Our DNA sequence data point to the presence of diverse and abundant assemblages of organisms previously not recorded on Trindade Island. The presence of some organisms may indicate the consequences of human influence while others are consistent with the known local diversity. However, we also recognize that the detection of fragments of an organism’s DNA does not confirm the presence of living organisms or viable propagules, as the finding can be related to encysted forms, spores, pollen or even single cells, while sometimes dead tissue can also provide detectable DNA. Sequence assignment also relies on the quality and completeness of data available in existing sequence databases.
Bacteria and Archaea
The present study detected few sequences representing the domain Archaea in samples PD5 and PF7, belonging to the class Thermoplasmata, phylum Euryarchaeota. Recent studies have shown that the relative abundance of these Archaea among all archaeal sequences in soil can be higher than previously reported [63]. Themoplasmatales is the order of Archaea with most organisms in culture and the only one validated under the rules of the International Code of Nomenclature of Bacteria. They comprise extreme acidophiles that lack a cell wall [64] and are usually found in volcanic continental areas; they derive their energy from aerobic respiration but can grow anaerobically using iron or sulfur in their metabolism, which contributes to their prevalence in acid mine drainage communities [65]. The sequences identified here could not be classified at the order level, and the physiology of these microbes remains unknown.
The bacterial community was dominated by Actinobacteria, Proteobacteria, Acidobacteria, Chloroflexi and Verrucomicrobia. This dominance is generally typical of soil bacterial community composition in soil samples [66].
Among the representatives of the phylum Actinobacteria, the class Acidimicrobiia stands out, as it is typically not as dominant as the classes Actinobacteria and Thermoleophilia in soils. Most cultured members of the class Acidimicrobiia are extreme acidophiles, originally obtained from geothermal or iron-rich mining sites and belonging to the family Acidomicrobiaceae [67]. However, these taxa were not detected in the current study and most sequences belonging to this group could not be further classified, indicating that they belong to unknown groups. The bacterium IMCC26256 was the most abundant identified sequence of Acidimicrobiia found in the Trindade Island samples, a taxon first described in a freshwater study and that forms a separate branch within this class [67]. Among the genera identified here were Luedemannella and Mycobacterium, taxa found in soil, and Acidothermus, found in acidic geothermal springs [67].
Only the classes alpha and gamma proteobacteria were identified in Trindade Island soil samples. However, it should be noted that the classification system used in the present study combines the class formerly known as betaproteobacteria with the gamma proteobacteria. Members of alpha proteobacteria are metabolically very diverse and include nitrogen-fixing bacteria [68]. Although the majority of the sequences assigned here belong to the order Rhizobiales, none of the identified taxa are nitrogen-fixing, but are genera found in soil and the rhizosphere. Sequences assigned to the orders Elsterales and Micropepsales were also abundant, but there are no cultured representatives of these orders and their physiology is unknown. Members of the gamma proteobacteria mostly represented the orders Burjkholderiales and Pseudomonadalles, groups that are common in soils. All three soil samples examined here contained high numbers of the genus Acidibacter, an acidophile known from iron-rich mine sites [69].
The Acidobacteria sequences assigned in this study belong to taxa usually found in acidic environments, such as members of the class Acidobacteriia (former subgroups 1, 2, 3, 5, 11, 12, 13, 14, 15, 24) [70]. Trindade Island soil samples included members of the class Acidobacteriia along with the Class Vicinamibacteria, which are heterotrophic aerobic bacteria, and class Holophagae, an anaerobic group. Their presence is consistent with bacterial communities from acidic, iron-rich soils, and possibly associated with geothermal sources. Many of the soil bacterial taxa assigned here are unknown and further research is necessary to understand their roles in Trindade Island soils.
Fungi
The most dominant sequences assigned in the giant fern forest soils were Sclerotiniaceae sp., Antarctomyces psychrotrophicus, Pseudogymnoascus sp., Apiotrichum sp. and Mortierella humilis, which represent fungi from different phyla. The family Sclerotiniaceae includes 47 genera and 284 species, many of which are pathogenic or saprophytic taxa, which able to infect various plant species and tissues. These fungi are characterized by the formation of sclerotia and stalked apothecia located within the colonized host plant tissue [61]. The genus Antarctomyces includes only two known species, A. psychrotrophicus and A. pellizariae, which were originally described from Antarctica. Antarctomyces psychrotrophicus was originally described in soil samples from King George Island [71] and later reported from other Antarctic habitats [72] but not elsewhere, this is the first record outside Antarctica. Pseudogymnoascus (anamorphic form-genus Geomyces) has a wide distribution globally [73, 74] and has been reported in soils from Arctic, alpine, temperate, and Antarctic regions [75, 72]. Pseudogymnoascus taxa are capable of colonizing and utilizing different carbon sources and can be particularly abundant at lower temperatures [76]. Pseudogymnoascus has received attention due the pathogenic species, P. destructans, the causative agent of white-nose syndrome (WNS) in bats in temperate regions [77].
Apiotrichum is an anamorphic basidiomycetous yeast genus containing 20 species distributed widely globally, including a number of soil-associated species [78, 79]. The genus Mortierella includes approximately 100 species [80], occurring mainly in different soil types [61]. Some members of Mortierella appear to assist crops and mycorrhizal fungi in phosphorus acquisition [81]. In addition, they can decompose plant debris and degrade polyaromatic hydrocarbons [82]. Mortierella humilis has been isolated from plant tissues, the rhizosphere and roots in different tropical and temperate ecosystems and is reported as a producer of fatty acids useful in medicine, pharmacology, cosmetics, agriculture and in the food industry [83]. In addition, M. humilis produces enzymes capable of degrading xylans, sugars (such as sucrose, galactose, fructose, mannose and maltose), paraffin, chitin, cellulose and lignin [84, 85, 86].
Metazoa
None of the Metazoa sequences detected here were found in both sampling locations. Desejado Hill samples generated the greatest diversity, but we suggest that other markers more widely used animal studies, such as COX1 would identify a more diverse community of metazoans living in Trindade Island soils. The Collembola (springtails) are one of the most common, widespread and abundant arthropod groups, with a wide global distribution [87], and are particularly abundant in leaf litter and soil. Despite their low biomass, Collembola do influence soils. Kubiena (1953) [88] reported a layer of humus 15-20 cm deep composed entirely of their feces and Lagerlöf & Andren (1991) [89] highlighted the importance of Collembola in agricultural soils. Desoria is a genus of about 100 species, with a wide distribution in the Northern Hemisphere, including in glacial-influenced habitats, while some species have affinity to anthropogenically disturbed areas. Having limited dispersal abilities, the genus may have been introduced in Trindade by humans, as is the case for D. trispinata, which is found in the Azores [90] and colonized the island in the 18th century. Lepidocyrtus is one of the largest collembolan genera worldwide [90]. Lepidocyrtus koreanus is an Asiatic species, but this specific assignment is likely an instance of database incompleteness. The presence of Collembola on Trindade Island was cited by Alves [6], but this may represent an error as the species cited is a true insect rather than a collembolan. However, it is also extremely unlikely that the island does not host a native, and likely highly endemic, collembolan community, as do other remote volcanic islands in the Atlantic [91, 92, 93, 94]. Both of the taxa assigned here were obtained from the Desejado Hill sample (PD6) and have not been recorded from Trindade Island previously.
The nematode genus Heterocephalobellus includes only three species worldwide, all terrestrial, and was originally described from Brazil [95]. To date, only marine nematodes have been investigated in Trindade Island [96]. In the current study, assigned sequences were only obtained from Desejado Hill (PD5). The platyhelminth Rhynchoscolex simplex is a common species and has been recorded from São Paulo, mainland Brazil [97]. The assigned sequence was obtained only from Fazendinha (PF7). Both the nematode and platyhelminth are new records for Trindade Island, but appear likely candidates as anthropogenic introductions. Again, certainly the nematodes are likely to have a diverse native community on Trindade Island, requiring application of both appropriate survey techniques and molecular probes.
Chromista and Protozoa
Most of the reads representing these groups were assigned to Kingdom rank, excepting that of Sellaphora pupula, a cosmopolitan freshwater diatom [98], including in Brazil, Africa and the Azores. In Brazil this species is found in coastal regions and the Atlantic Rainforest [99]. The assignment here is the first record from Trindade Island, where it was found only at Desejado Hill (PD5).
Cercomonadida was found only at Desejado Hill (PD6) and is the second most commonly recorded zooflagellate in soils globally [100, 101]. Within this group, Eocercomonas is a less known genus segregated from the widespread Cercomonas [101]. Eocercomonas echina is a freshwater and soil species previously reported only from the United Kingdom and South Korea [102] and was found here only at Fazendinha (PF7). Vahlkampfia is a poorly known genus including about 12 species that is abundant worldwide in a wide variety of aquatic and terrestrial habitats [103] and potentially pathogenic for humans [104]. It was found at both Desejado Hill (PD6) and Fazendinha (PF7). Thaumatomonas is another poorly known Northern Hemisphere genus commonly found in lake sediments [105], and assigned here only at Fazendinha only (PF7). All the protist sequences assigned in this study have not been previously reported from Trindade Island.
Viridiplantae
The four green algae (Chlorophyta) reported have not previously been recorded from Trindade Island [98]. Eremochloris sphaerica is a North American species common in brackish waters. Trebouxia is a cosmopolitan and widespread genus including more than 40 species [98] and has been found in almost every environmental condition including terrestrial and aerial. It is commonly found on tree bark in humid forests and is also a common photobiont of lichens [106]. Chlamydomonas is a genus including more than 200 species and is widely distributed in both fresh and sea water as well as in soil and snow [98]. Asterochloris is a genus with more than 19 cosmopolitan species and is one of the most common lichen photobionts [98]. All the green algae recorded here were present only at Desejado Hill (PD5 and PD6).
The moss Campylopus oerstedianus (Bryophyta) is a Central and North American species not previously reported from Trindade Island. Faria et al. [11] recorded two other species in this genus from Trindade Island, with further discussion of the identify of these representatives given by Gama et al. [20]. We recorded it only at Desejado Hill.
The fern Alsophila gigantea (=Cyathea gigantea) is an Asiatic species. Alves et al. [21] reported spores of a different species of Cyathea in air samples obtained on the island. However, the assignment generated in the current study is likely to reflect a lack of completeness in the available database, and mostly likely refers to C. delgadii, the single and dominant tree fern on the island. It is important to note that GenBank contain only 8 ITS sequences of Cyathea, not including C. delgadii, and that some of these sequences are very short (less than 300 bp). This further reinforces the importance of well curated and complete databases being available to support metabarcoding studies. Fern sequences were obtained from both sampling locations, Desejado Hill (PD5) and Fazendinha (PF7).
The two flowering plants reported here have not previously been reported from Trindade. Begonia cathayana (Begoniaceae) is an ornamental Chinese species, however other members of the genus are common in the Brazilian Atlantic Rainforest [107]. Begonia propagules could reach the island by means of aerial transfer or with human assistance. In the current study on the sequences were detected at both Desejado Hill (PD5) and Fazendinha (PF7). Vachellia gummifera (Fabaceae) is a leguminous plant native to Morocco and the Mediterranean Sahara. Again, some related species, V. caven, V. farnesiana, V. ibirocayensis and V. seyal, are native to Brazil, where they occur in coastal regions [108, 109]. The sequence was identified only at Fazendinha (PF7). It is important to note that, over time, many species have been introduced to Trindade Island with human assistance [6]. The number of these still present is not clear, but there remains the possibility that detectable DNA could remain and be detected using metabarcoding approaches.