Introduction

Mangroves form a diverse plant community that thrive in tropical and subtropical estuaries.The main attribute of mangroves is in their ability to live under constant physiological stress(Chaudhuri 1996)through morphological,reproductive and physiological adaptations(Zimmermann 1983;Das 1999).The most important ability of mangroves is their tolerance of seawater levels of NaCl(～500 mM)(Takemura et al.2000;Yan and Guizhu 2007;Hogarth 2007).The coastal environment and inhabitants are largely dependent on mangrove forests for its enormously productive and protective ecosystem.Barbier(2007)opined that the economic annual value of mangrove forest is around US$12,392 ha-1a-1,when we include all production and protection by coastal and estuarine areas.

However,mangrove forest areas all over the world have confronted a major threat as the climate(precipitation and temperature),sea level and incidence of UV-β radiation are clearly changing(Spalding et al.1997;Spiers 1999).Mangrove ecosystems are the front-line defense against the adverse consequences of changes in sea level and also can absorb a considerable amount of UV radiation(Hiraishi and Harada 2003;Hogarth 2007).

In the Indian subcontinent,the Sundarbans delta is a mangrove swamp of approximately 2152 km2(FSI 2009)with the world’s richest species diversity.This mangrove forest is cradle of 36 true mangrove species,28 associates and 7 obligatory mangrove species belonging to 29 families and 49 genera(Naskar and GuhaBakshi 1983).Chanda and Datta(1986)commented that a disparity in the salinity level prevails between the western and eastern zones of the Sundarbans;freshwater in fl ux in the west(Indian Sundarbans)is much lower;thus,the salinity is higher than in the east(Bangladesh Sundarbans).The cumulative effects of the geomorphology,demographic interference,siltation in the riverbed and decrease in freshwater in fl ux(Hopson and Webster 2010;Chowdhury and Ward 2007)have been detrimental for some economically important plant species including Heritiera fomes,Nypa fruticans,Xylocarpus mekongensis and X.granatum in the Indian Sundarbans(IUCN Red List 2014;Upadhyay et al.2002).

Genetic diversity has a major effect on biodiversity(Templeton 1993).Schaal et al.(1991)commented that genetic diversity is the most important criteria for any species to cope with changing environments or evolving competitors and parasites.Molecular markers can offer vital information to develop strategies for genetic conservation(Newbury and Ford-Lloyd 1993).The study of genetic diversity is a prerequisite to design the most suitable strategy for a conservation program.In the present study,the genetic polymorphisms of three true mangrove species have been studied by RAPD and ISSR markers.Excoecaria agallocha and Phoenix paludosa grow profusely in the Indian part of the Sundarbans forest,and the other,Xylocarpus granatum,is on the verge of extinction.In the background of the high salinity in Indian Sundarbans(Dasgupta et al.2010),this study aims to assess the sustainability of the three taxa in respect to their genetic diversity.Although genetic polymorphisms in Avicennia(Parani et al.1997;Schwarzbach and McDade 2002;Kader et al.2012)and Rhizophoraceae(Sheue et al.2009;Gurudeevan et al.2012)have been studied over the years,very limited work has been done on the three taxa presently studied in this paper.Parida et al.(1995)reported a high level of genetic polymorphism among E.agallocha.Das and Jena(2008)reported eight ecotypes of X.granatum in the Bhitarkanika mangrove forest,using RAPD markers.But so far there has been no report on the genetic polymorphism of X.granatum,E.agallocha and P.paludosa from the Indian Sundarbans.The present study on the comparative basis of genetic polymorphism of these three taxa might be helpful on understanding their current status in the Indian Sundarbans and inform strategies to conserve these taxa.

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Materials and methods

Collection sites

LeafbudsofX.granatum,E.agallocha,andP.paludosawere collected from fi ve naturally occurring islands in the Sundarbans mangrove swamps.All sites are included in the core region,at the northeastern part of the western Sundarbans(Indian part)to the southern extremity toward the sea(Bay of Bengal),without any demographic interference,namely Sajnakhali (22°07′26′N, 88°49′51′E), Sudhanyakhali(22°06′05′N, 88°48′06′E), Sonargaon (22°06′29′N,88°46′23′E),Jharkhali(22°01′13′N,88°41′10′E)and Dobanki(21°59′04′N,88°44′38′E)(Fig.1).The substrate salinityof the collection sites occurred an increasing gradient(11.77±2.1,12.25±1.96,12.4±1.18,13.98±2.29 and 15.23±2.16 mg kg-1)(Dasguptaetal.2012).Fromeachof the sites mentioned,samples were collected from at least six plants10to30 mapart.Thecontrolledmesophyticreplicasof the studied species,well-matured to 15–17 years old,collected from the premises of the Indian Statistical Institute,Kolkata,where the soil salinity was 2 mg kg-1,(Dasgupta et al.2012)were also taken for a comparative study.

Isolation of genomic DNA

Young leaf buds were used to isolate total genomic DNA for all plant samples.The desired amount of leaf bud was weighed,washed with double-distilled water.Qiagen DNeasy Plant Mini Kits were used to extract DNA from these samples after they were aseptically ground in liquid nitrogen with a mortar and pestle.

2.3.3 术中出血量 纳入了4个研究，共210例(SuperPATH组105例，传统入路组105例)，经χ2检验，研究间有异质性 ( I2>50%)，采用随机效应模型进行Meta 分析。结果显示SuperPATH组术中出血量少于传统入路组，且差异有统计学意义(WMD=-202.28，95%CI=-266.01～-138.54，P<0.00001)。见图4。

RAPD and ISSR analysis

For RAPD and ISSR analysis,20 ng genomic DNA was used for ampli fi cation in a Primus 25 Advanced Thermocycler(Peqlab,Erlangen,Germany);26 RAPD primers and 20 ISSR primers were screened to assess the genetic diversity of the plant species based on better reproducibility(Tables 1,2).The 25 μL reaction mixture contained 1.5 μL 10×reaction buffer,25 mM MgCl2,20 pM RAPD primer,2 mM dNTPs,1 U Taq polymerase(Bioline,Taunton,MA,USA)and Millipore water to volume.The DNA in the reaction mixture was subjected to denaturation at 94°C for 5 min;45 cycles of denaturation for 45 s,primer annealing for 1 min and primer extension for 1.30 min at 72°C.The chemical concentrations and PCR condition were same for RAPD and ISSR analysis except for primer annealing.The temperature for primer annealing was adjusted based on the melting temperature(Tm)of the individual RAPD and ISSR primers and varied from 38 to 42°C for RAPDs and 50–54 °C for ISSRs.One last cycle of primer extension at 72°C for 7 min was followed by a temperature drop of 10°C.PCR products were separated by electrophoresis using 1.5% agarose gel containing ethidium bromide(0.5 μg/mL)in Tris–borate-EDTA(TBE)buffer.The sizes of the amplicons were determined using size standards(Hyper Ladder II,Bioline).DNA bands were visualized with a UV transilluminator and photographed for later analysis using various statistical programs.The PCR experiment was done thrice for each experiment.

In RAPD and ISSR analysis,the presence or absence of the bands were scored as 1 or 0,respectively,to create a binary matrix using the multivariate analysis program NTSYS-PC(Rohlf 1993).Jaccard’s similarly matrix was constructed using the binary matrix and Jaccard’s coef ficient(Jaccard 1998).Following the unweighted pair group with arithmetic mean(UPGMA)method(Sneath and Sokal1973),a phylogenetic dendrogram was obtained from the similarity matrix by cluster analysis,using NTSYS version 2.1(Exeter Software,NY,NY,USA).POPGENE 32(Yeh et al.1997)statistical software was also used for calculating mean of observed number of alleles per loci(Na),mean effective number of allele per loci(Ne),Nei’s gene diversity value(h)(Nei 1973),and Shannon’s information index(I).

Fig.1 Location of the study area

Statistical analyses

1.1 切割金属假体磨块 从人工关节翻修术中取出关节假体，采用进口线切割机（Sodic日本产型号AQ360Ls）将其切割成不同大小的磨块，以增加球磨时磨块之间的接触面积，增加研磨效率。将磨块清洗干净以后用高压蒸汽消毒备用[9]。

Table 1 List of RAPD primers and bands ampli fi ed using DNA from Excoecaria agallocha(Ea),Phoenix paludosa(Pp),and Xylocarpus granatum(Xg)

Primer Nucleotide sequence Polymorphic bands/Total no.bands Primer Nucleotide sequence Polymorphic bands/Total no.bands Ea Pp Xg Ea Pp Xg OPA 1 5′CTGGGCAACT3′ 1/9 2/8 1/8 OPA 14 5′GTGACGTAGG3′ 2/9 2/8 2/7 OPA 2 5′GTCTTGCGGA3′ 2/8 2/9 1/7 OPA 15 5′GGGTAACGCC3′ 3/8 1/7 1/9 OPA 3 5′ACTGAACGCC3′ 3/10 3/8 2/8 OPA 16 5′GTGATCGCAG3′ 2/8 2/7 1/8 OPA 4 5′TCGGCACGCA3′ 2/8 2/10 1/8 OPA 17 5′CAATCGCCGT3′ 1/9 2/8 1/7 OPA 5 5′TCGCCCAGTC3′ 3/9 3/7 1/7 OPA 18 5′TCGGCGATAG3′ 2/7 2/8 1/9 OPA 6 5′AACGGCGACA3′ 2/9 2/8 1/9 OPA 19 5′CAGCACCCAC3′ 3/10 1/6 1/7 OPA 7 5′CAGGCCCTTC3′ 3/8 3/9 1/7 OPA 20 5′TCTGTGCTGG3′ 1/8 3/8 2/8 OPA 8 5′TGCCGAGCTG3′ 2/7 1/8 1/9 RAPD21 5′TTCCGAACCC3′ 2/7 2/7 1/8 OPA 9 5′AGTCAGCCAC3′ 2/10 2/9 2/7 RAPD22 5′AGCCAGCGAA3′ 3/9 2/6 1/9 OPA 10 5′AATCGGGCTG3′ 2/11 2/7 1/9 RAPD23 5′GACCGCTTGT3′ 2/8 1/7 1/8 OPA 11 5′AGGGGTCTTG3′ 2/8 2/7 1/7 RAPD24 5′AGGTGACCGT3′ 2/10 2/8 1/8 OPA 12 5′GGTCCCTGAC3′ 3/7 1/6 1/8 RAPD25 5′CAAACGTCGG3′ 2/8 2/6 1/9 OPA 13 5′GAAACGGGTG3′ 1/9 3/7 1/6 RAPD26 5′GTTGCGATCC3′ 2/9 2/8 1/9

Table 2 Statistical analysis of DNA polymorphism by RAPD and ISSR molecular markers for the three mangrove species

Species Marker No.loci No.polymorphic loci Percentage polymorphic loci Na Ne h I E.agallocha RAPD 223 55 24.66 1.1671 1.1358 0.0794 0.1162 P.paludosa 197 52 26.4 1.1753 1.1381 0.0818 0.1187 X.granatum 206 30 14.56 1.1135 1.0624 0.0637 0.0857 E.agallocha ISSR 189 47 24.87 1.1679 1.1369 0.0799 0.1197 P.paludosa 187 38 20.32 1.1469 1.1147 0.0688 0.1023 X.granatum 193 23 12.92 1.0847 1.0498 0.0583 0.0763

Results

The PCR-based RAPD technique is used very frequently by biologists because small amount of DNA is adequate and no prior knowledge about the DNA sequence of the species is needed(Williams et al.1990).It is a relatively fast,cheap method for estimating the genetic variation compared with morphological and allozyme methods.The molecular basis for RAPD was reported by Hadrys et al.(1992).This technique gives clear knowledge about the single base pair changes in the primer-binding sites or deletion in the areas between them.The only drawback of the RAPD technique is its lack of reproducibility for some ampli fi cation products,which can lead to faulty,controversial results.

As in the RAPD analysis,polymorphism was relatively higher for the profusely growing E.agallocha and P.paludosa.The 20 primers were selected for E.agallocha produced 24.87%polymorphism(Table 2)with 47 polymorphic among 189 ampli fi ed bands.Total number of bands per primer ranged from 7 to 12.ISSR1,ISSR4,ISSR7,ISSR12,ISSR 13,ISSR 15,ISSR 17,ISSR 18 AND ISSR 20 produced relatively more polymorphic bands(Table 3).P.paludosa produced 187 bands,of which 38 were polymorphic,for 20.32%polymorphism(Table 2).Eight primers(ISSR2,ISSR3,ISSR7,ISSR9,ISSR13,ISSR15,ISSR16 and ISSR20)each produced three polymorphic bands(Table 3).Total number of bands per primer ranged between 7 and 12.X.granatum had relatively less polymorphism with 12.92%,with 23 polymorphic bands of the 93 total bands(Table 2).ISSR4,ISSR7 and ISSR12 produced relatively more polymorphism among the samples(Table 3).Total number of bands expressed ranged between 8 and 12 per primer.Figure 2d–f shows a gel of the DNA bands ampli fi ed with the two ISSR primers for each of three species.

Fig.2 a–c RAPD gel,d–f ISSR gel of ampi fi ed DNA from three mangrove species from six sites:1,Mesophytic;2,Sajnekhali;3,Sudhanyakhali;4,Sonargaon;5,Jharkhali;6,Dobanki.a(OPA3,OPA 5)and d(ISSR1,ISSR4)Excoecaria agallocha;b(OPA3,OPA7)and e(ISSR7,ISSR 9)Phoenix paludosa;c(OPA9,OPA12)and f(ISSR4,ISSR12)Xylocarpus granatum

ISSR analysis

The 26 primers used for this study produced 223 bands in E.agallocha,of which 55 were polymorphic.Seven primers produced relatively more polymorphic bands:OPA3,OPA5,OPA 7,OPA12,OPA 15,OPA 19 and RAPD 22(Table 1).Of 223 bands generated for P.paludosa,55 were polymorphic(24.66%polymorphism)(Table 2).The total number of bands ranged between 7 and 11 per primer.P.paludosa produced 52 polymorphic bands among 197 bands,with primers OPA3,OPA5,OPA7,OPA13 and OPA20 produced the most polymorphic bands(Table 1)for 26.4%polymorphism(Table 2).Total number of bands ranged from 6 to 10 bands using these primers.The endangered X.granatum had the least polymorphism(14.56%)(Table 2);of 206 bands generated,30 were polymorphic.Primers OPA3,OPA9,OPA14 and OPA 20 primers were noteworthy(Table 1)for this species for producing a relatively higher number of polymorphic bands.Figure 2a–c shows a gel with the DNA bands ampli fi ed by two RAPD primers for each of the three species.

Dissimilarity found in the regions between microsatellites forms the basis of inter-simple sequence repeat(ISSR)analysis,which is frequently used to identify individuals,mostly plant species.Comparative studies of genetic variability of mangrove populations across large geographical ranges have often been done using ISSRs.Triest(2008)found signi fi cant differences in genetic diversity estimates between regions,despite the occasional low sample size of a population(10–20 individual mangrove trees).Genetic diversity within and among 10 mangrove and nonmangrove populations of Heritiera littoralis collected from three sites in China and one site in Australia was studied using ISSR by Jian et al.(2004),who reported a relatively high genetic diversity at the species level.Li and Chen(2004)estimated the genetic diversity of Sonneratia alba from China using ISSR marker;103 polymorphic bands were obtained among 133 bands(77.44%polymorphism)using 11 ISSR primers,indicating a signi fi cant genetic variation at the species level.Tan et al.(2005)estimated the genetic diversity both within and among populations of a prevalent viviparous mangrove,Ceriops decandra,using ISSRs.

Statistical analyses

ISSR analysis of E.agallocha produced 47 polymorphic bands among 189 bands;Na,Ne,h and I were 1.1679,1.1369,0.0799 and 0.1197,respectively.P.paludosa produced 187 bands,38 of which were polymorphic;1.1469,1.1147,0.0688 and 0.1023 were the respective Na,Ne,h and I values.Na,Ne,h,I values for X.granatum were 1.0847,1.0498,0.0583 and 0.0763,respectively.This species produced 23 polymorphic bands among a total of 193.For ISSR analysis,the UPGMA dendrogram based on the similarity matrix(Fig.3d–f)again showed that sites I,V and VI clustered together for X.granatum,whereas they were in distant clusters for the other two taxa.The same phenomenon was seen when a dendrogram was constructed using the combined RAPD and ISSR data(Fig.4a–c).

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RAPD analysis of E.agallocha produced 55 polymorphic bands among 223 total bands.The mean observed number of alleles per locus(Na)was 1.1671,and the mean effective number of alleles per locis(Ne)was 1.1358.Nei’s gene diversity value(h)was 0.0794 and Shannon’s information index(I)was 0.1162.P.paludosa produced 52 polymorphic bands among 197 bands expressed(Na=1.1753,Ne=1.1381,h=0.0818 and I=0.1187).For X.granatum,Na=1.1135,Ne=1.0624,h=0.0637 and I=0.0857.The 30 of 206 bands were polymorphic.The UPGMA dendrogram based on the similarity matrix constructed for the RAPD analysis(Fig.3a–c)showed that for X.granatum the non-saline site(site I)and the two most saline sites(site V and VI)clustered together,whereas in the other two taxa,the sites were in distant clusters.

Table 3 List of ISSR primers and bands ampli fi ed using DNA from Excoecaria agallocha(Ea),Phoenix paludosa(Pp)and Xylocarpus granatum(Xg)

Primer Nucleotide sequence Polymorphic bands/total no.bands Primer Nucleotide sequence Polymorphic bands/total no.bands Ea Pp Xg Ea Pp Xg ISSR 1 5′AGAGAGAGAGAGAGAGT3′ 3/8 1/11 1/11 ISSR 115′CACACACACACACACARC3′ 2/9 1/8 1/8 ISSR 2 5′AGAGAGAGAGAGAGAGG3′ 2/10 3/9 1/9 ISSR 125′CTCCTCCTCCTCCTCCTC3′ 3/10 1/9 2/9 ISSR 3 5′AGAGAGAGAGAGAGAGC3′ 1/9 3/10 1/10 ISSR 135′DBDACACACACACACAC3′ 3/11 3/10 1/8 ISSR 4 5′GAGAGAGAGAGAGAGAT3′ 3/11 1/8 2/8 ISSR 145′AGAGAGAGAGAGAGAGCTC3′ 1/9 1/9 1/11 ISSR 5 5′GAGAGAGAGAGAGAGAC3′ 2/7 2/7 1/9 15 ISSR 5′AGAGAGAGAGAGAGAGCTA3′ 3/12 3/11 1/9 ISSR 6 5′CACACACACACACACAG3′ 2/10 2/9 1/11 ISSR 165′AGTACGAGTTCTCTCTCTCTCTC3′ 2/9 3/8 1/12 ISSR 7 5′ACACACACACACACACT3′ 3/9 3/8 2/9 ISSR 175′ACACACACACACACACYA3′ 3/11 1/10 1/9 ISSR 8 5′AGAGAGAGAGAGAGAGYT3′ 2/11 1/9 1/11 ISSR 185′ACACACACACACACACYT3′ 3/9 1/9 1/11 ISSR 9 5′AGAGAGAGAGAGAGAGYC3′ 2/9 3/10 1/9 ISSR 195′ACACACACACACACACCTG3′ 2/8 1/9 1/9 ISSR 105′GAGAGAGAGAGAGAGAYG3′ 2/7 1/12 1/10 ISSR 205′ACAGTGTGTGTGTGTGT3′ 3/10 3/11 1/9

Discussion

RAPD analysis

Studies on mangroves have also been bene fi tted from these techniques. Lakshmi et al. (2000) reported intraspeci fi c genetic variation in E.agallocha.Both intraand interspeci fi c genetic variation in Avicennia was revealed by Parani et al.(1997).In addition,the parentage of a Rhizophora hybrid was analyzed using a RAPD marker.Teixeira et al.(2003)studied genetic polymorphism of 16 individuals of Avicennia alba from the Mekong Delta in Vietnam using 20 primer pairs for microsatellites.Six of the primers ampli fi ed polymorphic loci,seven of the loci ampli fi ed appeared to be monomorphic,and the other six pairs did not yield satisfactory products.Sheue et al.(2009)worked on the morphology and molecular basis of segregation of two Ceriops species(C.zippeliana and C.decandra)from southeastern Asia using RAPD marker.Pawar et al.(2013)assessed the genetic diversity of X.granatum and X.mekongensis collected from different localities of the states of Tamil Nadu and Andhra Pradesh in India using the RAPD technique,where the authors reported that the two species of Xylocarpus had lower levels of similarity.

Fig.3 Dendrogram analysis.a–c RAPD analysis,d–f ISSR analysis,e–f RAPD and ISSR combined.a,d Excoecaria agallocha,b,e Phoenix paludosa,c,f Xylocarpus granatum

资产存储量对于企业的发展来说是比较重要的，资产储存量能够显示出一个企业的综合实力，并且也会影响到企业发展的稳定性和安全性，企业要是资产存储量比较大，产生金融风险的时候，企业应对风险的能力也就越高。所以为了能够有效地识别金融风险，银行这些金融结构在管理金融的过程中需要充分掌握信贷企业的资产存储量，这样可以更加方便地判断所存在的信贷风险，如此也能够有效地识别以及控制金融风险。

Fig.4 Dendrogram analysis.a–c RAPD and ISSR combined.a,d Excoecaria agallocha,b,e Phoenix paludosa,c,f Xylocarpus granatum

The present work on genetic polymorphism analysis using RAPD and ISSR revealed that relatively fewer polymorphicsms exist in X.granatum than in the other two species.Kader et al.(2012)worked with a set of 10 RAPD and 10 ISSR markers to analyze the genetic diversity of the genus Avicennia from Sundarbans,India.In terms of polymorphism detection,polymorphic bands content per primer and total number of loci detected per primer,they found that ISSR markers were more ef fi cient than RAPD markers.In our study,although the level of polymorphism was in the same range in the ISSR and RAPD analyses of all three plant species,the total number of bands produced per primer was found to be high using ISSR.The result indicated that the percentage polymorphism was relatively low(14.56%for RAPD,12.92 for ISSR)in X.granatum,the stressed plant in the Indian Sundarbans compared with the other two taxa,which might be ascribed to the passive sustainability of this plant in the elevated saline regime.The profusely growing species,E.agallocha and P.paludosa had a relatively higher percentage polymorphism(24.66 and 26.4%for RAPD,24.87 and 20.32%for ISSR,respectively).From a genetic perspective,a higher level of genetic diversity results in a greater ability to adapt and evolve(Li and Chen 2004).Low genetic diversity can result in reduced adaptability and increased occurrence of less bene fi cial genes,leading to eventual extinction of the species;thus,the lower level of diversity within populations should receive special attention.The level of genetic diversity can provide very important information about the status of a species,an assessment of its conservation value,and ex situ conservation.

The salinity of Indian Sundarbans has risen over the years(Nandy(Dutta)et al.2009).Suf fi cient genetic polymorphism is a prerequisite for the plant to adapt to its environment through internal changes in its anatomy,physiology and biochemistry.The relatedness(proximity or distance)of genetic polymorphic expression of the studied taxa is evident from the prepared dendrogram(based on the similarity matrix).In the dendrogram prepared for the stressed species,X.granatum,from RAPD data,the extreme saline zone and mesophytic zone grouped into the same clade.The same phenomenon was observed in the dendrogram based on the ISSR data.On the contrary,in the two profusely growing species,the dendrograms prepared from RAPD and ISSR data showed that the most saline zones clustered in one clade.

The result depicted from this study agrees well with our previous study showing that the antioxidant activity of X.granatum is much weaker than P.paludosa and E.agallocha based on enzymatic and non-enzymatic variables(Dasgupta et al.2013)and the genetic polymorphism of another IUCN Red-listed species Heritiera fomes was relatively lower than in the profusely growing species Bruguiera gymnorrhiza (Dasgupta et al.2015). The antioxidative response in plants is intricate,and scavenging of ROS takes place using enzymatic and non-enzymatic machinery.So the higher level of genetic polymorphism of P.paludosa and E.agallocha might be attributed to superior protection against ROS;thus,growth of these two species is more sustainable in the increasing salinity of the present-day Sundarbans.

（2）大数据时代下技术手段的革新带来城乡规划技术运用的转型。以往的城乡规划课程体系中，原有数据分析的理念已经跟不上时代发展。采用传统方式获取规划数据（如居民出行问卷调查等），因采用小样本抽样方式，统计结果容易出现偏向性，“以偏概全”而导致不能反映出总体的真实情况。抽样分析是信息收集手段不完善时代的产物（维克托·迈尔 2013）。

Conclusion

The relatively low polymorphism shown by the RAPD and ISSR analyses for X.granatum might be attributed to its relatively poorer ability to scavenge ROS in areas of higher salinity,which might be attributed to one of the major reasons for its gradual natural decline in the Indian Sundarbans.Currently,there are very limited resources in the natural habitats of X.granatum populations in the Indian Sundarbans;thus,the most important aspect in protecting the resources,is maintaining higher genetic diversity in the species and thus its the ability to adapt and evolve,and it also provides very important information on the status of a species and conservation value.When selecting the best provenance for introduction and transplantation,quality groups,and individuals for genetic improvement,special attention should be paid to populations and individuals that come from special habitats or demonstrate irreplaceable uniqueness.

Acknowledgements The authors are sincerely indebted to the Director,Sundarbans Biosphere Reserve and Chief Principal Conservator of Forest and Wildlife,Government of West Bengal,for granting the required permission to conduct fi eldwork in the Sundarbans Reserved Forest.The authors thank the Indian Statistical Institute for funding this work.

References

Barbier EB(2007)Valuing ecosystem services as productive inputs.Econom Policy 22:177–229

Chanda S,Datta SC(1986)Prospects and problems of a mangrove ecosystem in western Sundarbans(India).Trans Bose Res Inst 49(3):47–57

Chaudhuri JK(1996)Mangrove forest management.Mangrove rehabilitation and management project in Sulawesi.p 297

Chowdhury MR,Ward MN(2007)Seasonal fl ooding in Bangladesh—variability and predictability.Hydrol Process 21:335–347

Das S(1999)An adaptive feature of some mangroves of Sundarbans,West Bengal.J Plant Biol 42(2):109–116

Das AB,Jena S(2008)Chromosome stability and interpopulation genetic variability in a tree mangrove Xylocarpous granatum Koen.(Meliaceae)as revealed by RAPD markers.Cytologia 73:105–113

Dasgupta N,Nandy P,Tiwari C,Das S(2010)Salinity-imposed changes of some isozymes and total leaf protein expression in fi ve mangrovesfrom two differenthabitats.J Plant Int 5(3):211–221

Dasgupta N,Nandy P,Sengupta C,Das S(2012)Protein and enzymes regulations towards salt tolerance of some Indian mangroves in relation to adaptation.Trees 26(2):377–391

Dasgupta N,Nandy P,Das S(2013)Salt stress:a biochemical and physiological adaptation of some Indian halophytes of Sundarbans.In:Molecular stress physiology of plants.Springer India,pp 155–177

Dasgupta N,Nandy P,Sengupta C,Das S(2015)RAPD and ISSR marker mediated genetic polymorphism of two mangroves Bruguieragymnorrhizaand Heritierafomesfrom Indian Sundarbans in relation to their sustainability.Physiol Mol Biol Plants 21(3):375–384

Forest Survey of India(FSI)(2009)State forest report 2009.Ministry of Environment and Forests,Government of India,226p

Gurudeevan S,Satyavani K,Ramanathan T(2012)Genetic identiif cation of Ceriops decandra(ChiruKandal)using tRNA(Leu)molecular marker.Asian J Plant Sci 11:91–95

Hadrys H,Balick M,Schierwater B(1992)Applications of random ampli fi ed polymorphic DNA(RAPD)in molecular ecology.Genome 1(1):55–63

Hiraishi T,Harada K(2003)Greenbelt tsunami prevention in South-Paci fi c Region.http://eqtap.edm.bosai.go.jp/

Hogarth PJ(2007)The biology of Mangroves and sea grasses.Oxford University Press,New York,p 273

Hopson TM,Webster PJ(2010)A 1–10-Day ensemble forecasting scheme for the Major River Basins of Bangladesh:forecasting severe fl oods of 2003–07.J Hydrometeorol 11(3):618–641

Jaccard P(1998)Nouvelles researches sur la distribution forale.Bull Soc Sci Nat 44:223–270

Jian S,Tang T,Zhong Y,Shi S(2004)Variation in inter-simple sequence repeat(ISSR)in mangrove and non-mangrove populations of Heritiera littoralis(Sterculiaceae)from China and Australia.Aquat Bot 79(1):75–86

Kader A,Sinha SN,Ghosh PD(2012)Evaluation of genetic diversity of Avicenniaceae family in Indian Sundarban by using RAPD and ISSR markers.Iranian J Genet Plant Breed 1:22–27

Lakshmi M,Parani M,Ram N,Parida A(2000)Molecular phylogeny of mangroves VI.Intraspeci fi c genetic variation in mangrove species Excoecaria agallocha L.(Euphorbiaceae).Genome 43(1):110–115

Li H-S,Chen G-Z(2004)Genetic diversity of Sonneratia alba in China detected by Inter-simple Sequence Repeats(ISSR)analysis.Acta Bot Sinica 46:512–521

Nandy(Dutta)P,Dasgupta N,Das S(2009)Differential expression of physiologicalandbiochemicalcharactersofsomeIndianmangroves towards salt tolerance.Physiol Mol Biol Plants 15(2):151–160

Naskar KR,GuhaBakshi DN(1983)A brief review on some less familiar plants of the Sundarbans India.J Eco Taxon Bot 4(3):699–712

Nei M(1973)Analysis of gene diversity in subdivided populations.Proc Nat Acad Sci 70(12):3321–3323

Newbury HJ,Ford-Lloyd BV(1993)The use of RAPD for assessing variation in plants.Plant Growth Reg 12:43–51

Parani M,Lakshmi M,Elango S,Ram N,Anuratha CS,Parida A(1997)Molecular phylogeny of mangroves II.Intra and interspeci fi c variation in Avicennia revealed by RAPD and RFLP markers.Genome 40(4):487–495

Parida A,Anuratha CS,Lakshmi M,Parani M,Kurjen J(1995)Application of molecular markers in assessing genetic diversity in Indian mangroves.In:Use of Induced Mutations and Molecular Techniques in Crop Improvement(FAO/IAEA,Vienna,Austria,595–600)

Pawar UR,Baskaran J,Ajithkumar IP,Panneerselvam R(2013)Genetic variation between Xylocarpus species(Meliaceae)as revealed by Random Ampli fi ed Polymorphic DNA(RAPD)markers.Emirates J Food Agric 25(8):597–604

Rohlf FJ(1993)Ntsys-PC.Numerical taxonomy and multivariate analysis system Version I.80-Setauket,NY,Exeter Software

Schaal BA,Leverich WJ,Rogstad SH(1991)Comparison of methods for assessing genetic variation in plant conservation biology.Genetics and conservation of rare plants.Oxford University Press,New York,pp 123–134

Schwarzbach AE,McDade LA(2002)Phylogenetic relationships of the mangrove family Avicenniaceae based on chloroplast and nuclear ribosomal DNA sequences.Syst Bot 27:84–98

Sheue CR,Liu HY,Tasi CC,Rashid SMA,Yong JWH,Yang YP(2009)On the morphology and molecular basis of segregation of two species Ceriops zippeliana Blum.and C.decandra(Griff.)Ding Hou(Rhizophoraceae)from Southeastern Asia.Blumea 54:220–227

Sneath PH,Sokal RR(1973)Numerical taxonomy.The principles and practice of numerical classi fi cation

Spalding M,Blasco F,Field C(1997)World Mangrove Atlas.The International Society for Mangrove Ecosystems.Okinawa,Japan,p 178

Spiers AG(1999)Review of International continental wetland resources.Global review of wetland resources and priorities for wetland inventory.Finlayson,CM and Spiers,AG(eds.),supervising Scientist Report 144.Canberra,Australia:63–104

Takemura T,Hanagata N,Sugihara K,Baba S,Karube I,Dubinsky Z(2000)Physiological and Biochemical Responses to Salt Stress in the Mangrove,Bruguiera gymnorrhiza.Aquat Bot 68:15–28

Tan F,Huang Y,Ge X,Su G,Ni X,Shi S(2005)Population genetic structure and conservation implications of Ceriops decandra in Malay Peninsula and North Australia.Aqua Bot 81(2):175–188

Teixeira S,Arnaud-Haond S,Duarte CM,Serrao E(2003)Polymorphic microsatellite DNA markers in the mangrove tree Avicennia alba.Mol Ecol Notes 3:544–546

Templeton AR (1993)Translocation as conservation tool.In:Biodiversity in mangrove landscapes,theory and practice(ed.Szaro R.I.N)(Oxford University Press)

The IUCN Red List of Threatened Species.Version(2014)＜www.iucnredlist.org＞.Downloaded on 22 March 2015

Triest L(2008)Molecular ecology and biogeography of mangrove trees towards conceptual insights on gene fl ow and barriers:a review.Aqua Bot 89(2):138–154

Upadhyay VP,Ranjan R,Singh JS(2002)The Human Mangrove Con fl icts-The way out.CurrSci 83:1328–1336

Williams JGK,Kubelik AR,Livak KJ,Rafalski JA,Tingey SV(1990)DNA polymorphisms ampli fi ed by arbitrary primers are useful as genetic markers.Nucleic Acids Res 18:6531–6535

Yan L,Guizhu C(2007)Physiological adaptability of three mangrove species to salt stress.Acta Ecol Sinica 27(6):2208–2214

Yeh FC,Yang RC,Boyle TB,Ye ZH,Mao JX(1997)POPGENE,the user-friendly shareware for population genetic analysis.Molecular biology and biotechnology center.University of Alberta,Canada,p 10

Zimmermann MH(1983)Xylem structure and the ascent of sap.Springer,Berlin