Plasticity of leaf morphology of Bruguiera sexangula to salinity zones in Bangladesh’s Sundarbans

Md.Salim Azad · Abdus Subhan Mollick ·Rawnak Jahan Khan Ranon ·Md. Nabiul Islam Khan · Md. Kamruzzaman

Abstract Bruguiera sexangula (Lour.) Poir is an exclusive evergreen mangrove species to the Sundarbans of Bangladesh. It grows well in moderate saline zones with full sunlight. This study presents leaf morphological plasticity in B. sexangula to saline zones. Leaves were sampled from diff erent saline zones and various morphological traits were measured. The results exposed a wide deviations of leaf size parameters: leaf length varied 6.6-17.3 cm; width 2.7-7.8 cm; upper quarter width 2.2-6.5 cm; down quarter width 2.5-7.3 cm; and petiole length 0.17-1.43 cm. Leaf length was signif icantly larger in fresh water than in other salinity zones, whereas, leaf width, upper and lower leaf quarters were signif icantly larger in medium saline zone. Leaf shape parameters showed a large variation among saline zones.Leaf base angle was signif icantly larger in both medium and strong salinity zones. Tip angle was signif icantly greater in medium salinity zone. Leaf perimeter was signif icantly larger in fresh water but leaf area was signif icantly bigger in medium saline zone. Leaf index and specif ic leaf area were maximum in moderate saline zone. Plasticity index was the highest in moderate saline for almost all the parameters presented. The ordination (PCA) showed clusters of leaf samples although there were some overlap among them which suggested a salt-stress relationship among salinity zones. The results indicate that B. sexangula had a plasticity strategy on leaf morphological parameters to salinity in the Sundarbans. This study will provide basic information of leaf plasticity of this species among saline zones which will help for site selection of coastal planting and will also provide information for policy makers to take necessary steps for its conservation.

Keywords Salinity stress · Phenotypic variation ·Plasticity index · Specif ic leaf area · PCA

Introduction

Forest tree species display considerable phenotypic plasticity to withstand changing environmental conditions (Neale and Kremer 2011; Parent et al. 2015; Chen et al. 2022).Mangroves, salt- and f lood-tolerant trees and shrubs, occur in intertidal zones of tropical and subtropical coastlines,endure saline conditions during their lifetime (Feller et al.2010; Donato et al. 2011; Tomlinson 2016). Soil salinity has long been acknowledged as an important growth regulator and distribution of mangroves (Ball 2002; Krauss et al.2008; Wang et al. 2011). Mangroves demonstrate plasticity to salinity compared to many terrestrial species (Feller et al.2010; Naidoo 2016).

Leaves are the most important organs of plants that exhibit variations in shape, size, and color (Tsukaya 2002a;Li et al. 2019 ). Contrasting environments cause phenotypic diff erences in leaf morphological traits known as phenotypic plasticity, the ability to alter its morphology / physiology with environmental changes (Schlichting 1986; Sultan 2000). Phenotypic plasticity in leaf shape is widespread in aquatic plants (Li et al. 2019). Many plants very often produce sudden and dramatic alterations in leaf morphology ref lecting environmental inf luences (Zotz et al. 2011). Such plants show leaf morphological plasticity permitting adaptation to an unpredictable environment (Wells and Pigliucci 2000).

Soil salinity particularly inf luences leaf plasticity in mangrove ecosystems (Alam et al. 2018; Nasrin et al. 2019; Khan et al. 2020; Mollick et al. 2021). Accordingly, mangrove vegetation adapts various salt stress techniques for growth and development (Feller et al. 2010). Leaves show higher plasticity than other plant organs (Klan?nik and Gaber??ik 2015) and play signif icant roles to adjusting life styles and strategies (Wright et al. 2004), particularly in photosynthesis(Knight and Ackerly 2003; Suwa 2011). Mangrove species often modify leaf shape, size and arrangement to balance leaf energy. In general, upright oriented leaves are small and thick in shape which helps them to avoid direct sunlight and thereby minimize the rate of transpiration (Feller et al.2010).

Generally, leaf shape and size are very much linked with physiological functions, as narrow leaves are less capable of absorbing sunlight than wider leaves. Leaf shape is also linked with cuticle status, number of stomata and their closing and opening (Wells and Pigliucci 2000), which is directly related with primary productivity, biomass accumulation and carbon sequestration (Kathiresan and Bingham 2001).

The Sundarbans is a productive and dynamic mangrove area in the delta formed by the conf luence of three major rivers, having wide range of species (Azad and Matin 2012;Rahman et al. 2015; Sarker et al. 2016, 2019; Azad et al.2019, 2020a). Among them,Bruguiera sexangula(Lour.)Poir, the upriver orange mangrove, is an important species and a member of the Rhizophoraceae family distributed in throughout Southeast Asia and Oceania (Bangladesh, India,Myanmar, Sri Lanka, Malaysia, Thailand, Indonesia, Vietnam, Cambodia, Singapore, Papua New Guinea, the Philippines, Solomon Islands, northern New Caledonia and Australia). The genusBruguierais represented by six species in Asia and the Pacif ic, and East Africa (Siddiqi 2001).Two species,B. sexangula(known locally as Kakra) andB. gymnorrhiza(known locally as Lal Kakra) are found in the Bangladesh Sundarbans.B. sexangula, a true mangrove species, can grow up to 30 m with a buttressed stem with pneumatophores or knee roots present. Its bark is generally uneven and grey in color. Leaves are simple and opposite,glossy green upper surfaces pale green undersides. This species is viviparous and produces 5 - 10 cm long propagules,purple when mature and green when immature (Hossain 2015). The bark of this species is very important for tannin production. The wood is used for fuel, poles, and charcoal production. The leaves and roots have medicinal properties(Das and Siddiqi 1985; Hossain 2015). The bark has antitumor activities (Bandaranayake 1998). This species also acts as shelter belts along rivers (Das and Siddiqi 1985). Thus,the conservation of this species is very important.

With increasing salinity in the mangroves over the past few years,B. sexangulahas shown a several hundred percent increases compared to other dominant tree species in the Sundarbans (Aziz and Paul 2015). It has become important tree species, particularly in moderate and strong saline zones.B. sexanguladisplays variability in leaf size and shape in response to salinity. Therefore,it is important to understand the phenotypic plasticity in leaf morphological traits ofB. sexangulain order to understand the impacts of climate change-induced salinity in the Sundarbans. Until this study, no initiatives have been undertaken to identify the leaf plasticity mechanism inB.sexangulaamong the salinity zones in the Sundarbans.These results should provide a base for future mangrove conservation, management and will also provide information for site selection of coastal planting of this species.Considering the distinct diff erences in leaf shape and size of many mangrove species, it was hypothesized thatB.sexangulamay show leaf morphological plasticity among saline zones in the Bangladesh Sundarbans. The objectives of our study are to: (1) assess leaf morphological traits to diff erent salinity zones; and, (2) quantify plasticity of those traits among salinity zones.

Material and methods

Study sites

The research was carried out in the Bangladesh Sundarbans located 21°31′ and 22°30′ N and 89°00′ and 89°55′E (Fig. 1). The Sundarbans are approximately 60% in Bangladesh and 40% in India with a wide range of species(Azad and Matin 2012; Rahman et al. 2015; Sarker et al.2016; Azad et al. 2019, 2020a, 2021).

The area in Bangladesh has three distinct saline zones,namely a fresh water or oligohaline zone (＜ 5 ppt of soil water), a medium mesohaline zone (5-18 ppt of soil water)and a strong polyhaline zone (＞ 18 ppt of soil water)(Wahid et al. 2007; Iftekhar and Saenger 2008). This mangrove forest is crisscrossed by several rivers, canals and channels. The study sites have the three saline zones noted. Regular inundation occurs usually twice daily, with large volumes of freshwater discharged by the Baleshwar,Sibsha and Passur rivers. Soil of the study sites is clay( Azad et al. 2020b).The climate of these study sites is characterized by a long-wet period (June - September) with a short dry period (December - January). Mean annual temperatures,rainfalls, and relative humidity vary between 25 °C to 35 °C, 1800 to 2790 mm, and 80% to 85%, respectively,74% of the total rainfall occurs during the monsoon period(Chowdhury et al. 2016).

Fig. 1 Map of diff erent sites in diff erent salinity zones of Bangladesh Sundarbans

Plant materials and data acquisition

Twenty-f ive healthy trees were selected within each saline zone, and fresh leaves collected from different canopy heights. Leaves of mangroves may show diff erences in size on the vertical prof ile (Anderson 1986) and thus leaves were systematically collected from diff erent canopy heights, 0.5 and 1.0 m below the top height. A total of 300 leaves were collected from the three saline zones (oligohaline, mesohaline and polyhaline), 100 leaves from each zone (Fig. 1).Height and diameter of sample trees, and positions of leaves were indiff erent among the saline zones. Fresh leaves were placed in polybags and taken to the laboratory of Khulna University.

A digital scanner was used to scan the leaves and NIH ImageJ software used to measure diff erent leaf quantitative parameters (Fig. 2). Numerical leaf parameters were selected to define leaf morphological variations among saline zones. Leaf area and perimeter were calculated and leaf index according to Mollick et al. ( 2011). Leaf mass was used to calculate specif ic leaf area (SLA cm 2 g -1 ) as leaf area (cm 2 ) per unit oven dry leaf mass (gm). SLA was estimated according Petruzzellis et al. ( 2019). A digital caliper was used to measure leaf thickness.

Statistical analysis

Fig. 2 Explanations of leaf morphological parameters of Bruguiera sexangula

Descriptive statistics for all parameters were carried out to show differences within morphological parameters among the saline zones. ANOVA and Tukey’s post hoc test were conducted for data analysis. One-way ANOVA was done to examine significant differences of leaf morphological parameters among saline zones. Tukey’s post hoc test was also done to compare mean leaf morphological parameters. The sensitivity of leaf morphological traits was investigated through developing plasticity indexes (PI) according to Valladares et al. ( 2006). These were determined using PI = (Z - z)/Z, where Z is the largest value of a specific parameter and z the smallest for the same parameter. Plasticity indexes ranged from 0 to 1.PCA was conducted to construct an ordination plot of leaf morphological traits. Raw data were log-transferred prior to PCA to meet normalize data (Hammer et al. 2001). R programming software (R Core Team 2019) was applied to execute statistical and graphical analysis.

Results

Leaf size parameters related to saline zones

These parameters showed large diversity among saline zones (Table 1). Leaf length (LL) varied 2.6-times(6.6-17.3 cm); width (LW) 2.8-times (2.7-7.8 cm);upper quarter width (LUQW) 2.9-times (2.2-6.5 cm);down quarter width (LDQW) 2.9-times (2.5-7.3 cm); and petiole length (PL) 8.4-times (0.17-1.43 cm). One-wayANOVA showed significant differences (P＜ 0.05) of leaf size traits (LL, LW, LUQW, LDQW and PL). LL was significantly greater in the oligohaline zone than that of other salinity zones, whereas LW, LUQW and LWQD were significantly larger in the medium salinity zone than the other zones (Table 1).

Table 1 Mean and standard deviation of leaf shape and size parameters of B. sexangula in Bangladesh Sundarbans

Leaf shape parameters related to saline zones

Fig. 3 Association of leaf length against leaf width showing leaf index of B. sexangula in diff erent salinity zones of Bangladesh Sundarbans. Note: FW: fresh water or low saline; MS: moderate saline;and SS: strong saline

Leaf shape parameters also showed diversity among saline zones (Table 1). Leaf base angles varied 2.6-times (55.4-143°); tip angles 1.9-times (71.6-137.2°);area 6.9-times (14.8-103.2 cm 2 ); perimeter 3-times(17.6-53.7 cm); leaf index 2.4-times (1.4-3.3); ratio between area and perimeter, 5.9-times (0.4-2.3); ratio between leaf length and petiole length, 18.7-times(3.3-61.3); ratio between base angle and tip angle, 2.5-times (0.5-1.3); and ratio between LUQW and LDQW,2.1-times (0.7-1.6) (Table 1). One-way ANOVA showed signif icant diff erences (P＜ 0.05) of leaf shape traits among salinity zones (Table 1). Base angle was signif icantly larger in both mesohaline and polyhaline zones than in the oligohaline zone, whereas, tip angle and leaf index were signif icantly greater in the mesohaline zone than in the other two zones (Table 1). However, leaf perimeters were signif icantly larger (P＜ 0.05) in the oligohaline zone(Table 1).

Leaf index

Leaf indexes represent the relationship between leaf width to leaf length. The scatter plot of leaf indexes showed major clusters in diff erent saline zones although there was some overlap between the mesohaline and oligohaline zones (Fig. 3). The scattered plot also revealed that leaf index in the mesohaline zone appeared at the top of the f igure and in the polyhaline zone at the bottom. It is understandable that leaf growth and development in different salinity zones show signif icant diff erences (results not shown).

Leaf plasticity index

The results revealed that the plasticity index (PI) of each trait showed large variations (Fig. 4). PI revealed thatalmost all the parameters were maximum in the mesohaline zone. In the oligohaline zone, the plasticity index of LL/PL was maximum and LUQW/LDQW minimum. PI of leaf area in the mesohaline zone was maximum and LUQW/LDQW minimum. The plasticity index of petiole length in the polyhaline zone showed was maximum and leaf index minimum (Fig. 4).

Specif ic leaf area (SLA) and leaf thickness (LT)

SLA is a signif icant functional trait of plant growth and was maximum in the mesohaline zone. Leaf thickness also varied among saline zones and was maximum in the polyhaline zone and minimum in the mesohaline zone (Fig. 5).

Fig. 5 Specif ic leaf area and leaf thickness of B. sexangula in diff erent salinity zones of Bangladesh Sundarbans. Note:-FW: fresh water or low saline; MS: moderate saline; and SS: strong saline

Ordination of saline zones for various morphological parameters

With regards to multivariate ordinations among the morphological parameters measured, PCA showed that PC1 (47.8%)and PC2 (15.8%) explained 63.6% of the total variance for morphological traits with a large diff erence in eigenvalues(7.16 for PC1 and 2.37 for PC2). The ordination plot showed the pattern of plasticity of leaf morphological traits among saline zones (Fig. 6). Leaf morphological parameters ofB. sexangulathat specif ied three diff erent clusters of leaf samples measured from study areas were clearly varied among saline zones, though there were overlap among them(Fig. 6). Morphological parameters (leaf size) viz. LL, LW,LUQW and LDQW were signif icantly correlated (P＜ 0.01)with PC1. Leaf shape traits viz. leaf area, leaf perimeter and ratio between leaf area and perimeter were also signif icantly correlated (P＜ 0.01) with PC1. Leaf base angle and ratio between leaf base and tip angle were signif icantly (P＜ 0.01)correlated with PC2 (Table 2).

Discussion

Fig. 6 Ordination of diff erent saline zones for several leaf morphological traits of B.sexangula in diff erent salinity zones of Bangladesh Sundarbans. Note: LL: leaf length;LUQW: leaf upper quarter width; LDQW: leaf down quarter width; PL: petiole length;FW: fresh water or low saline;MS: moderate saline; and SS:strong saline

Table 2 Correlations of variables with PC1 and PC2 with eigenvalues and corresponding variance

This study provides clear indications of leaf morphological variation ofB. sexangulaamong saline zones. All leaf morphological parameters, especially size (length, width, middle width, LUQW, LDQW and petiole length) and shape (base and tip angle; area and perimeter) provide evidence that the species has developed plastic strategies among saline zones.All leaf shape parameters (except length) were maximum in the mesohaline zone and lower in the polyhaline zone. Adaptive responses of leaf morphological traits to salinity has been documented in several mangrove ecosystems in which mangroves cope with environmental heterogeneity (Sultan 2000; Khan et al. 2020). Leaf size parameters are the most adaptive traits to environmental variations (Tsukaya 2002b).Adaptive responses to salinity of mangrove species were also reported forHeritiera fomesBuch.-Ham. (Khan et al. 2020),Avicennia offi cinalisL. (Alam et al. 2018) andSonneratia apetalaBuch.-Ham. (Nasrin et al. 2019). Abbruzzese et al.( 2009) suggested that size of leaf traits ref lect an excellent biomarker for climate change adaptation.

The present study also found that leaf area maximized in the mesohaline zone. Mollick et al. ( 2021) documented leaf area of three mangrove species (H. fomes,Excoecaria agallochaL. andCeriops decandra(Griffi th) Ding Hou decreased with increasing salinity. Hoppe-Speer et al. ( 2011)found that leaf area ofRizophora mucronataLam. decreased due to salt stress. They also noticed that leaf cell expansion decreased with increasing salinity.

Leaf index ref lects leaf morphological adaptation to a certain salinity gradient that might be characterized as an evolutionary novelty (Tsukaya 2002b; Smith 2006). This study found a higher leaf index plasticity in the mesohaline zone than in the other zones (Fig. 4) because salt stress in the moderate saline zone is favorable forB. sexangula(Kathiresan and Bingham 2001; Hossain 2015). In contrast, Khan et al. ( 2020) documented higher leaf index plasticity in low saline zone inH. fomes. They also noticed that the picture is contradictory in high salinity zone due to salt stress ofH.fomesis not favorable in medium or in strong salinity zones(Kathiresan et al. 2010; Hossain 2015; Sarker et al. 2019).Therefore, leaf index may be used for leaf morphological traits plasticity regarding environmental change (Mollick et al. 2011).

Similarly,B. sexangulashowed high leaf morphological plasticity in moderate saline zone (except petiole length and the ratio between leaf length and petiole length) than that of fresh water zone or in strong saline zone in the Sundarbans. In contrast, Khan et al. ( 2020) stated thatH. fomesshowed high leaf morphological traits in fresh water zone than in medium or in strong saline zone. The diff erence of leaf morphological plasticity among mangrove species is due to capability of adaptive responses to salinity stress of respective species (Sarker et al. 2019; Khan et al. 2020;Mollick et al. 2021).H. fomesis a climax and main species in the Sundarbans, Bangladesh. It prefers fresh water for growth and development (Kathiresan et al. 2010; Hossain 2015; Azad et al. 2019, 2020c). Whereas,B. sexangulagrows well in moderate saline zone with full sunlight but it can tolerate strong saline condition (Kathiresan and Bingham 2001; Hossain 2015). With increasing salinity over the past few years, the mangrovesB. sexangulashowed a several hundred percent increase comparing to other dominant tree species in the Sundarbans (Aziz and Paul 2015). Salinity stress may inf luence species colonization in diff erent zonation in mangrove forest (Kathiresan and Bingham 2001),and thereby, mangrove species showed adaptive responses of leaf morphological plasticity to saline zones (Khan et al.2020). ThusB. sexangulashowed high leaf morphological plasticity in moderate saline zone than in fresh water or in strong saline zone. Generally, mangroves are subjected to salinity stress which might inf luences adaptive responses even within a short distance (Schmitz et al. 2007; Feller et al.2010; Arrivabene et al. 2014; Tomlinson 2016). Salinity is a limiting factor for mangroves habitat (Ball 2002; Gopal and Chauhan 2006; Alam et al. 2018; Nasrin et al. 2019;Khan et al. 2020). Thus, mangroves are distributed diff erentially in a banded shape along salinity regimes (Nguyen et al. 2015) and show leaf morphological plasticity among saline zones (Khan et al. 2020) in comparisons to terrestrial plants (Naidoo 2016). Therefore, our study provides clear evidence of leaf morphological plasticity of all parameters measured among salinity zones forB. sexangula.

High level of leaf specif ic area (SLA) ref lects increasing productivity (photosynthesis) and broader ecological niche to a particular species (Ishii et al. 2018).B. sexangulashowed maximum SLA plasticity in moderate saline zone due to salt stress and growing ability in full sun light(Hossain 2015). Nandy et al. ( 2007) documented that SLA ofB. gymrorrhizawas decreased by 14% in high saline habitat in Indian Sundarbans. Mollick et al. ( 2021) also documented that SLA was reduced inH. fomes,E. agallochaandC. decandrain high saline zones. The opposite scenario was noticed regarding leaf thickness ofB. sexangulain this study. Leaf thickness was maximum in strong saline zone. Camilleri and Ribi ( 1983) found that leaf thickness of Rhizophora mangle was higher in higher salinity in south Florida. They also mentioned that thicker leaves contain water storing tissues than thin leaves of mangrove species.Although the function of water storage tissue is unknown,but it is likely to gauge that species containing salt excreting glands seems to be less developed than the species having no salt glands. Thus, water storage tissue has signif icant roles in osmoregulation, transpiration and other physiological activities. Thus, the degree of leaf thickness and succulence depends on diff erent saline habitats.

The PCA ordination provided clusters of salinity zones indicated plastic behaviors of leaf morphological traits to salinity, although there was some overlap among them.The leaves ofB. sexangulashowed changes in phenotype among saline zones (Fig. 6) which is an indication of adaptive responses to salinity of this species. Plasticity of leaf traits develops very often due to changes in environment(Tsukaya 2002b; Khan et al. 2020), but genetic factors may also be accountable (Mollick et al. 2011; Tsukaya 2013).

Conclusion

The study investigated leaf morphological traits plasticity forB. sexangulain Bangladesh Sundarbans, based upon all parameters measured of the sample leaves collected among saline zones. We believe that this is one of the pioneer study of leaf morphological plasticity of mangrove tree species in the Sundarbans. Results suggest that the mangrovesB.sexangulashows variation of leaf size and shape parameters among saline zones. Leaf size parameters (leaf middle width, upper and down quarter width) were maximum in moderate saline zone except leaf length. Leaf area was also maximum in moderate saline zone. The results exhibited that leaf index (so called bio-marker of leaf morphology)of the studied species was higher in moderate saline zone.Specif ic leaf area was also higher in moderate saline zone,but the scenario was opposite regarding leaf thickness. Leaf thickness was minimum in moderate saline zone. Leaf shape,size, specif ic leaf area and leaf thickness ref lect adaptation of mangrovesB. sexangulaamong the saline zones in the Sundarbans. The results also show that leaf morphological plasticity index for all the measured parameters (except petiole length and the ratio between leaf and petiole length)is higher in moderate saline zone. Plasticity index exhibited plastic approaches to salt stress. Leaf morphological variation in diff erent salinity zones could be better understandable through plasticity index. The study predicts to understand that adaptive responses to salinity may shift morphological traits of mangrove species in future in the world to climate change situations. This study also provides a basement on leaf morphological plasticity ofB. sexangulawith respect to salinity regimes in Sundarbans. The results of this study will also inspire further investigation to develop ecological conservation and management strategies for this species.

Acknowledgements We thank to the field staffs for their help throughout f ield work. We also thank Bangladesh Forest Department for their valuable support to conduct f ield work with logistics. The authors are also thankful to FWT Discipline, Khulna University, Bangladesh for lab support. The authors are also thankful to Journal of Forestry Research Editing Service for English language editing.