Mangrove is a type of wetland found in the intertidal zone of tropical and subtropical coasts (Odum et al. 1985, Spalding et al. 2010), at the interface between the influence of the saline seawater and discharges of continental freshwater (Moreno-Casasola et al. 2006, Rzedowski 2006). In tropical zones, mangrove forests are vital to the maintenance of coastal ecosystems’ health (Adeel & Pomeroy 2002). Plant species that develop in mangrove ecosystems are classified into two types: true mangroves and associated species (Tomlinson 1986).
Conocarpus erectus L. (Combretaceae) is a tree or shrub mangrove associated species (López-Portillo & Ezcurra 2002) that establishes in a transitional manner in this ecosystem, between the true mangrove species and the plant communities further inland (Odum 1985, Krauss et al. 2008). Conocarpus erectus represents an important component of the Caribbean mangroves (López-Portillo & Ezcurra 2002), since the transition areas in which it establishes typically present soil with high percentage of sand and conditions of salinity and flooding in which other tree species do not prosper (Tovilla-Hernández & De La Lanza-Espino 1999, Thom 1967, Carter et al. 1973, Ellison & Farnsworth 1996, Rzedowski 2006). C. erectus possess importance by its medicinal and ornamental uses and as timber species (Tovilla-Hernández & De La Lanza-Espino 1999, Al-Humaid & Moftah 2007, Abdel-Hameed et al. 2012, Hussein 2016, Raza et al. 2018).
Among the biotic factors that could influence the functioning of mangrove ecosystems we found the arbuscular mycorrhizal association; however, little is known about its importance in these wetlands. Arbuscular mycorrhizal fungi (phylum Glomeromycota; Schüßler et al. 2001) are obligate symbionts recognized for providing nutritional benefits to their hosts and conferring tolerance to various stresses, such as elevated salinity in the substrate (Solaiman & Hirata 1996, Sokri & Maadi 2009, Borde et al. 2011). For this reason, it is likely that C. erectus benefits from association with these symbionts in its natural environment.
Despite the fact that elevated salinity and flooding can be detrimental to arbuscular mycorrhizal fungi (AMF) development (Juniper & Abbott 1993, Juniper & Abbott 2006, Le Tacon et al. 1983), mangrove plants present arbuscular mycorrhizal colonization in their roots (Lingan et al. 1999, Kumar & Ghose 2008, D’Souza & Rodrigues 2013b, Hu et al. 2015, Sengupta & Chaudhuri 2002, Wang et al. 2014, Gupta et al. 2016) and some assays have shown that the plants that establish in these ecosystems receive nutritional benefits (increased absorption of phosphorus, nitrogen and potassium; Wang et al. 2011, Xie et al. 2014, Dsouza & Rodrigues 2017) when associated with AMF. This suggest that not only are the AMF present in mangrove ecosystems, but also are effective symbionts for the species that establish there, where they likely perform as important a function as they do in terrestrial ecosystems (Ramírez-Viga et al. 2018).
With the aim to achieve an approach of the mycorrhizal status of C. erectus under natural conditions, roots and rhizospheric soil were collected from this species in mangroves of the Ría Lagartos Biosphere Reserve in Mexico. To determine temporal and spatial variation of some relevant components of the association in mangrove ecosystems, sampling was conducted in sites with different one-year typical salinity and, due to mangroves being seasonally dynamic environments (Zhang et al. 2016), in two contrasting climatic seasons (dry and cold northerly fronts or northwind).
Due to the detrimental effect that salinity and flooding can have on the AMF, we expected that in zones classified as more saline and in the wet season (when flooding may increase due to rain input), the fungal variables of AMF percentage of root colonization, spore density and species richness presented lower values than in zones classified as less saline and in the dry season (when flooding may decrease in de absence of rain input).
Materials and methods
Study area. The study area is found in the Ría Lagartos Biosphere Reserve, in the state of Yucatán, Mexico, between the coordinates: 21° 37’ 29.56’’ and 21° 23’ 00.96’’ N, 88° 14’ 33.35’’ and 87° 30’ 50.67’’ W. The study area represents a mangrove pertaining to the physionomic kind of “Dwarf mangrove”, characterized by being an extreme environment with highly saline and low in nutrient availability soils, strong winds and transient flooding in rain and northwind seasons, where trees heights ranges between 1 and 2 meters (Flores-Guido & Espejel-Carbajal 1994, CONANP 2007)
Three locations, consisting on Conocarpus erectus forests, were selected in the study area. These sites were classified into three categories according to salinity data recorded in the zone by the National Commission of Protected Natural Areas (CONANP, by its Spanish acronym) in the year 2009: Higher Salinity (HS), with a mean salinity of 86 ppt (parts per thousand), Medium Salinity (MS) with mean salinity of 70 ppt and Lower Salinity (LS) with mean salinity of 62 ppt.
Three main climatic seasons occur in the State of Yucatán: (1) the rainy season, which occurs from June to October, and in which the coast of the Yucatán Peninsula receives the majority of its annual mean precipitation (< 700 mm); (2) the season of northwind, which occurs from November to February and is characterized by precipitation (20-60 mm), strong winds (> 80 km/h) and relatively low temperatures associated with polar fronts; and (3) the dry season or drought season, characterized by the absence of precipitation, which occurs from March to May (Jiménez & Orellana 1999, Vidal-Zepeda 2005). Sampling for this study was conducted in northwind (December 2009) and drought (May 2010) seasons.
Collection and processing of roots and soil. In each of the three collection sites (HS, MS and LS), 20 individuals of Conocarpus erectus were selected and georeferenced. Fine roots were taken from each of these individual plants in order to quantify the percentage of arbuscular mycorrhizal colonization. In addition, four rhizospheric soil samples were taken (one from each cardinal point around the plant: 1 kg of soil in total, used as a composite sample per tree) to determine spore density, identify the AMF species from the field samples and for use in of propagation pots. The soil moisture content at each site was evaluated from the rhizospheric soil collected from each individual. For this, a subsample of the soil was oven-dried at 60 °C until reaching constant weight and the difference between the fresh and dry soil weight calculated.
Root staining and evaluation of the percentage of mycorrhizal colonization. Collected roots were washed, stained with trypan blue and mounted on slides, following the procedure of Phillips and Hayman (1970), modified by Hernández-Cuevas et al. (2008). The total percentage of mycorrhizal colonization in these roots was quantified following the method of McGonigle et al. (1990), modified by Hernández-Cuevas et al. (2008).
Spore extraction. The rhizospheric soil was dried at ambient temperature and the spores extracted using the techniques of wet sieving and decantation (Gerdemann & Nicolson 1963) and centrifugation with a saccarose gradient (Daniels & Skipper 1982), modified by Hernández-Cuevas et al. (2008). The spores extracted from each sample were placed on slides with polyvinyl alcohol + lactophenol (PVLG) and Melzer reagent for subsequent identification. These spore samples extracted from the rhizosphere were used to quantify the spore density in 50 g of soil and to determine AMF richness. The numbers of potentially viable and non-viable spores were estimated in each sample, categorizing as potentially viable the spores that presented cellular content and apparently undamaged cell walls.
Spore propagation. Arbuscular mycorrhizal fungi spores were propagated in order to obtain samples in better condition than those obtained from field sampling and thus to facilitate the identification of species. Propagation of spore communities was conducted in culture pots, following the method of Hernández-Cuevas & García (2008) and using Zea mays as a host species. At the end of this bioassay, the soil was processed with the technique of wet sieving and decantation (Gerdemann &Nicolson 1963) and centrifugation with a sucrose gradient (Daniels & Skipper 1982), modified by Hernández-Cuevas et al. (2008). The spores extracted from each sample were placed on slides with PVLG and Melzer reagent.
Identification of AMF species. Determination of the AMF species present in the rhizosphere of C. erectus was conducted using the AMF spores extracted from the rhizosphere of the mangroves and from the propagation pots. From these propagules, the arrangement, consistency, shape, size, color, wall texture, number of layers that comprise the wall, ornamentation, type of hyphae, auxiliary structures and scars were recorded. This was conducted using an optical microscope with a ruler reticle and objectives of 10X, 40X, 60X and 100X. The spore descriptions were compared with those of Schenck & Pérez (1988), those of West Virginia University (2019) International Culture Collection of (vesicular) Arbuscular Mycorrhizal Fungi (INVAM, by its Spanish acronym) (invam.wvu.edu/), those of the website of Janusz Blaszkowski (2003) (www.zor.zut.edu.pl/Glomeromycota/) and those of Arthur Schüßler (2020) (www.amf-phylogeny.com/).
Statistical analyses. Soil moisture content, arbuscular mycorrhizal colonization and AMF spore density were analyzed using two-way analysis of variance, with season and collection site as factors. This analysis was conducted with the software SigmaStat 3.2. Correlation analysis was performed to know the strength and direction of the relation between soil water content and total percentage of colonization.
Soil moisture content. Soil moisture content (Figure 1) differed significantly among sites and between seasons of sample collection (F 2, 114= 7.888; p < 0.001). The highest values of moisture content in the sites HS and MS were recorded during northwind season.
Soil moisture content in the sampling sites in the two periods sampled: northwind and drought seasons. LS = lower salinity, MS = medium salinity, HS = higher salinity
Arbuscular mycorrhizal colonization and AMF spore density. In the roots of C. erectus, arbuscular mycorrhizal colonization (Figure 2) of types Arum and Paris were recorded. The most and least frequent structures were hyphae and arbuscules, respectively. These structures were recorded in the roots from all collection sites and in both seasons. Analysis of variance revealed significant differences for the percentage of total colonization between collection seasons (F 1, 114 = 5.687; p = 0.019) and the interaction of these with the collection sites (F 2, 114 = 6.185; p = 0.003). The percentage of total colonization (Table 1) varied significantly between seasons only in site HS, decreasing during the drought season. Of the three sites, HS presented the highest values of colonization in the northwind season. During the drought season, no significant differences were found among sites. Correlation analysis showed a significant relationship between soil water content and total percentage of AMF colonization (p < 0.05000) with a correlation coefficient of -0.2994.