Wood anatomy of Laguncularia racemosa ( Combretaceae ) in mangrove and transitional forest , Southern Brazil

Mangroves represent an environment of great heterogeneity and low diversity of plant species that have structural and physiological adaptations linked to a high salinity environment. Laguncularia racemosa is a typical tree species in mangroves and transitional zones. This study aimed to compare the wood anatomy of L. racemosa (Combretaceae) in two different forests (mangroves and transitional forests), which have different soil conditions. For this, we obtained wood and soil samples in March 2016. We analyzed soil nutritional contents in one 15 cm deep soil sample per forest type. In addition, we selected five mangrove trees in each formation for wood anatomy analysis and took one wood sample per individual, per area. We prepared histological slides and separated materials following standard methods for wood anatomy studies. Soil analysis showed that mangrove soils had higher phosphorus, potassium and calcium contents. The transitional soil had lower pore water salinity and soil pH, probably due to high aluminum levels. Anatomical attributes differed between different forest populations. In the different wood aspects evaluated, we obtained higher values in mangrove individuals when compared to the transitional forest population: vessel elements length (375.79 mm), tangential vessels diameter (75.85 mm), frequency of vessels (11.90 mm) and fiber length (889.89 mm). Moreover, parenchyma rays height was larger in the samples of the transitional forest (392.80 mm), while the mangrove population presented wider rays (29.38 mm). The structure of the secondary xylem in the studied species apparently responds to edaphic parameters and shows variations that allow it to adjust to the environmental conditions. The population of the transitional forest showed a secondary xylem that invests more in protection than the mangrove population. Rev. Biol. Trop. 66(2): 647-657. Epub 2018 June 01.

Mangroves are coastal ecosystems in the transition areas between marine and terrestrial environments, of well-known ecological, economic and social importance (Madi, Boeger, & Reissmann, 2015).Mangroves occur in tropical and subtropical regions.They constitute authentic coastal forests, in which plant structure and ecology are highly adapted to topographic and geomorphological differences, saline fluctuations and tidal amplitudes (Santos et al., 2012).Mangroves forests may cover estuarine areas, bays, inlets, river mouths, lagoons, or simply be exposed to the coastline (Schaeffer-Novelli, 1995).
Along the Brazilian coast, mangrove forests have different structure and species distribution (Lima & Tognella, 2012).For example, although L. racemosa distribution ranges over the coast, its representativeness is greater in Southern mangroves (Schaeffer-Noveli et al., 1990).Usually, L. racemosa can be found in open areas, as well as in transitional areas of restinga and lowland forests (Bernini & Rezende, 2003).The transitional forests are less affected by tides regime and are subject to less frequent flooding located inland (EMBRAPA, 2013).
Mangrove tree species may present three different mechanisms to cope with high salinity and to control salt concentration in their tissues (Parida & Jah, 2010).L. racemosa shows an ecophysiological mechanism of salt exclusion, which may be associated with a wide range of tolerance to salinity: L. racemosa occurs at contrasting salinities, in areas of salinity close to fresh water towards salt water (Sobrado, 2005).This mechanism is also reflected in its morphological and anatomical adaptations.For example, Bartz, Melo Jr., and Larcher (2015) suggests mangrove individuals are smaller, with lower trunk diameter height and crown density and higher leaf area, than individuals of transitional forests.
The high evaporative demand combined with the vulnerability to embolism or cavitation, due to the high concentration of ions in the environment, were previous associated to adaptations in the secondary xylem of mangrove species (Lovelock et al., 2007;Krauss et al., 2008).At high salinity, the low soil water potential requires a decrease in the plant water potential to maintain water uptake by roots, leading plants to decrease water conductivity in secondary xylem (Reef & Lovelock, 2015).The anatomical characteristics and, consequently, the physical-mechanical properties of this tissue, is affected by environmental variations (Baas, Wheeler, & Fahn, 1983;Carlquist, 2001;Marcati, Angyalossy-Alfonso, & Benetati, 2001;Cosmo, Kuniyoshi, & Botosso, 2010).
We expected mangroves species, as L. racemosa, to express several secondary xylem phenotypes in response to variation of environmental factors, such as precipitation, temperature and water availability.Thus, this study aimed to evaluate the wood anatomy of L. racemosa populations occurring in mangrove and transitional forests between mangrove and restinga forest.We hypothesize that mangrove individuals may have anatomical adaptations to deal with higher pore water salinity and lower soil water potential, while transition mangroves are less conservative in water use.

Study area:
The Caieira Municipal Natural Park was located in the center-east portion of the city of Joinville/SC, Brazil (Fig. 1).Inside the park, there was the Lagoa of Saguaçu (26°18'24'' S -48°47'36'' W), with approximately 1 000 km² of mangrove remnants and transitional forest, from mangrove towards restinga and lowland forest.The transitional forest occured between the mangrove zone and restinga forest; is not directly subject to the tides regime (not frequently flooded), and is characterized by the presence of the species Talipariti tiliaceum (L.) Fryxell (Malvaceae) as a schrub (EMBRAPA, 2013).L. racemosa had high absolute density in both mangrove and transitional forests.
The holomorphic soils of mangrove forests were developed by the deposition of marine and fluvial sediments, with the presence of organic matter, under constant influence of the sea.Usually, mangrove soils occur in regions of flat topography in the coastal strip.The soil of the transitional forest was a Neosoil composed by thin organic material or mineral material (EMBRAPA, 2013).
The climate is hot and temperate (Cfa in the Köeppen and Geiger classification), with average annual temperature around 21.1 °C and with 1 706 mm annual rainfall to the year of 2016 (Climate-Data, 2016).The sampling period (March/2016) corresponded to the end of the summer season, with average temperature of 24.1 o C and a mean precipitation of 204 mm.

Soil analysis:
In both formations, we evaluated soil chemical characterization from one composed sample, collected 15 cm deep and homogenized, according to the methodology proposed by EMBRAPA (2013).We performed soil characterization using the Soil Analysis Laboratory (LAS) of the Agricultural Research and Rural Extension Company of Santa Catarina (Epagri), considering nutritional status and texture (EMBRAPA, 2013).We collected interstitial water using a sterile collecting device after digging holes into the soil of both study areas with the aid of a hollow tube.The salinity was measured using a refractomer (Vodex).The collections took place in March/2016.Wood analysis: A total of ten wood samples -one sample per tree, from the outer wood region (close to the vascular cambium) -from mature populations, were taken at breast height (1.30 m high) from five individuals per population, during low tide following the Tides Tables (Epagri/Ciram, 2014), on the same period of March of 2016, which corresponded to the driest period of the year.Mature individuals were defined as adult trees which have been through reproductive periods.In the mangrove area, individuals were sampled near the fringe.Samples were registered in the Wood Library JOlw under the numbers JOlw 1 129 to 1 138 (collection number JCM 1 401 to 1 409 and 1 010) for mangrove samples and JOlw 1 119 to 1 128 (collection number JCM 1 411 to 1 419 and 1 020) for transitional mangroves samples.
Descriptions terminology followed the recommendations of the IAWA List of Microscopic Features for Harwood Identification (IAWA Committee, 1989).Thirty measurements of wood attributes were measured: tangential diameter (μm) and vessel frequency (n/mm²), vessel and fiber length (μm), fiber wall thickness (μm), height and width (number of cells and μm).
We calculated mean values and standard deviations for all anatomical attributes.We compared mean values by Student's t-test, with a significance level of 5 %, in "R" (R Development Core Team, 2011) with the "labdsv" package (Roberts & Roberts, 2007), following the normality test and homogeneity of variance when necessary.
Wood anatomical description (Fig. 2): L. racemosa presented indistinct growth rings and diffuse porous wood, without specific vessel arrangement.Vessels were solitary and multiples of 2 to 3, rare 4-5, with simple perforation plate.Bordered intervessel pits were alternate; radio-vessel bordered pits were larger than the intervessel pits.Fibres had minute to simple bordered pits; layers of thin-walled fibres alternate with layers of thick-walled fibres.Axial paratracheal parenchyma was aliform lonsangular to confluent; starch grains were present in the axial parenchyma.Rays were heterogeneus and uniseriate, with body composed of procumbent cells, and margins of erect and squared cells.
Regarding quantitative data, populations of L. racemosa presented differences for all studied wood anatomical parameters, except for mean fiber wall thickness (Table 2, Fig. 3).Mangrove population had the highest values for mean vessel diameter, length and frequency, ray width and fiber length.The transitional forest population had the mean higher rays.Mean fiber wall thickness was equal in both populations.

DISCUSSION
The higher variation of sodium content between the areas can be explained by the proximity to salty waters (Prada-Gamero, Vidal-Torrado, & Ferreira, 2004).Sodium is one of the cations associated to saline soils (Maksimovic & Ilin, 2012).The transitional forest area is not directly affected by salty water flooding, so its concentration of calcium was lower than in mangrove area.Another cation directly affected by salinity is calcium, which presents higher concentrations in more saline soils (Maksimovic & Ilin, 2012).In higher salinity soils with unconstrained sediments, calcium may act as a secondary messenger in cellular processes adjustments (Madi et al., 2015).The porewater salinity, linked to tides dynamics, vary between areas closer to the salt water or to the restinga forest (Kathiresan & Bingham, 2001).Soils cation exchange capacity in mangroves is correlated to the flooding amplitude and tide duration (Ukpong, 2000;Naidoo, 2010;Urrego, Molina, & Suárez, 2014), resulting in salinity gradients and ions concentration along the flooding plateau.Both population samples were collected in the dry season, in low tides, and in the transitional forests floodings are less frequent and the influence of tides is low.
We found lower values of pH in the transitional area, which may influence the availability of nutrients (Ukpong, 2000;Krauss et al., 2008;Lima & Tognella, 2012).It is known that the pH changes the availability of important nutrients such as phosphorus, and non-essential like aluminium (Hossaim & Nuruddin, 2016).This situation was observed in the mangrove study area, where we found most of the nutrients in higher concentration and the pH is near to neutral.On the other hand, phosphorus is one nutrient highly sensitive to pH variation, in more acidic or alkaline soils, phosphorus tends to be less available.In this study, the contents of phosphorus in the transitional forests were much lower than in the mangroves, reflecting this situation.
Mangrove species are subject to extremely low water potential in the soil, because of high salinity, constraining hydraulic conductivity by the xylem (Lovelock et al., 2007).The presence of salt reduces the availability of water to the plant, known as physiological drought (Maksimovic & Ilin, 2012).This leads to a constant trade-off between safety against cavitation and efficient hydraulic conductivity (Sobrado, 2007;(Robert, Koedam, Beeckman, & Schmitz, 2009;Schmitz, Robert, Verheyden, Kairo, Beeckman, & Koedam, 2008).In order to minimize effects of cavitation due to a physiological drought, it is expected that plants present narrower and shorter vessel elements in higher densities (Sobrado, 2007;Schmitz et al., 2008).However, in the present study we found a higher vessel frequency of larger diameter in the higher salinity population (mangrove area), indicating that the individuals are investing more in hydraulic conduction than safety.Schmitz, Verheyden, Beeckman, Kairo, and Koedam (2006) demonstrated the effect of salinity in vessel frequency, the higher the salinity, the higher the frequency of vessels.The authors showed that vessel diameter was less affected by salinity.Yáñez-Espinosa, Terrazas, Lopez-Mata, and Valdez-Hernandez (2004) and Robert et al. (2009) observed the same relation between salinity and higher vessel frequency for Rhizophora mucronata Lam.(Rizophoraceae).Schmitz et al. (2006) suggested that a higher vessel frequency optimizes water transport under stressful conditions (high salinity), allowing the maintenance of functional and embolized vessels proportion, as showed in other studies (Baas et al., 1983;Mauseth & Plemons-Rodriguez, 1997).
Fiber wall thickness was also linked to water conduction, as a safety strategy in conditions of highly negative pressures within the secondary xylem, gave support to the vessels, and avoid collapse and cavitation (Hacke & Sperry, 2001;Jacobsen, Ewers, Pratt, Paddock III, & Davis, 2005).Chimelo and Mattos-Filho (1988) observed wider rays in environments with larger water restrictions.Our results showed that the two populations have nearly the same amount of radial parenchyma, compensating the lower width with height and vice-versa.According to Tyree, Salleo, Nardini, Lo Gullo, and Mosca (1999), radial parenchyma is probably related to embolism repair, which is importantly fundamental for mangrove species subject to a constant physiological drought.Zheng and Martínez-Cabrera (2013) showed that rays size might vary in function: wider rays are more related to conductivity while narrower to mechanical strength.
This study showed that L. racemosa presents distinct wood anatomical features that  demonstrate the plasticity of the tissue face to soil conditions, mainly those related to hydraulic conductivity (vessels).Possibly the of transitional area is subject to a more unstable situation, although presenting less saline soils, the very low pH and nutrients availability are possible factors that affect xylem structure and conductivity.

Fig. 2 .
Fig. 2. Comparative wood anatomy of Laguncularia racemosa (Combretaceae).A -B: cross section showing vessel diameter and vessel frequency in mangrove (A) and transitional forest (B).C -D: radial section showing height and width of rays in mangrove (C) and transitional forest (D).E: vessel length in mangrove (Mg) and transitional forest (Tr).F: fiber height in mangrove (Mg) and transitional forest (Tr).Scale bars = 100 μm.

Fig. 3 .
Fig. 3. Comparison of wood anatomy attributes in populations of Laguncularia racemosa (Combretaceae) in mangrove and transitional forest at the Caieira Municipal Natural Park, Joinville, Santa Catarina, Brazil.

TABLE 2
Mean values and standard deviation of wood attributes of Laguncularia racemosa (Combretaceae) populations in mangrove and transitional forest populations of the Caieira Municipal Natural Park, Joinville, Santa Catarina, Brazil (n= 30)