Distribution of arbuscular mycorrhizal fungi (AMF) in Terceira and São Miguel Islands (Azores)

Abstract Background The data, presented here, come from samples collected during three research projects which aimed to assess the impact of land-use type on Arbuscular Mycorrhizal Fungi (AMF) diversity and community composition in pastures of Terceira Island (Azores, Macaronesia, Portugal) and also in the native forest of two Azorean Islands (Terceira and São Miguel; Azores, Macaronesia, Portugal). Both projects contributed to improving the knowledge of AMF community structure at both local and regional scales. New information Little is known on the AMF communities from Azores islands and this study reports the first survey in two Azorean Islands (Terceira and São Miguel). A total of 18,733 glomeromycotan spores were classified at the species level from 244 field soil samples collected in three different habitat types – native forests (dominated by Juniperus brevifolia and Picconia azorica), semi-natural and intensively-managed pastures. Thirty-seven distinct spore morphotypes, representing ten glomeromycotan families, were detected. Species of the family Acaulosporaceae dominated the samples, with 13 species (38% of the taxa), followed by Glomeraceae (6 spp.), Diversisporaceae (4 spp.), Archaeosporaceae (3 spp.), Claroideoglomeraceae (3 spp.), Gigasporaceae (3 spp.), Ambisporaceae and Paraglomeraceae, both with the same number of AMF species (2 spp.), Sacculosporaceae (1 sp.) and Entrophospora (family insertae sedis). Members of the family Acaulosporaceae occurred almost exclusively in the native forests especially associated with the Picconia azorica rhizosphere, while members of Gigasporaceae family showed a high tendency to occupy the semi-natural pastures and the native forests of Picconia azorica. Members of Glomeraceae family were broadly distributed by all types of habitat which confirm the high ecological plasticity of this AMF family to occupy the more diverse habitats.


Introduction
Arbuscular mycorrhizal fungi (AMF) are one of the most important groups of below-ground biota (Jeffries et al. 2003; . These obligate symbionts live in association with approximately 80% of vascular plants and have essential ecological roles, namely, they facilitate plant growth through enhancing uptake of several macro-and micro-nutrients of low mobility (e.g. P, Zn, Cu) in soil (Brundrett and Tedersoo 2018). Arbuscular mycorrhizas can also provide other ecological functions such as influencing the microbial and chemical environment of the mycorrhizosphere, stabilising soil aggregates (Rillig and Mummey 2006) and conferring plant tolerance to several abiotic (Göhre and Paszkowski 2006;Li et al. 2013;Chitarra et al. 2016) and biotic (Vos et al. 2012;van der Heijden et al. 2015) stresses.
AMF are, therefore, beneficial for plant performance, playing a crucial role for the sustainability of natural and agricultural ecosystems ) and important ecosystem services (Chen et al. 2018). However, despite their ecological role, little is known about how their community structure varies in relation to habitat type in the Azores archipelago.
The Azores archipelago has an extended area of grasslands (Martins 1993), including natural grasslands, semi-natural pastures and intensive pastures (Cardoso et al. 2009). It also has the unique native forest, Laurisilva, which has more endemic plants and animals than any other habitat in the region. In the last 500 years, as a consequence of human activity, much of this native forest has been replaced by man-made habitats and has been subjected to fragmentation (Borges et al. 2005). Thus, immediate action to restore and expand native forest is required to avoid the ongoing loss of endemic species (Terzopoulou et al. 2015). AMF play an important role in habitat restoration, by improving plant nutrition and performance under environmental stress by facilitating plant adaptation in both nursery and field conditions (Babu and Reddy 2011). Therefore, understanding the AMF diversity in the native forest will help to define strategies for management and restoration of such endangered forests. An important step in restoration strategies is the re-establishment of adapted native plant species (Ferrol et al. 2004). A good understanding of mycorrhizal associations in undisturbed localities could then be used to provide information about AMF inoculum production for use in the rehabilitation of degraded ecosystems.
In this contribution, we list the species of Arbuscular Mycorrhizal Fungi (AMF) found in ecological studies, comparing anthropogenically disturbed pastures and forests of Terceira Island (Azores, Macaronesia, Portugal) and also in the native forests of São Miguel Island (Azores, Macaronesia, Portugal).

General description
Purpose: In this contribution, we list the AMF species found in pastures from different landuse types of Terceira Island to investigate the effect of disturbance on AMF community structure. Native forests from Terceira and São Miguel Island were also sampled to observe patterns of AMF species composition and distribution, in order to provide baseline information for later use in establishing strategies for conservation of Picconia azorica and Juniperus brevifolia, in particular and native Azorean forests, in general.

Project description
Study area description: All data used in this study came from surveys about AMF diversity and composition in different ecosystems (pasturelands and native forests) conducted in two Islands of the Azorean archipelago, Terceira and São Miguel (Melo et al. 2014, Melo et al. 2017 (Fig. 1). The sampling areas were cattle-grazed upland pastures of two different types from Terceira and four fragments of native forests from each Island (Table 1) (Fig. 2). The two pasture types include semi-natural pastures with low grazing intensity and frequency (managed for more than 50 years, with low stocking density, grazed only in summer and with a relatively high diversity of grasses and forbs) and intensively-managed pastures with high grazing intensity and frequency (managed for more than 30 years, with high stocking density, grazing during all year and characterised also by a depauperate vascular flora of five or fewer dominant species) (Melo et al. 2014). The semi-natural pastures, Pico Galhardo (TER_SP_PG) and Terra Brava (TER_SP_TB) (Fig. 1) are included in Terceira Natural Park and are dominated by the perennial grasses Holcus lanatus and Agrostis castellana, have a high floristic diversity (Dias 1996, Borges 1997, often including other grasses such as Anthoxanthum odoratum, Lolium multiflorum, Holcus rigidus and Poa trivialis and non-forage species including Lotus uliginosus, Rumex acetosella ssp. angiocarpus, Potentilla anglica, Hydrocotyle vulgaris, Plantago lanceolata and Lobelia urens (For more details see Melo et al. 2014). The intensively-managed pastures, Agualva 1 (TER_IP_R1) and Agualva 2 (TER_IP_R2) (Fig.  1) resulted from the conversion of undisturbed native forest to wood production of nonnative trees and then to permanent pastures. They are now surrounded by an exotic eucalyptus plantation. The vegetation is dominated by Holcus lanatus and Lolium perenne, but also has high populations of Trifolium repens, P. lanceolata, Cyperus esculentus, Mentha suaveolens, Cerastium fontanum and Rumex conglomeratus (Dias 1996, Borges 1997.

Site
Longitude Latitude In Terceira Island, the native forests included two fragments from Natural Park -Pico Galhardo (TER_NF_PG) and Lagoinha (TER_NF_LA) (Fig. 1)  azorica -Terra Brava (TER_NF_TB) and Serreta (TER_NF_SE) (Fig. 1). Terra Brava is located in the very wet Laurisilva at 650 m altitude ( Fig. 1) 1) and is characterised by a low diversity of plants, dominated by Morella faya and P. azorica and, occasionally, by L. azorica. These forests are located in the most thermophilic areas of Azores and are almost extinct (Dias 1996). The highest canopy is dominated by a dense cover of P. undulatum and, rarely, by L. azorica. This forest is mixed with other invasive woody species, including Metrosideros excelsa, E. globules, A. melanoxylon, Sphaeropteris cooperi, Fuchsia magellanica and Rubus inermis. The herbaceous stratum is dominated by Dryopteris azorica, Hedera helix var. azorica, Smilax aspera and Gomphocarpus fruticosus (Dias 1996).

Sampling methods
Sampling description: In semi-natural and intensively-managed pastures, the soil samples with associated roots were randomly collected with a shovel, from the rooting zone of the dominant plant species, H. lanatus, to a depth of 0 -20 cm. In native fragments of P. azorica, the distance between samples taken on each site was a minimum of 25 m and maximum of 40 m and the distance between sample sites was about 20 km in Terceira and 15 km in São Miguel. Each soil sample was geo-referenced and consisted of four subsamples collected from different points (approximately N, S, E and W) around the rooting zone of each P. azorica plant with a shovel to a depth of 0 -20 cm or 0 -30 cm, depending on the soil conditions and the depth of rhizosphere system. The litter layer was removed during sampling and replaced afterwards. Subsequent samples were taken from the same marked plants following the cardinal points. In the case of native fragments of J. brevifolia, the distance between samples taken on each site was between 25 m and 40 m and the distance between sample sites was about 5 km in Terceira and 24 km in São Miguel. The sample collection followed the same procedure as for P. azorica (Melo et al. 2017). For all habitat types, each soil sample consisted of approximately 2 kg of rhizosphere soil. In the lab, the soil samples were air-dried, sieved through a 2 mm mesh and stored at 4ºC before analysis.
Quality control: Frequently, spores directly extracted from the soil are low in number and contaminated by other organisms, which makes their identification difficult. Consequently, it is necessary to establish trap cultures to promote sporulation and provide specimens for detailed examination. Open pot-trap cultures (Gilmore 1968) were established from each soil sample collected at semi-natural and intensive pastures with one-week-old Zea mays seedlings (Melo et al. 2014). Soil samples collected from the native forests were used to establish two of such cultures, one with one-week-old Z. mays seedlings and another one with micropropagated J. brevifolia and P. azorica seedlings (Melo et al. 2017). Establishment of single or multi-spore cultures of the different AM fungal morphotypes with Plantago lanceolata as host plant was attempted in pots of river sand. Spores with a healthy appearance (oily contents; without evidence of contamination by non-AMF) of each AM fungal morphotype were used as inoculum by placing them on a seedling root system under a dissecting microscope, immediately before transplanting into the pot (Melo et al. 2017. Specimens were given a voucher number, linked to their culture attempt number. Individual microscope slides were numbered serially so that photographic images could be traced back to their specimen of origin and details were recorded in a database to allow complete tracking of culturing history and linkage of related voucher specimens. The new cultures were placed in a climate-controlled plant growth chamber. When needed, cultures were watered with deionised water. Individuals of morphologically characterised spore types, extracted from field soil, trap cultures or single spore cultures, were used for DNA analysis. Molecular characterisation, including DNA extraction, PCR, cloning, RFLP, sequencing and phylogenetic analyses, is published in Melo et al. (Melo et al. 2017).
Step description: Glomeromycotan spores were extracted from 50 g of air-dried soil from each sample (field soil, trap cultures and single or multi-spore cultures) by wet sieving and sucrose centrifugation (Walker 1992) and stored at 4ºC in autoclaved water, pending examination. Different spore types were initially separated in water under a stereomicroscope. Representatives of each morphotype were identified through a compound microscope in a 4:1 mixture of polyvinyl alcohol lacto-glycerol (PVLG) and Melzer's reagent, photographed and stored as semi-permanent slide preparations. Counts were made for the total number of spores of each morphotype under a dissecting microscope after classification into either known species or types that could not be placed in a current species, based on colour, size, surface ornamentation, hyphal attachment, reaction to Melzer's reagent and wall structure. Identification of spores was carried out by use of primary literature and experience from more than 40 years of taxonomic study of the Glomeromycota by C. Walker (e.g., Walker and Trappe 1981, Walker et al. 1984, Koske and Walker 1985, Walker et al. 1986, Walker and Diederichs 1989, Walker 1992

Description:
The following data table includes all the records for which a taxonomic determination of the species was possible. The dataset submitted to GBIF (Melo et al. 2019) is structured as a sample event dataset, with two tables: event (as core) and occurrences. The data in this sampling event resource have been published as a Darwin Core Archive (DwCA), which is a standardised format for sharing biodiversity data as a set of one or more data tables. The core data table contains 226 records (eventID). One extension data table also exists with 665 occurrences. An extension record supplies extra information about a core record. The number of records in each extension data table is illustrated in the IPT link. This IPT link archives the data and thus serves as the data repository. The data and resource metadata are available for downloading in the downloads section.