugia oligodon was described by Ehrenberg (1844) on the basis of samples from Kurdistan and a 10-word diagnosis. Later, with the Challenger expedition that took place from 1872 to 1876 and which had a brief passage to Sao Miguel Island, the Irish naturalist Archer (Archer 1874) studied samples from Lake Furnas and reported six new taxa. By the end of the nineteenth century, Gerne (1888) and Barrois (1896) published several papers on freshwater biota of the Azores including some additions to testate amoebae fauna (Table 1). Table 1. Historical records of testate amoebae from Azores Archipelago Taxa (Current name) Ehrenberg — Archer 1874 Gerne Barrois Gadea 1854 1888 1896 1979 Arcella vulgaris Ehrenberg, 1830 X Centropyxis aculeata (Ehrenberg, 1830) as Echinopyxis X X Stein, 1857 aculeata Centropyxis constricta (Ehrenberg, X Xx 1841) Penard, 1890 Centropyxis sp. Xx Difflugia acuminata Ehrenberg, 1838 X X Difflugia globulosa Dujardin, 1837 as D. globularis Difflugia mitriformis Wallich, 1864 X Difflugia oligodon Ehrenberg, 1844 X Difflugia pyriformis Perty, 1849 as D. azorica X X Euglypha sp. X Euglypha acanthophora (Ehrenberg, as E. alveolata as E. 1841) Perty, 1849 alveolata Nebela collaris (Ehrenberg, 1848) Leidy, Xx X 1879 s.l. Quadrulella symmetrica (Wallich, 1863) as Difflugia X Kosakyan et al., 2016 : symmetrica Trinema enchelys (Ehrenberg, 1838) X as T. acinus X X Leidy, 1879 Almost 100 years later, Gadea (1979), in his work on nematodes living in mosses, recorded three genera (Centropyxis, Euglypha and Plagiostoma). Of the three genera reported, Plagiostoma seems to be a misprint and refers to Centropyxis plagiostoma or genus Plagiopyxis. Since that time, no studies have been carried out on testate amoebae in the Archipelago. The main objective of this data paper is to provide a record of the diversity and detailed distribution of testate amoebae in Sao Miguel Island (Azores Archipelago, Portugal). 4 Souto M etal Additional information on species ecology is also discussed. Our purpose is to release this valuable dataset since no similar datasets have been previously published for Azores and it constitutes a relevant tool for comparison for ecologists studying, for example, biogeographic patterns or climate change and as modern analogues for environmental reconstructions on oceanic islands in paleoecological studies. Project description Title: Records of testate amoebae in Sao Miguel Island (Azores Archipelago) Personnel: Collections were undertaken and occurrence data recorded during 2020 in Sao Miguel Island. The collectors were Martin Souto, Vitor Gongalves and Pedro Miguel Raposeiro. Identification was done by Martin Souto and Xabier Pontevedra-Pombal. Production and analysis of scanning electron microscopy images was done by Xabier Pontevedra-Pombal. 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). 37°S0'N 25°50'W 25°40'W 25°30'W 25°20'W 25°10'W Figure 1. EES] Geographical location of the study localities. At top left - The Azores Archipelago in the Atlantic Ocean highlighted by a square; at top right, Sao Miguel Island in the Azores Archipelago highlighted by a square; and, at the bottom, the 16 study localities. 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 (Guimaraes and Olmeda 2008). Dominant tree species of this endemic forest are Distribution of testate amoebae in bryophyte communities in Sao Miguel ... 5 Juniperus brevifolia (Seub.) Antoine, Laurus azorica (Seub.) Franco and //ex azorica Loes. with a close canopy in which a great diversity of ferns and mosses is found (Elias et al. 2016). Cryptomeria japonica (Thunb. ex L.f.) D. Don forest occupies about 22% of the land area in the Azores (representing 60% of forest plantation area (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 jJuniperoideum (Brid.) Mull. 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 Sohagnum and other bryophytes (Dias and Mendes 2007). Apart from their ecological importance, peatlands are, together with lakes (e.g. Hernandez et al. 2017, Raposeiro et al. 2017, Vazquez-Loureiro et al. 2019), the best paleoecological archives available in the Azores. Due to the existence of active volcanoes, Sao 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 (Elmarsdottir et al. 2015), such as lawn communities of Sohagnum spp, Calliergon sp. and Polytrichum sp. According to (Porteiro 2000), Sao Miguel Island has 33 lakes, located at a range of between 260 m (Azul and Verde) and 830 m in altitude (Eguas Norte). In general, the dominant vegetation of lake shores are J. effusus, Osmunda regalis L. Agrostis sp., surrounded by hygrophyte shrub communities of Cal/una vulgaris. The most abundant bryophytes are Rhytidiadelphus squarrosus (Hedw.) Warnst, H. cupressiforme and several Sphagnum species. The presence of dense carpets of Fissidens sp. or Campylopus sp. is more common in open areas. Funding: This work was funded by FCT-— Foundation for Science and Technology, the European Union, QREN, FEDER, COMPETE programmes (PMR - DL57/2016/ICETA/ EEC2018/25; MSS - ICETA/EEC2018/25; DiscoverAzores project - PTDC/CTA-AMB/ 28511/2017; CIBIO/InBIO - UID/BIA/50027/2013 and POCI-01-0145-FEDER-006821) and Consolidation and Structuring project 2018 GRC-ED431C 2018/32 of the government of the Xunta Galicia. Sampling methods Study extent: This study covers 16 sampling locations on Sao Miguel Island (Fig. 1, Table 2), encompassing several habitat types (native forest, Cryptomeria forest, lake shore, peatland and hydrothermal vents). Within these habitats, different species of bryophytes were subsampled in triplicate, from a total of 46 moss subsampling locations, comprising a total of 138 samples. 6 Souto M etal Table 2. Habitat characteristics and location of the sixteen studied localities in Sao Miguel Cod. Locality Latitude Longitude’ Alt. Date Habitat (N°) (W°) (m) Loc. 1 Lagoa Azul 37.97833 -25.84638 260 04-Feb. Lake shore with degraded aquatic vegetation Loc. 2. Lagoa Verde 37.89056 -25.80194 260 04-Feb. Strongly altered and degraded aquatic vegetation Loc. 3 Lagoado Canario 37.83540 -25.75898 755 04-Feb. Aquatic vegetation with Sohagnum and Thuidium communities Loc. 4 Lagoa do 37.82340 -25.75049 775 04-Feb. Lake shore and shrub communities rich Caldeirao Norte in Sphagnum Loc.5 Lagoado Carvao 37.82360 = -25.74241 700 15-May. Lake shore and shrub communities Loc. 6 Lagoa da Prata 37.80652 -25.73641 520 15-May. Peatlands with Sohagnum and Cryptomeria forest Loc. 7 Alto da Barrosa 37.76333 -25.50056 800 27-May. Shrub communities of Calluna with epiphytic bryophytes Loc. 8 Lagoa do Fogo 37.76669 -25.48024 580 22-Jun. Lake shore and Cryptomeria forest Loc. 9 Ribeira da Praia 37.72583 -25.46822 180 05-May. Fissidens communities Loc. |Lombadas 37.77745 = -25.46167 600 11-May. Hydrothermal, native and Cryptomeria 10 forest Loc. Lagoa de Sao 37.79306 -25.41278 600 27-Feb. Aquatic vegetation surrounded by 11 Bras Cryptomeria forest Loc. LagoadoAreeiro 37.76324 -25.42743 580 13-Feb. Aquatic vegetation surrounded by 12 Cryptomeria forest Loc. LagoadoCongro 37.75522 -25.40737 700 15-May. Lake shore and Fissidens communities 13 Loc. _Ribeira do 37.81769 -25.26294 600 27-May. Native and Cryptomeria forest 14 Caldeirao Loc. Planalto dos 37.80056 -25.23944 900 10-Jun. Native forest and peatlands 15 Graminhais Loc. _Ribeira do 37.79972 -25.20194 580 = 09- Native forest 16 Guilherme March. 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 Distribution of testate amoebae in bryophyte communities in Sao Miguel ... if, 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 x 10 cm) were chosen, defining a total 138 sampling points (Table 2). Figure 2. EESl Different habitats sampled in this study: 1) native forest, 2) Cryptomeria forest, 3) lakeshore, 4) hydrothermal vent and 5) peatlands. Step description: Testate amoebae were sorted by fragmenting, washing and stirring of 10 x 10 cm of a wet mass of moss material into 1 litre of distilled water and then sieved through a 300 wm 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:/Awww.theplantlist.org (June 2020). 8 Souto M et al 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). The Archipelago is comprised of nine volcanic islands that are divided into three groups: the western group (Corvo and Flores Islands), the central group (Faial, Pico, Graciosa, Sao Jorge and Terceira Islands) and the eastern group (Sao Miguel and Santa Maria Islands). Sao Miguel is the largest Island, with an area of 746 km? and approximately 45% of the Island is between 300-800 m a.s.I., with a maximum elevation of 1103 m. The climate in the Azores is temperate oceanic, with regular and abundant rainfall, with high levels of relative humidity (up to 95% in high elevation native forests), ensuring moderate thermal variations throughout the year (Brito de Azevedo et al. 1999). Mean annual temperatures range between 14 and 18°C, while the mean annual precipitation is between 740 and 2400 mm (Marques et al. 2008, Hernandez et al. 2016). Coordinates: 37.704 and 37.917 Latitude; -25.857 and -25.125 Longitude. Taxonomic coverage Description: Testate amoebae found on Sao Miguel Island Taxa included: Rank Scientific Name order Arcellinida family Arcellidae species Arcella arenaria Greeff, 1866 species Arcella catinus Penard, 1890 family Netzeliidae species Cyclopyxis eurystoma Deflandre, 1929 species Cyclopyxis kahli Deflandre, 1929 infraorder Incertae sedis Sphaerothecina species Trigonopyxis arcula (Leidy, 1879) Penard, 1912 family Difflugiidae species Difflugia bacillifera Penard, 1890 species species family species species species species family species species species species species species family species species family species family species family species order species species species order family species species Distribution of testate amoebae in bryophyte communities in Sao Miguel ... Difflugia glans Penard, 1902 Difflugia elegans Penard, 1890 Centropyxidae Centropyxis aerophila Deflandre, 1929 Centropyxis constricta (Ehrenberg, 1841) Penard, 1890 Centropyxis discoides (Penard, 1890) Deflandre, 1929 Centropyxis elongata (Penard, 1890) Tomas, 1959 Hyalospheniidae Alabasta militaris (Penard, 1890) Duckert, Blandenier, Kosakyan & Singer, 2018 Nebela collaris (Ehrenberg, 1848) Leidy, 1879 Padaungiella lageniformis (Penard, 1890) Lara & Todorov, 2012 Padaungiella tubulata (Brown, 1911) Lara & Todorov, 2012 Planocarina carinata (Archer, 1867) Kosakyan et al., 2016 Quadrulella symmetrica (Wallich, 1863) Kosakyan et al., 2016 Heleoperidae Heleopera rosea Penard, 1890 Heleopera sphagni Leidy, 1874 Microchlamyidae Pyxidicula cymbalum Penard, 1902 Phryganellidae Phryganella acropodia (Hertwig & Lesser, 1874) Hopkinson, 1909 Cryptodifflugiidae Cryptodifflugia oviformis Penard, 1890 Incertae sedis Arcellinida Argynnia caudata (Leidy, 1879) Argynnia dentistoma (Penard, 1890) Physochila griseola Penard, 1911 Euglyphida Euglyphidae Euglypha acanthophora (Ehrenberg, 1841) Perty,1849 Euglypha cristata Leidy, 1874 10 Souto M et al species Euglypha filifera Penard, 1890 species Euglypha laevis (Ehrenberg, 1845) Perty, 1849 species Euglypha rotunda Wailes, 1911 species Euglypha strigosa (Ehrenberg, 1871) Leidy, 1879 species Tracheleuglypha dentata (Vejdovsky, 1882) Deflandre, 1928 family Assulinidae species Assulina muscorum Greeff, 1888 family Cyphoderiidae species Cyphoderia ampulla (Ehrenberg, 1840) Leidy, 1879 family Sphenoderiidae species Sphenoderia fissirostris Penard, 1890 species Trachelocorythion pulchellum (Penard, 1890) Bonnet, 1979 family Trinematidae species Corythion constricta (Certes, 1889) Jung, 1942 species Corythion dubium Taranek, 1881 species Playfairina valkanovi Golemansky, 1966 species Trinema complanatum Penard, 1890 species Trinema enchelys (Ehrenberg, 1838) Leidy, 1879 species Trinema lineare Penard, 1890 Temporal coverage Notes: 04-02-2020 through to 22-06-2020 Usage licence Usage licence: Open Data Commons Attribution License IP rights notes: This work is licensed under a Creative Commons Attribution (CC-BY) 4.0 Licence. Data resources Data package title: Checklist of testate amoebae in Sao Miguel Island (Azores Archipelago) Distribution of testate amoebae in bryophyte communities in Sao Miguel ... 11 Resource link: http://ipt.gbif.pt/ipt/resource?r=tecamebas Alternative identifiers: https:/Awww.gbif.org/dataset/13c79ceb-Oceb-424f-b000- 7991d7c49834 Number of data sets: 1 Data set name: Checklist of testate amoebae in Sao Miguel Island (Azores Archipelago) Data format: Darwin Core Archive Description: This paper presents data distribution of testate amoebae in Sao Miguel Island (Azores Archipelago) collected during 2020. The dataset has been published as a Darwin Core Archive (DwC-A), which is a standardised format for sharing biodiversity data as a set of one or more data tables (Souto et al. 2021). The core data table contains 16 events (eventID), 499 occurrences (occurrencelD) with 43 taxa (taxonID). The number of records in the data table is illustrated in the IPT link. This IPT archives the data and thus serves as the data repository. The data and resource metadata are available for downloading in the downloads section. Column label Column description id Identifier of the record, coded as a global unique identifier locality Name of the locality where the event occurred continent Continent of the sampling site country Country of the sampling site island Island from the Island Group of the sampling site islandGroup Island group of the sampling site eventID Identifier of the event, unique for the dataset occurrencelD Identifier of the occurrence, coded as a global unique identifier type The nature of the resource Habitat Habitat sampled basisOfRecord The specific nature of the data record samplingProtocol Sampling protocol recordedBy Person who collected the specimens identifiedBy Person who identified the specimens eventDate Time interval when the event occurred taxonID The identifier for the set of taxon information (data associated with the Taxon class). Specific identifier to the dataset 12 Souto M etal scientificName The name with authorship applied on the first identification of the specimen Kingdom Kingdom name Phylum Phylum name Class Class name Order Order name Family Family name Genus Genus name specificEpithet The name of the first or species epithet of the scientificName scientificNameAuthorship The specimen accepted name, with authorship taxonRank The taxonomic rank of the most specific name in the scientificName minimumElevationInMetres Elevation in metres decimalLatitude The geographic latitude of the sampling site decimalLongitude The geographic longitude of the sampling site coordinateUncertaintylInMetres The indicator for the accuracy of the coordinate location in metres, described as the radius of a circle around the stated point location. geodeticDatum The spatial reference system upon which the geographic coordinates are based countryCode Code of the country where the event occurred Additional information Analysis This study presents 499 testate amoebae (Protista) occurrences in 46 sampled sites (16 localities) in Sao Miguel Island, belonging to 43 species from 25 genera, 14 families and two orders. The order Euplyphida, represented by five families, accounted for 58.5% of the total occurrences and the order Arcellinida 41.5% of the total occurrences. The families with the highest number of occurrences were Euglyphidae and Trinematidae (121), Hyalospheniidae (54), Heleoperidae (38), Centropyxidae (35) and Assulinidae (32). Additionally, the families with the highest number of taxa were Euglyphidae and Trinematidae (7), followed by Hyalospheniidae (6) and Centropyxidae (5). The families with lower occurrences (< 5) were Cyphoderiidae (4) and Microchlamyidae (1). The genera with the highest number of occurrences were Euglypha (106), Trinema (82), Heleopera (38) and Centropyxis (35). The other 21 genera had less than 35 occurrences. The genera with the highest number of taxa were Euglypha (6) and Centropyxis (4). Euglypha strigosa, Trinema lineare and Euglypha rotunda were the most frequent species occurring in 39, 37 and 34 sites, respectively. Assulina muscorum (32 sites), Cyclopyxis eurystoma (27 sites), Distribution of testate amoebae in bryophyte communities in Sao Miguel ... 13 Corythion dubium (26 sites), Trinema complanatum (25 sites) and Euglypha laevis (24 sites) were amongst the most ubiquitous testate amoebae (Figs 3, 4). Figure 3. EESl SEM images of most frequent testate amoebae found in Sao Miguel: a) Trinema lineare, b) Corythion constricta, c) Assulina muscorum, d) Padaungiella lageniformis, e) Heleopera sphagni, f) Cyclopyxis eurystoma, g) Euglypha laevis, h), Euglypha strigosa and i) Nebela collaris 50 um Figure 4. EES] Most frequent testate amoebae found in Sao Miguel: a) Trinema lineare, b) Corythion constricta, c) Assulina muscorum, d) Padaungiella lageniformis, e) Heleopera sphagni, f) Cyclopyxis eurystoma, g) Euglypha laevis, h), Euglypha strigosa and i) Nebela collaris. A total of six taxa occurring at only one sampling site were considered rare (Fig. 5). These included Cryptodifflugia oviformis; Euglypha filifera; Physochila griseola; Planocarina carinata; Pyxidicula cymbalum and Trachelocorythion pulchellum. Another 14 taxa were 14 Souto M etal considered occasional, occurring in two to five sampling sites. These included species, such as Alabasta militaris, Argynnia dentistoma, Cyclopyxis kahli, Argynnia caudata, Corythion constricta, Difflugia bacillifera, Difflugia elegans, Euglypha acanthophora, Arcella catinus, Centropyxis constricta, Cyphoderia ampulla, Euglypha cristata, Padaungiella tubulata and Trigonopyxis arcula. Species richness significantly differed amongst habitats (one-way ANOVA, P < 0.05). Overall species richness was in the order Natural forest > Peatland > Cryptomena forest > Lake > Hydrothermal, but significant differences were found only between Native forest and Lake habitats (Fig. 6). Euglypha strigosa | rinema lineare | Euglypha rotunda | Assulina muscorum | Cyclopyxis eurystoma _ Corythion dubium Trinema complanatum | Euglypha laevis Heleopera rosea Trinema enchelys Heleopera sphagni ebela collaris Tracheleuglypha dentata Quadrulella symmetrica Padaungiella lageniformis Centropyxis aerophila Sphenoderia fissirostris | Playfairina valkanovi Arcella arenaria Difflugia glans | Phryganella acropodia | Centropyxis elongata Centropyxis discoides Trigonopyxis arcula | Padaungiella tubulata | Euglypha cristata | Gentropysis constricts Cyphoderia ampulla Arcella catinus Euglypha acanthophora Difflugia elegans yclopyxis kahli |Wail my) Natural bach Bea res i ahd | ME Cryptomeria rachelocorythion pulchellum Pyxidicula cymbalumn MH Peatland lanocarina carinata | TP Hydrothermal Physochila griseola Euglypha filifera ME Lake Cryptodifflugia oviformis | 0 10 20 30 40 50 60 70 80 90 % Occurences of sampling sites Figure 5. EES Percentage of occurrence of each species for the 46 samples and their distribution amongst habitats (proportion of occurrences is represented in colour bars) 18 16 14 12 10 Species richness (S) Native Cryptomeria Peatland Hydrothermal Lake Figure 6. EESl Boxplot of species richness in each sampled habitat. Different letters indicate significant differences (Tukey Test, p < 0.05). Distribution of testate amoebae in bryophyte communities in Sao Miguel ... 15 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 Sao 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—collans species complex is a problematic group having very similar species (Heal 1964, Gilbert et al. 2003, 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, Satkauskiené 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 Annobon (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 Annobon (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 Sao Miguel Island were composed mainly by genus with a cosmopolitan distribution which are also known from other oceanic islands. For example, in the comparable Annobon 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 16 Souto M etal habitat diversity surveyed. The information regarding protist communities cames from Sphagnum moss on peatlands (Glime 2017, Lamentowicz and Mitchell 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 (Sj6gren 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 (Sj6gren 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 (Sjégren 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 Sao 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 Distribution of testate amoebae in bryophyte communities in Sao Miguel ... 17 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-T6oha 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 Sao 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. 18 Souto M etal Acknowledgements This work was funded by FCT — Foundation for Science and Technology, the European Union, QREN, FEDER, COMPETE programmes (PMR - DL57/2016/ICETA/EEC201 8/25; MSS - ICETA/EEC2018/25; DiscoverAzores Project - PTDC/CTA-AMB/28511/2017; UID/ BIA/50027/2020 and POCI-01-0145-FEDER-006821), AZORESBIOPORTAL — PORBIOTA (ACORES-01-0145-FEDER-000072) and Consolidation and Structuring Project 2018 GRC-ED431C 2018/32 of Xunta de Galicia government. We would like to thank Rafael Carballeira for his collaboration in the realisation of the SEM photographs. A special thanks to Mr. Matthew James Mole for revising this article. 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