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, Melo et al. 2018) (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 non-native 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).

Figure 1.  

Location of sampling sites in Terceira and São Miguel Islands (Azores).

Figure 2.  

Habitat types in Terceira and São Miguel Islands, Azores: a. native forest of Juniperus brevifolia; b. native forest of Picconia azorica; c. semi-natural pastures; d. intensively-managed pastures.

In Terceira Island, the native forests included two fragments from Natural Park – Pico Galhardo (TER_NF_PG) and Lagoinha (TER_NF_LA) (Fig. 1) (Melo et al. 2017), both dominated by the Azorean cedar Juniperus brevifolia, a rare conifer species that is endemic to Azores, which dominates at high-elevation (> 650 m), with subdominant endemic woody perennials, including Laurus azorica (Lauraceae), Ilex perado azorica (Aquifoliaceae), Erica azorica (Ericaceae), Vaccinium cylindraceum (Ericaceae) and Frangula azorica (Rhamnaceae) (Elias et al. 2016). Nevertheless, in Lagoinha, invasive woody species, including Cryptomeria japonica, Pittosporum undulatum (Pittosporaceae), Eucalyptus globules and Acacia melanoxylon (Fabaceae), have begun to establish. The remaining two native fragments from Terceira Island include two populations of Picconia 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) being dominated by endemic woody plants, predominantly L. azorica, I. azorica, Frangula azorica, V. cylindraceum, E. azorica, Myrsine africana and, occasionally, by J. brevifolia and P. azorica. Serreta (NFSE) is located at low altitude (95 m) (Fig. 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).

In São Miguel Island, the four native fragments are two populations of J. brevifolia up to 700 m altitude (Lombadas and Tronqueira) and two populations of P. azorica in the lowlands around 95 m altitude in Lombo Gordo and Ribeira Quente (Fig. 1). Lombadas (SMG_NF_LO) is included in the Natural Reserve of Lagoa do Fogo in São Miguel (Fig. 1). Although the surrounding vegetation is dominated by the introduced species C. japonica, Clethra arborea, A. melanoxylon and E. globulus, this forest still retains several endemic elements, including J. brevifolia, V. cylindraceum (Ericaceae), L. azorica, Euphorbia stygiana (Euphorbiaceae), F. azorica, E. azorica, I. azorica and Culcita macrocarpa (Culcitaceae) (Silva 2001). Tronqueira (SMG_NF_TR) is located in hyperhumid native forest (Fig. 1), a type of forest that has been largely replaced by other land uses (Moreira et al. 2012), resulting in an abundance of exotic plants, such as C. japonica and C. arborea. The tree layer is composed of the endemic woody plants J. brevifolia, I. azorica, L. azorica, M. africana (Myrsinaceae) and E. azorica, while the shrub layer is mostly formed by V. cylindraceum and Viburnum treleasei (Adoxaceae). Lombo Gordo (SMG_NF_LG) is covered by a coastal scrubland (Fig. 1) where P. azorica dominates in certain areas, but is mixed with other native and invasive woody species including M. faya, E. azorica, P. undulatum, Arundo donax, Hedychium gardnerianum and Phormium tenax (Martins et al. 2011). Ribeira Quente (SMG_NF_RQ) is also a coastal scrubland (Fig. 1) dominated by the endemic plants L. azorica and P. azorica but also associated with other native and invasive woody species, such as P. undulatum, M. faya and A. melanoxylon.

Arbuscular mycorrhizal fungi (AMF) diversity and composition were investigated at three habitat types: native forests of J. brevifolia and P. azorica, semi-natural pastures and intensively-managed pastures. Each habitat type was represented by two sites. At each site from semi-natural (Pico Galhardo (10.39 ha) – TER_SP_PG; Terra Brava (8.81 ha) – TER_SP_TB) and intensively (Agualva 1 (5.03 ha) – TER_IP_R1; Agualva 2 (3.06 ha) – TER_IP_R2) managed pastures, ten soil samples were collected in August 2007 (i.e. a total of 40 soil samples) (Project CD_Melo_PhD). In natural forests of J. brevifolia from Terceira (Pico Galhardo (13.97 ha) – TER_NF_PG; Lagoinha (3.05 ha) – TER_NF_LA) and from São Miguel (Lombadas (37.42 ha) – SMG_NF_LO; Tronqueira (51.70 ha) – SMG_NF_TR), 21 soil samples were collected from seven marked J. brevifolia plants in each site at three different sampling times (September 2012; May 2013; September 2013) in both islands, resulting in a total of 84 soil samples (2 islands × 2 sites/island × 7 samples/site × 3 sampling dates) (CD_Melo_Postdoc; FCT - PTDC /AGR-ALI/122152/2010). In natural forests of P. azorica from Terceira (Terra Brava (9.72 ha) - TER_NF_TB; Serreta (7.67 ha) – TER_NF_SE) and from São Miguel (Lombo Gordo (41.69 ha –SMG_ NF_LG; Ribeira Quente (6.32 ha) – SMG_NF_RQ), 30 soil samples were collected from ten marked P. azorica plants in each site, during the same sampling times for J. brevifolia, in both islands resulting in 120 soil samples (2 islands × 2 sites/island × 10 samples/site × 3 sampling dates) (CD_Melo_Postdoc; FCT - PTDC /AGR-ALI/122152/2010).

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.

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, Melo et al. 2018). 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, Melo et al. 2018). 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, Melo et al. 2018).

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, Walker and Vestberg 1998, Schüßler et al. 2011, Krüger et al. 2012, Redecker et al. 2013, Walker et al. 2018, Wijayawardene et al. 2018, Schüßler and Walker 2019) and joint authorship with A. Schüßler of the website amf-phylogeny.com, which lists all accepted species in the phylum. The illustrated manual of Blaszkowski (2012) was also used. Comparisons were made, where possible, with the type and the authenticated specimens and with other literature such as original species descriptions, websites (e.g. http://invam.wvu.edu) and examination of other well-documented specimens, when available.

Terceira and São Miguel Islands, the Azores, Macaronesia, Portugal.

37.423 and 38.959 Latitude; -27.532 and -24.917 Longitude.