Introduction Top

The big-leaf mahogany (Swietenia macrophylla King) is a tree mainly found in natural wet and dry tropical forests worldwide, in a wide variety of climatic and edaphic conditions (Mayhew & Newyon, 1998; Navarro-Martínez et al., 2018). Big-leaf mahogany is a highly appreciated fine timber that is an economically important and emblematic species from the Neotropics (Langbour et al., 2011; Navarro-Martínez et al., 2020). Its natural distribution includes fragmented populations from southeastern Mexico along the Atlantic coast of Central America and northern South America, occupying a large geographical arc south of the Amazon, between Brazil, Colombia, Peru and Bolivia (Lamb, 1966; Snook, 1996). This tropical tree has been intensively exploited and subjected to international trade for over 300 years, showing, therefore, a decline in its population size and increased fragmentation in several areas of its natural distribution (Navarro & Hernández, 2004; Grogan et al., 2010). Mexico reports a loss of 76% of the tropical evergreen forest areas containing mahogany trees by the end of the 20th century (Calvo et al., 2000). The original distribution of mahogany in Peru and Bolivia decreased by 4% and 8%, respectively, while a region between Venezuela and Bolivia, underwent 58 million hectares of deforestation until 2001 (representing 20% of the original distribution) (Kometter et al., 2004). In contrast, the Yucatan Peninsula, specifically in protected areas and forest ejidos in Quintana Roo and Campeche, harbors semievergreen and semideciduous forests with abundant and conserved populations of mahogany (Navarro Martínez et al., 2018, 2020). Currently, mahogany is a preferred species for reforestation and the establishment of commercial plantations throughout tropical America (Negreros-Castillo et al., 2018).

Vascular plants host a great variety of soil fungi, being susceptible to soil-borne pathogens, but plant roots are also colonized by non-pathogenic fungi like arbuscular mycorrhizal fungi (AMF) and dark septate fungi (DSF) (Mandyam & Jumpponen, 2005). Remarkably, AMF (belonging to Glomeromycota phylum) are an important ecological and economics group of soil fungi forming symbiotic associations with the vast majority of plants (Wang & Qiu, 2006; Brundrett & Tedersoo, 2018; Chen et al., 2018), including most forest tropical species (Stürmer et al., 2018). In this association, these fungi receive their carbon sources from the plants in exchange for water and minerals (e.g., P, N). As such, they play critical roles in the biogeochemical cycle of C, N and P (van der Heijden et al., 2015). Most tropical forest species have different grades of dependence on AMF, depending on successional stages or soil fertility (Danieli-Silva et al., 2010; Schüßler et al., 2016).

The AMF symbiosis deserves more attention in tropical ecosystems, especially in degraded tropical regions where the availability of nutrients such as phosphorus is a limiting factor in plant growth. Different studies have addressed the identification of AMF spores within the rhizosphere and root colonization (i.e., vesicles and arbuscules) of seedling and mahogany trees in Neotropical natural areas (Herrera & Ferrer, 1980; Rodriguez-Morelos et al., 2014), young plantations in the Amazon region (Noldt & Bauch, 2001; Pereira et al., 2014) and agroforestry systems and tropical forests of Southeast Asia where mahogany was introduced for cultivation (Dhar & Mridha, 2006, 2012; Shi et al., 2006, 2007; Mridha & Dhar, 2007; Nandi et al., 2014). However, the taxonomic identity of the specific AMF species colonizing the roots of S. macrophylla remains unknown. An increasing number of case studies report Glomero mycota molecular diversity from ecosystems worldwide (Husband et al., 2002; Lara-Pérez et al., 2020. A unique molecular operational taxonomic unit (MOTU) nomenclature – virtual taxa (VT) –, was performed to classify AMF rRNA sequences, as implemented in a public database MaarjAM (http://www.maarjam.botany.ut.ee; Öpik et al., 2010, 2014). Then a consistently named system of small-subunit (SSU) rRNA gene sequence phylogroups can be used as a proxy for species and/or higher-level organism identification in ecological research (Öpik et al., 2014).

According to Rodriguez et al. (2009), the DSF (Class 4 endophytes) are distinguished as a functional group based on the presence of darkly melanized septa, and their restriction to plant roots, primarily ascomycetous fungi that are conidial or sterile and that form melanized structures such as inter- and intracellular hyphae and microsclerotia in the roots. DSF are found worldwide and coexist often with different mycorrhizal fungi. They have been reported from 600 plant species including plants that have been considered non-mycorrhizal (Jumpponen & Trappe, 1998). However, studies of endophytic fungi carried out in tropical forests are limited to fungal species that colonize the above ground part of the plant (Arnold et al., 2000; Cannon & Simmons, 2002; Silva et al., 2018). Lately, 55 endophytic fungi (Class 2 endophytes) were isolated from the roots of mahogany monoculture and identified by their rDNA ITS1 region (Rodriguez et al., 2009; Maulana et al., 2018). A close relationship between DSF and AMF with P availability and uptake in plants was suggested. Whereas DSF increases the pool of P in the rhizosphere, AMF are responsible for P transfer to the host, with co-colonization of plants by dual fungal colonization suggesting a synergistic outcome (García et al., 2012; Della Monica et al., 2015).

In the present study, the specific objectives were to (i) describe the diversity of AMF in the roots of S. macrophylla, based on rDNA sequences and (ii) evaluate the dual colonization by AMF and DSF, showing the types of fungal colonization patterns.

Material and methods Top

Study area and sampling design

This study was conducted in permanent plots of S. macrophylla seed trees, within a natural tropical rainforest from Ejido Laguna Om (18°25’60”N & 89°7’60”W), municipality of Othón P. Blanco, Quintana Roo, Mexico. The dominant accompanying plant species include Manilkara zapota (L.) P. Royen, Vitex gaumeri Greenm, Lysiloma latisiliquum (L.) Benth, Brosimum alicastrum Sw. and Acacia collinsii Saff. The climate is warm subhumid with rains in summer and winter. The average temperature is 26°C and the annual precipitation is 1290 mm (INEGI, 2016).

Ten adult seed trees of mahogany were selected, at a distance of at least 100 m between individuals, for sampling mycorrhizal roots. Select trees had ages of 32.6 ± 4.6 years, basal diameter (± SD) of 1.4 ± 0.43 m and height (± SD) of 12.3 ± 3.21 m. Samples were obtained during the dry season of 2016, from February to April, removing the organic matter and digging up to 20 cm depth; secondary roots anchored to the supporting mahogany roots were collected. After removing adhering soil, samples were deposited in hermetic bags and microtubes of 1.5 mL;10 cm of additional roots were placed in cetyltrimethyl ammonium bromide (CTAB) buffer solution as described by Harrison et al. (1994), for their temporary preservation and subsequent laboratory analysis.

Mycorrhizal and DSF colonization

To determine the degree of mycorrhizal and DSF colonization, we used the method of Phillips & Hayman (1970) as modified by Kormanik et al. (1980). Secondary root segments of 1-2 cm were used, KOH (10% w/v) was added to permeate and clarify the cells, then root segments were autoclaved for 10 min at 121 °C (68977.59 Pa), then H₂O₂ (10% v/v) was added for 10 min to remove pigments, then roots were acidified with HCl (10% v/v) for 3 min, and stained with trypan blue in an autoclave for 10 min at 121 °C.

To evaluate fungal colonization, three replicates of 15 root segments of 1 cm each (45 cm total per tree), were placed in parallel on a slide and fixed with glycerin for observation under a microscope a 10x and 40x. We analyzed a total of 450 cm of root for this study, and only stained cenocitic hyphae, coils, vesicles, and arbuscules were counted to determine AMF colonization. The mean percentages of these fungal structures in all root segments were used in our analysis. To quantify dark septate fungal colonization, we counted the presence of hyphae that were both septate and melanized with thick walls, and microsclerotia. The fungal structures observed were recorded with a Nikon D850 camera. The Figure 1. Relative abundance (%) of virtual taxa of arbuscular mycorrhizal fungi associated with secondary roots of mature trees of Swietenia macrophylla.presence of intracellular and intercellular hyphae, as well as Arum-type arbuscules and Paris-type arbuscules, and the percentage of total colonization was quantified. The Arum-type colonization is characterized by intercellular hyphae and well-defined arbuscules; Paris-type consists of intracellular hyphae, the presence of coils or coils with rudimentary arbuscules; and the intermediate is the combination of the two patterns of colonization (Dickson, 2004). The data were analyzed with the non-parametric U-Mann Whitney test (p < 0.05), in the PAleontological STatistics (PAST) program.

Figure 1. Relative abundance (%) of virtual taxa of arbuscular mycorrhizal fungi associated with secondary roots of mature trees of Swietenia macrophylla.

DNA extraction and bioinformatics

Genomics DNA was extracted from 300 mg of a composite sample of mahogany roots (three trees) using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer´s instructions with 50 mL of elution buffer. DNA was sent to the Research and Testing Laboratory (Lubbock, TX, USA) for Illumina MiSeq sequencing, targeting a partial sequence of the small subunit (SSU) 18S rRNA gene. To perform sequencing reactions, the methodology reported by Lara-Pérez et al. (2020) was followed.

Denoising, homopolymers, and chimeric sequences were removed using UCHIME (Edgar et al., 2011). Virtual taxa (VT) were assigned with Blast search against the MaarjAM AMF database with sequence similarity ≥ 97%, and choosing the sequences with the highest values for the phylogenetic tree. We used the VT concept that allows standardization, as well as binomial taxonomic nomenclature, and comParison between studies where phylogenetically defined sequence variations correspond roughly to species-level taxa. We employed MaarjAM database described by Öpik et al. (2014) for the identification of environmental sequences. Application of VT is becoming widespread, and the MaarjAM database is increasingly used as a reference for environmental sequence identification.

Representative sequences for each VT were chosen for phylogenetic analysis. The phylogenetic tree was performed using the Neighbor-joining methodology with the MEGA 7 program (Tamura et al., 2007) with the Kimura-2 model (Kimura, 1980), and the bootstrap method (Felsenstein, 1985) with 1000 replicates as support for the branches. Sequences of Paraglomus laccatum (AM295493) and Paraglomus brasilianum (AJ301862) were obtained from the NCBI database (www.ncbi.nlm.nih.gov) and used as outgroup. Representative sequences of each VT were submitted to the NCBI database under accession numbers from MK511799 to MK511809.

ResultsTop

Metabarcoding

In total, we obtained 2840 reads and designated VT based on sequence similarity with a minimum identity ≥ 97%. Eleven VTs were obtained, that correspond to Glomus sp. (10) and Diversispora sp. (1). The four most abundant VT were VT186, VT126, VT129, and VT87 in order of priority (Figs. 1 and 2).

Figure 2. Phylogenetic tree of representative sequences of arbuscular mycorrhizal fungi virtual taxa associated with secondary roots of Swietenia macrophylla. Reference sequences from the MaarjAM database (Öpik et al., 2010). Bootstrap support values > 50 (999 iterations) are shown. Sequences from the present study are indicated with gray circles. New sequences have been submitted to the NCBI database (accession numbers from MK511799 to MK511809).

Colonization by fungal groups

In the present study, the co-occurrence of interactions of fungi like AMF and DSF were observed in mahogany roots. Different AMF structures such as hyphae, coils, vesicles, and arbuscules were identified (Fig. 3). Septate hyphae and microsclerotia corresponding to DSF were also observed (Fig. 4). We found significantly (p < 0.05) higher colonization of AMF than DSF. The roots of adult mahogany trees presented 80% of AMF colonization, while DSF fungal structures were present in 66% of root-length colonization (Fig. 5a). Hyphae were the most frequent structures in mahogany roots with 80% colonization, followed by vesicles with 18%, coils 2%, and arbuscules with 0.5%. Septate hyphae were observed in 60% and microsclerotia in 12 % of root length colonization (Fig. 5b).

Figure 3. Arbuscular mycorrhizal fungi structures in secondary roots of mature trees of Swietenia macrophylla. A, arbuscules; V, vesicles; C, coils; Ih, intracellular hyphae. Bar: 50 µm.

Figure 4. Dark septate fungi structures on secondary roots of mature trees of Swietenia macrophylla: mc, microsclerotia; sh, septate hyphae. Bar: 50 µm.

Figure 5. Percentages of root length colonization on secondary roots of Swietenia macrophylla mature trees by: (a) arbuscular mycorrhizal fungi (AMF) and dark septate fungi (DSF); (b) AMF (hyphae, vesicles, coils and arbuscules) and DSF (septate hyphae and microsclerotia); (c) AMF colonization types; intermediate colonization is the combination of Arum-type and Paris-type colonization. Values are presented as means ± SE (n = 10). Different letters above the histogram bars indicate significant differences between groups (p<0.05, U-Mann Whitney test).

Mycorrhizal colonization

Mycorrhizal colonization on mahogany roots was mainly Paris-type, characterized by intracellular hyphae and arbuscules, with 62% of root length colonization. The intermedia type colonization was 7% and the Arum-type was detected in only 4% of the root length (Fig. 5c.