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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.
Results
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.
Figure 1 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.
Figure 2 A-D. Arbuscular mycorrhizal fungi structures colonizing roots of C. erectus. h = hyphae, v = vesicles, s = spores, c = coil and a = arbuscule are pointed out with arrows in the different images. Photographs taken in optic microscope: A 10X, B 60X, C 10X, D 40X
Table 1 Total percentage of mycorrhizal colonization in the roots of Conocarpus erectus and AMF spore density in its rhizosphere (number of spores/50 g of soil) mean ± S.E., in each study site (HS = higher salinity, MS = medium salinity, LS = lower salinity) and sampling season (northwind, drought). Lower case superscript letters denote differences among sites in each season and upper-case superscript letters denote differences in each site between seasons, for each of the variables. Different letters indicate significant differences (p< 0.05). n = 20
| Variable | Season | Site | ||
|---|---|---|---|---|
| HS | MS | LS | ||
| Percentage of colonization | Northwind | 71.09 ± 5.12 a A | 48.87 ± 5.45 b A | 54.21 ± 3.95 ab A |
| Drought | 40.70 ± 5.24 b B | 45.49 ± 5.35 a A | 58.02 ± 5.50 a A | |
| Spore density | Northwind | 13.26 ± 1.45 a A | 15.22 ± 4.74 a A | 9.62 ± 1.01 a A |
| Drought | 9.67 ± 1.26 a B | 7.60 ± 2.02 a A | 8.29 ± 0.98 a A | |
A total of 94 % of the field spores presented signs of parasitation, had no content or were damaged, for which reason they were considered non-viable. Spore density (Table 1) did not vary significantly among sites but did between seasons in the site HS (F 1, 107 = 4.826; p = 0.030), with the highest values of density presented during the northwind season.
Richness of AMF species. A total of 16 AMF species associated with the rhizosphere of C. erectus (Table 2) were identified. These belonged to eight genera and five families. The species found in all of the sites were Acaulospora scrobiculata, Funneliformis geosporum, Claroideoglomus etunicatum, Glomus rubiforme and Scutellospora erythropus. In addition to these 16 species, a morphotype of the genus Gigaspora was recorded that could not be identified to species level.
Table 2 List of arbuscular mycorrhizal fungi identified in the rhizosphere of Conocarpus erectus in each sampling site (HS = higher salinity, MS = medium salinity, LS = lower salinity) and season (N: northwind D: drought).
| Species | Site - Season | |||||
|---|---|---|---|---|---|---|
| HS-N | HS-D | MS-N | MS-D | LS-N | LS-D | |
| Acaulospora foveata Trappe & Janos | * | |||||
| Acaulospora myriocarpa Spain, Sieverd. & N.C. Schenck | * | |||||
| Acaulospora rehmii Sieverd. & S. Toro | * | * | * | |||
| Acaulospora scrobiculata Trappe | * | * | * | * | * | * |
| Ambispora appendicula (Spain, Sieverd. & N.C. Schenck) C. Walker | * | |||||
| Claroideoglomus etunicatum (W.N. Becker & Gerd.) C. Walker & A. Schüßler comb. nov. | * | * | * | * | * | * |
| Gigaspora decipiens I.R. Hall & L.K. Abbott | * | * | * | * | * | |
| Gigaspora sp. | * | |||||
| Scutellospora erythropus (Koske & C. Walker) C. Walker & F.E. Sanders [as 'erythropa'] | * | * | * | * | * | * |
| Funneliformis coronatum (Giovann.) C. Walker & A. Schüßler comb. nov. | * | |||||
| Funneliformis geosporum (T.H. Nicolson & Gerd.) C. Walker & A. Schüßler comb. nov. | * | * | * | * | * | * |
| Glomus deserticola Trappe, Bloss & J.A. Menge | * | * | * | * | * | * |
| Glomus lacteum S.L. Rose & Trappe [as 'lacteus'] | * | |||||
| Glomus microaggregatum Koske, Gemma & P.D. Olexia | * | * | ||||
| Glomus microcarpum Tul. & C. Tul. [as 'microcarpus'] | * | * | * | * | * | |
| Glomus rubiforme (Gerd. & Trappe) R.T. Almeida & N.C. Schenck | * | * | * | * | * | * |
| Sclerocystis microcarpus S.H. Iqbal & Perveen | * | |||||
| Total AMF species richness: | 10 | 9 | 9 | 7 | 12 | 11 |
The site that presented the greatest richness in both seasons sampled was LS (16 species for both seasons), followed by HS (10 species for both seasons) and finally MS (9 species for both seasons) (Table 2). No different species were recorded in the propagation pots. Gigaspora sp. and Sclerocystis microcarpus were registered only in the drought season and Acaulospora foveata, A. myriocarpa, A. appendicula, Funneliformis coronatum, Glomus lacteum and G. microaggregatum were only registered in the northwind season. Also we found some unique species by study site: A. myriocarpa was only found in the MS site and the species A. foveata, A. appendicula, Gigaspora sp., F. coronatum, G. lacteum and S. microcarpus were only registered in LS site. We did not find unique species in the HS site (Table 2).
Discussion
According to Carmona et al. (2013) recordings, arbuscular mycorrhizal fungi was found colonizing the roots of Conocarpus erectus. AMF colonization were registered in both seasons and in all sampling sites, and a high AMF species richness were found in its rhizosphere.
Both Arum and Paris kind of AMF colonization were found in C. erectus roots, this being a common finding in other plant species that has been studied in mangrove ecosystems (D’Souza 2016). Sengupta & Chaudhuri (2002) report 58.6 - 81 % of AMF colonization in trees pertaining to different successional states of a mangrove ecosystem in India. The studies that register AMF radical colonization in mangrove systems point out that these percentages depend both on the soil characteristics and on the form of life, identity and phenology of the phytobiont (Kumar & Ghose 2008, Muthukumar & Udaiyan 2000, Sengupta & Chaudhuri 2002, Sosa-Rodríguez et al. 2009). In his thesis dissertation, Echeverría (2006) reports less than 20 % of AMF colonization in C. erectus growing in a Petén near Yucatan’s coast, lower percentage than the one registered in this study. This difference could emphasize the soil properties influence on the AM association, as the Petenes are a kind of wetland with distinct characteristics in relation to those typically found in dwarf mangroves, as the presence of vegetation islands conformed by medium forest and mangrove in the center, with exterior influence of sea water and interior influence of freshwater and with high organic matter content in its soil (Moreno-Casasola et al. 2006).
Conocarpus erectus is typically found in substrates with salinities of 0-90 ppt but can tolerate salinities of up to 120 ppt (Agraz-Hernández et al. 2006). The substrate conditions in the three sites analyzed in the present study are considered hypersaline (FAO 1994) and can cause damage through the toxicity of the ions themselves or by osmotic stress to both the plants and the AMF (Juniper & Abbott 2006, Munns & Tester 2008, Evelin et al. 2009). However, it has been reported that the AMF are capable of providing benefits to their hosts in conditions of elevated salinity (Kumar et al. 2015). In this study, arbuscules were recorded within the roots of C. erectus in all three sites, indicating that the association is functional (Espinosa-Victoria 2000) under the entire range of salinities analyzed. This suggests that this fungal mycelium is morphologically and physiologically adapted to extreme environments, as it has been pointed out by Klironomos et al. (2001) and Sosa-Rodríguez et al. (2009).
Salinity and flooding affect the mycorrhizal association depending on the level at which they are present and the interaction between these two factors (Carvalho 2003, Sosa-Rodríguez et al. 2009, Wang et al. 2011). Correlation coefficient showed a negative correlation between soil water content and total percentage of colonization, this means that as soil water content increases, total percentage of colonization tends to decrease. In addition, although we did not measure the salinity in each collection point, our data suggest that the variation in the fungal variables studied could be also related to the season (in relation to the change in soil water content) effect on the salinity soil concentration. The highest percentage of colonization was recorded in the site of highest salinity (HS) in the northwind season (when highest soil water content was registered). This site apparently represents the most contrasting environment of the three sampling sites between seasons and presented a statistically significant decrease in the percentage of colonization, as well as in the spore density and species richness in the drought season, compared to the northwind season. During this latter season, precipitation in the coastal zones can flush the excess salts from the soil and thus favor development of the soil biota (Moreno-Casasola et al. 2006), including AMF. In the drought season, on the other hand, salt commonly concentrates in the substrate due to the intense evaporation (Kozlowski 1997), thus raising the salinity and inhibiting the development and association of AMF.
AMF species richness reported in this study results lower than that reported by Kumar & Ghose (2008) and Muthukumar & Udaiyan (2000), who found 44 and 35 species respectively in India mangroves. For their part, Sengupta & Chaudhuri (2002), Kothamasi et al. (2006) (both mangroves in India) and Wang et al. (2010) (mangrove in China), report seven, five and six species respectively, being these quantities lower than our finding. This variation could be related with the extension of the collection or with edaphic or hosts diversity characteristics of the sites. The AMF species richness in our study was greater in the least saline site (LS) in both seasons. This site would be less stressful in terms of salinity, compared to the other sites, giving more sensitive AMF species the opportunity to develop and produce spores (6 unique species were recorded in this site. See table 2). The conditions of site LS would be even less stressful in the northwind season, when the highest species richness value of all of the sites and both seasons was recorded.
The spore density recorded in the C. erectus rhizosphere coincides with that found by Kumar & Ghose (2008), who reported between 5 and 60 spores per gram of soil in the rhizosphere of mangrove species. The distribution of the mangrove on the Ría Lagartos lough is discrete and fragmented and is associated with other ecosystems such as tular, grassland, tropical low flooding deciduous forest, coastal dune vegetation and peten (Andrade 1997). The higher AMF spore density recorded in the northwind season coincides with that reported by Ramos-Zapata et al. (2011) for coastal dune areas. Those authors propose that the spores are transported by water in the rainy season from adjacent terrestrial ecosystems, in addition to the fact that this season is characterized by strong winds that can carry spores from other sites (Warner et al. 1987, Egan et al. 2014). The fact that no differences were recorded among sites in terms of the number of spores as differences were recorded between seasons (with higher numbers in the northwind season), suggests that the pattern observed may be due to transport of spores by the rainwater (this spores not necessarily retaining viability, as we observed) rather than changes in AMF sporulation in the different sites. This coincides with that suggested by Xu et al. (2016), who state that many of the AMF propagules in the wetlands are transported by water and can remain in the soil after the flooding has subsided. A total of 17 AMF species were found in the rhizosphere of C. erectus, which coincides with the values of richness often found in mangrove vegetation, ranging from 5 to 45 species (Kothamasi et al. 2006, Gupta et al. 2016), depending on the sampling area.
Acaulospora scrobiculata, Funneliformis geosporum, Claroideoglomus etunicatum and Glomus rubiforme were present in the rhizosphere of C. erectus of all the three sites (lower, medium and higher salinity) and have been reported in other mangrove (Kumar & Ghose 2008, Wang et al. 2010, D’Souza & Rodrigues 2013a, b, Xie et al. 2014, Gupta et al. 2016) and “semi-mangrove” (Wang et al. 2015) ecosystems. According to Chagnon et al. (2013), these species could present ruderal (Glomeraceae) and stress resistance (Acaulosporaceae) strategies. Gupta et al. (2016) state that the presence of F. geosporum in zones of high salinity could indicate that it is adapted to such conditions. Arbuscular mycorrhizal communities within a host can change significantly over various seasons, being the primary driver of local adaptation of AM fungal species the edaphic factors (D’Souza 2016). The unique species found in the lower salinity (LS) site, particularly in the northwind season (Acaulospora foveata, A. appendicula, Funneliformis coronatum and Glomus lacteum), could be the most salinity vulnerable species, but also be especially adapted to flooding conditions. A. foveata, Gigaspora decipiens, F. coronatum and G. lacteum, have also been reported by other authors in mangrove ecosystems (Kumar & Ghose 2008, Wang et al. 2010, D’Souza & Rodrigues 2013a). It has been proposed that the AMF species that are found in certain ecosystems can be particularly adapted to the environmental conditions that domain in those systems, so given that previously named species have been recorded in various mangroves, it is hypothesized that they are adapted to survive and prosper in the flooded and/or the saline conditions that (in differing degree) prevail in these ecosystems (Krishna 2005, Saint-Etienne et al. 2006, Egerton-Warburton et al. 2007, Wang et al. 2010). Field data, as reported by Miller & Bever (1999) suggests that the AMF are not physiologically equivalent in their tolerance to wetland conditions, and for that reason we can find significant differences in species composition related to relative water depth, and although a reduction of AMF radical colonization along with salinity rising levels has been reported in wetlands (Saint-Etienne et al. 2006), it is recognized that some AMF species are more tolerant than others to elevated salinity (Borde et al. 2011).
The presence of AMF arbuscules in the roots of C. erectus indicates that there is an exchange of nutrients between these symbionts. On the other hand, the differences in the percentages of mycorrhizal colonization, spore density and AMF species richness found among the sites and seasons of collection suggest that variation in the edaphic environment could affect the dynamics of the association and both fluctuate according to seasonal variation. These dynamics require further study, in particular in relation to the substrate moisture content and the interstitial salinity and the interaction of both. Due to its potential adaptation to flooding and/or saline soil conditions, the AMF species reported in this study and in other mangrove ecosystems does have a potential use in the restauration of damaged wetlands.
