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Oil induces chlorophyll deficient propagules in mangroves
Marine Pollution Bulletin 150 (2020) 110667
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Oil induces chlorophyll deficient propagules in mangroves a
a
b
Dimitri Veldkornet , Anusha Rajkaran , Swapan Paul , Gonasageran Naidoo a b c
T c,∗
Department of Biodiversity and Conservation Biology, University of the Western Cape, South Africa Sydney Olympic Park Authority, Sydney, Australia University of KwaZulu-Natal, School of Life Sciences, Westville, South Africa
A R T I C LE I N FO
A B S T R A C T
Keywords: Albino Avicennia marina Chlorophyll deficiency Gene mutation Oil pollution Polycyclic aromatic hydrocarbons (PAHs)
In Australia, some trees of the mangrove, Avicennia marina, growing in a chronic oil polluted site, produce chlorophyll deficient (albino) propagules. We tested the hypothesis that albinism was due to an oil-induced mutant allele that controls photosynthesis. We determined whether there are genetic differences between normal and chlorophyll deficient propagules. Four gene regions (nuclear 18S–26S cistron; chloroplast - trnH-psbA, rsp16 and matK) were sequenced and analysed for normal and albino propagules. Mutations occurred in both nuclear (ITS) and coding chloroplast (matK) genes of albino propagules. There were 10 mutational differences between normal and albino propagules in the matK samples. Analysis of molecular variation (AMOVA) of the matK dataset indicated highly significant genetic differentiation between normal and albino propagules. Our study suggests for the first time that PAHs from a chronic oil polluted site resulted in mutations in both nuclear and chloroplast genes, resulting in the production of albino propagules.
1. Introduction Mangroves occur in low wave energy sheltered locations of the tropics and subtropics and have high ecological and conservation value due to their high productivity and multiple ecosystem services they provide (Barbier, 2016; Friess, 2016). Mangrove ecosystems occur in close proximity to urban and industrial areas and are therefore highly vulnerable to oil pollution (Lewis et al., 2011). Oil is composed of a variety of hydrocarbons (USEPA, 2008) such as alkanes, cyclohexenes and aromatics (polycyclic aromatic hydrocarbons, PAHs). PAHs degrade very slowly and persist in the environment for prolonged periods due to their insolubility in water and low rates of degradation (Lewis et al., 2011). PAHs are phytotoxic and affect plants at all stages of growth (Gomes et al., 2007; Naidoo et al., 2010). Oil contamination of mangroves reduces germination and growth, induces leaf abscission, decreases ion and water uptake, reduces electron transport rate (ETR) and quantum yield through Photosystem II (ΦPSII), disrupts membranes and organelles and increases mortality (Zhang et al., 2007; Naidoo et al., 2010; Naidoo and Naidoo, 2016, 2017, 2018). In several parts of the world, there have been reports of mangroves producing propagules which lack their normal green coloration and are either yellow, pink or red (Fig. 1). These mutants are chlorophyll deficient and are referred to as albinos. Albino propagules have been reported in Central America, South East Asia, Florida, Puerto Rico and
∗
Corresponding author. E-mail address: [email protected] (G. Naidoo).
https://doi.org/10.1016/j.marpolbul.2019.110667 Received 25 June 2019; Accepted 11 October 2019 Available online 02 November 2019 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.
Australia. Mutants have been observed in several genera of mangroves including Avicennia, Rhizophora, Kandelia, Ceriops and Pelliciera (Table 1). Mutants of Rhizophora, Ceriops and Kandelia can be observed whilst attached to the parent tree as they are large and contrast with the green foliage and normal sibling propagules. This allows for easy visual recognition of potential genotypic differences in the trees. Mutants of Avicennia propagules are more difficult to locate because of their small size (Fig. 1). Albino bearing parent trees appear normal but are heterozygous and possess a mutant recessive gene. Albino-bearing trees produce normal and albino propagules in some measurable ratio of frequencies and have been scored for the mutant trait (Klekowski and Godfrey, 1989; Lowenfeld and Klekowski, 1992; Klekowski et al., 1994 a,b,c; Chen et al., 1996). The chlorophyll deficient genotype is presumably due to a mutant allele in the nuclear genes that controls photosynthesis in the chloroplast. In fiowering plants, approximately 300 nuclear gene loci control the presence of chlorophyll in the chloroplasts (Klekowski, 1992; Corredor et al., 1995). Chlorophyll deficient mutants are recessive, with homozygotes germinating as white or yellow offspring. Albino propagules produce at least a pair of leaves before dying, usually within 3 months, after depletion of maternal reserves, (Paul and Young, 2005). Environmental conditions such as petroleum contaminated sediments may enhance the intraspecific mutation rates for R. mangle populations (Klekowski et al., 1994c). In many other localities where
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polluted site were identified as producing albino propagules.Yellow and red albino propagules survive for about 3 months and their growth is stunted with abnormal leaves and branches (Paul and Young, 2005). Most of the trees that bear albino propagules are over 30 years old and also exhibit abnormal adventitious roots that arise from the tree trunks (Fig. 1). These abnormal roots were induced experimentally by oil in field and laboratory studies (Naidoo et al., 2010). 2.2. DNA extraction and analysis DNA was extracted from freeze-dried propagules using the Qiagen DNeasy Plant Mini Kit (www.qiagen.com), following the kit's protocol and visualised on 0.8% agarose gels. Four gene regions were selected in this study because they readily amplify across a number of taxa. The nuclear 18S–26S cistron was sequenced using primers ITS4 (forward: TCC TCC GCT TAT TGA TAT GC) and ITS5m (reverse: GGA AGG AG AAG TCG TAA CAA GG) (Sang et al., 1995). The two non-coding chloroplast gene regions were trnH-psbA (trnH forward primer: CGC GCA TGG TGG ATT CAC AAT CC) (Tate and Simpson, 2003), and psbA3 (reverse primer: GTT ATG CAT GAA CGT AAT GCT C) (Sang et al., 1995), and rsp16 (using primers rps16F: AAA CGA TGT GGT ARA AAG CAA C and rps16R: AAC ATC WAT TGC AAS GAT TCG ATA), (Shaw et al., 2005); and the coding region matK using primers KIM 3F (CGT ACA GTA CTT TTG TGT TTA CGA G) -KIM 1R (ACC CAG TCC ATC TGG AAA TCT TGG TTC), (Cuénoud et al., 2002). PCR was performed using the EmeraldAmp PCR Master Mix (Takara Bio Inc., Shiga, Japan) with 0.5 mM of each primer. The PCRs were performed in volumes of 25 μl containing 1 μl of the DNA template of unknown concentration, 12.5 μl 2X EmeraldAmp® PCR Master Mix, 0.5 μl BSA, 0.5 μl of each primer and sterile distilled water. The PCR protocol consisted of an initial 2 min denaturing step at 94 °C; 40 cycles, each comprising 94 °C for 1 min 45 s, 55 °C for 30 s, 72 °C for 2 min; and a final 6 min extension step at 72 °C. All PCRs were performed on a GeneAmp 2700 PCR System (Applied Biosystems, Foster City, CA, USA). Sequencing was conducted by Inqaba Biotechnical Industries. Sequences generated were assembled with CODONCODE ALIGNER v5.4 (Codon Code Corp, Barnstable, MA, USA, http://www.codoncode.com). Each of the four gene regions were handled differently in alignment. Alignment of trnH-psbA and rps16 was unambiguous, because of the absence of indel variation, and all sequences were readily assembled. The MUSCLE function was used for ITS and matK, then checked manually and adjusted where necessary, with gaps positioned to minimize nucleotide mismatches. Gap penalties and mismatch scores were based on the default setting in Codoncode Aligner. Haplotype diversity (h, the probability that two haplotypes chosen at random in a sample are different) and genetic diversity (π, the mean number of differences between all pairs of haplotypes) (Nei and Li, 1979; Nei and Tajima, 1981) for all samples were calculated in DnaSP v5 (Librado and Rozas, 2009). Population genetic structure was investigated for the nuclear and chloroplast data using analyses of molecular variance (AMOVA) in Arlequin 3.5.2.2 to test for significance under the null hypothesis of panmixia with 10 000 permutations. Pairwise calculations of ФST, an analogue of FST, consider haplotype frequency and the extent of differentiation among haplotypes and assume that the mutation rate is negligible compared to the migration rate.
Fig. 1. Top left: Albino propagules of A. marina in Sydney; Top right: Albino propagules of Kandelia obovata in southern China; Bottom left: Albino propagules of Rhizophora apiculata in Palau; Bottom right: adventitious roots on stems of old A. marina trees in an oil-polluted site, Sydney, Australia. Pictures of Kandelia and Rhiziphora are courtesy of K. Krauss.
albino propagules were reported (Table 1), the soil had significant amounts of biogenic hydrocarbons, including PAHs, or a history of chronic oil contamination (Klekowski et al., 1994a,b; Duke and Watkinson, 2002; Paul and Young, 2005). These observations suggest that petroleum hydrocarbons may exacerbate lethal mutations in mangroves, suggesting a cause-effect relationship. Molecular responses to oil stress and effects of oil pollution on mutation in mangroves, however, have not been adequately investigated and are poorly understood (Huang et al., 2014; Guedes et al., 2018). Despite their ecological and economic relevance, mangrove plant sequences are poorly represented in biological databases. The objective of this study was to determine whether there were genetic differences between normal and chlorophyll deficient propagules of A. marina. We employed standard Sanger sequencing and statistical analyses and report mutational differences in nuclear and coding chloroplast DNA between normal and albino propagules of this pioneer mangrove species. As far as we are aware, this is one of a few studies to investigate the genetic effects of oil and provides novel insights on the long term dynamics of oil pollution on mangroves. 2. Materials and methods 2.1. Study site Albino and normal propagules of A. marina, were collected from Badu Mangroves, Sydney Olympic Park, Australia (33° 15′ 24.3″ S; 151° 04′ 48.1” E). This area has a long history of industrial pollution for about 30 years. The entire area, situated on the former mudflat of Homebush Bay, was infilled with industrial waste and refuse and continues to receive road and catchment runoff and is a highly polluted site. The mangrove sediment in this bay is highly contaminated with petroleum hydrocarbons. The concentration of total petroleum hydrocarbons in the sediment ranged from 385 to 554 pg g−1 (Paul and Young, 2005), which is more than 50-times that found in another polluted study site in Queensland, Australia, with albino propagules (Duke and Watkinson, 2002). Propagules of A. marina with green cotyledons are considered ‘normal’. Albino propagules (Fig. 1) were of two types: yellow-orange (with bright yellow cotyledons) and reddishgreen (with reddish-green cotyledons). In total, 30 trees in this oil
3. Results The non-coding chloroplast gene regions (rps16 and trnH-psbA) showed no differences between normal and albino samples and were consequently removed from further analysis. This was probably due to PAHs not affecting gene regions that do not code for any amino acids and are not under selection pressure (Shaw et al., 2005). Results from the genetic analysis indicated mutations in both nuclear (ITS) and coding chloroplast (matK) genes (Tables 2 and 3). 2
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Table 1 Reports of chlorophyll deficient mangrove propagules in different species and various localities. Mangrove species
Location
Pollutant
Reference
Rhizophora mangle
Florida Puerto Rico
oil oil oil oil oil oil oil oil oil oil, pesticides ? oil ? ? ?
Handler and Teas (1983) Handler and Teas (1983) Lowenfeld and Klekowski (1992) Klekowski (1988) Klekowski et al. (1994a),b,c Perry (1998) Duke and Watkinson (2002) Duke and Watkinson (2002) Duke and Watkinson (2002) Paul and Young (2005) Da Silva et al. (1997) Duke and Watkinson (2002) Duke and Watkinson (2002) Duke and Watkinson (2002) Chen et al. (1996)
Hawaii Central America Australia Australia
Rhizophora stylosa Avicennia marina Avicennia germinans
Brazil Australia Australia Central America China
Ceriops Pelliciera rhizophorae Kandelia candel
Nucleotide diversity was higher compared to haplotype diversity for ITS and matK datasets. In the ITS dataset, nucleotide diversity ranged from 0.0007 (normal) to 0.0020 (albino) for the nuclear dataset, whereas the haplotype diversity was higher for albino samples (0.667) compared to normal samples (0.500). There were two unique haplotypes with one base mutation for normal samples and two mutations for albino samples. AMOVA suggested no significant genetic differentiation between normal and albino samples (ФST = 0.26667; p > 0.05); but a high degree of variation within populations (73.33%) compared to variation among populations (26.67%). Sequences were more divergent in the matK (π = 0.0090; h = 0.571) compared to the ITS dataset and separated normal and albino samples into two distinct haplotypes. There were 10 mutational differences between normal and albino samples. AMOVA of the matK dataset suggests highly significant, genetic differentiation between normal and albino samples (ФST = 1.000; p < 0.05) with a high degree of variation among populations (100%).
Table 3 Summary statistics for 18S–26S cistron intergenic spacer (ITS) coding chloroplast DNA (matK) between normal and albino samples of Avicennia marina. π = nucleotide diversity, h = haplotype diversity. Gene region
Type
No. Base pairs
π
h
No. Haplotypes
Mutations
ITS
Normal Albino Combined Normal Albino Combined
670 670 670 633 633 633
0.0007 0.0020 0.0020 0.0000 0.0000 0.0090
0.500 0.667 0.607 0.000 0.000 0.571
2 2 3 1 1 2
1 2 3 0 0 10
MatK
populations and lower their evolutionary potential (Giska et al., 2015). Exposure to pollution may also result in reduced individual fitness and decreased size of wild populations. In addition, when populations have become small, genetic drift may remove variation faster. The genetic diversity is predicted to be low because genetic variation is increasingly lost through genetic drift (Simonsen and Klok, 2010; Hobbs et al., 2013; Giska et al., 2015). We speculate that the low genetic diversity and high haplotype diversity in albino mangrove propagules resulted in genetic drift which supports the genetic erosion hypothesis of van Straalen and Timmermans (2002). We suggest that exposure to chonic contaminants, such as PAHs, may lead to reduced genetic diversity. In a recent study, Arabidopsis thaliana seedlings exposed to the water-soluble fraction of marine fuel MF380 (WSF-MF380) exhibited responses similar to those of heat, hypoxia, osmotic and oxidative stresses (Nardeli et al., 2016). In the mangrove, L. racemosa, oil exposure modulated the expression of genes related to protein homeostasis, photosynthesis, hypoxia and ethylene response (Guedes et al., 2018). In other studies, adverse effects of PAHs on chloroplasts and on photosynthesis have been reported in mangroves (Naidoo et al., 2010; Naidoo and Naidoo, 2016, 2017; 2018). In A. thaliana, PAHs were
4. Discussion Data from this study suggest that PAHs from a chronic oil polluted site resulted in mutations in the mangrove A. marina. Chlorophyll-deficiency in mangrove propagules may be caused either by mutation of genes in the nuclear or chloroplast genome (Klekowski, 1992, Klekowski and Godfrey, 1989). Our data indicated that mutations occurred in both nuclear (ITS) and coding chloroplast (matK) genes. Previously, mutant alleles in albino propagules of Rhizophora were determined by their effects on chloroplast ultrastructure (Klekowski et al., 1994b). Genetic diversity can be influenced by a range of factors including population size, natural selection, mutation rates, gene flow between populations, introgression from hybridisation and historical effects on these factors such as population bottlenecks (Hobbs et al., 2013). Chronic exposure to pollution may decrease genetic diversity of
Table 2 Segregating sites in 18S–26S cistron intergenic spacer (ITS) and coding chloroplast DNA (matK) between normal and albino. samples of Avicennia marina. MatK Type Normal
Albino
Position Consensus N1 N2 N3 N4 A1 A2 A3 A4
ITS 81 C . . . . G . . .
311 G . . . . T . . .
355 A . . . . C . . .
547 A . . . . T . . .
562 A . . . . G . . .
575 A . . . . T T T T
3
579 A . . . . G . . .
581 T . . . . A . . .
590 T . . . . G . . .
597 C . . . . T . . .
191 C . . . . . . T T
525 G . . . . . . A A
625 T C . . . . . . .
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development, severe storms and chronic oil pollution. The more sensitive mangrove species might change in genetic makeup by direct mutation such as the production of albino propagules, while others may change by genetic selection over many generations for greater tolerance to chronic pollution (Duke and Watkinson, 2002). Mangroves subject to chronic oil pollution have relatively lower biodiversity and plants generally exhibit genetic abnormalities (Duke and Watkinson, 2002; Paul and Young, 2005; Naidoo et al., 2010; Naidoo and Naidoo, 2017). Chronic oil pollution leads to decline in ecosystem function and eventual collapse as there is little or no seedling recruitment. In such cases, the benefits and services of mangrove ecosystems will be lost. Shorelines would be subjected to greater erosion and there will be a reduction in estuarine and nearshore fisheries (Barbier, 2016; Friess, 2016).
reported to have caused the modulation of photosynthesis-related genes (Weisman et al., 2010). In A. marina, our results showed greater mutations in the chloroplast dataset compared to the nuclear dataset. This suggests that PAHs may have a greater effect on the chloroplast genome, which ultimately results in chlorophyll-deficiency in mangroves. Photosynthesis is a complex process that includes many potential target reactions, such as light harvesting, carbon fixation, photophosphorylation and electron transport (Wang et al., 2014). The nucleus plays a critical role in maintaining normal function of the chloroplast (Richly and Leister, 2004). The chloroplast, as the site of photosynthesis, requires adaptive changes in the expression levels of nuclear genes to respond to endogenous and environmental stimuli. These changes in gene expression are regulated by a complex signalling network (Wang et al., 2014). Richly and Leister (2004) reported that the nuclear genome encodes about 2090 predicted chloroplast proteins suggesting that the nucleus plays a critical role in the regulation of photosynthesis and environmental perturbations via gene expression. Conclusions about the identity of specific genes mutated in each of the genomes, however, require additional studies with greater sample size and advanced sequencing techniques. Detecting genetic responses to environmental change is difficult in the absence of a priori candidate loci with a large effect on the phenotype of interest.This suggests that genome-wide approaches allow for better insight into population genetic processes occurring across a whole genome (Giska et al., 2015). Next Generation Sequencing (NGS) techniques (Hofker et al., 2014) may be more appropriate in the identification of genes that are directly associated with chlorophyll-deficiency. Whole genome sequencing may be used to determine the mutation frequency for genomes and the genes responsible for coding proteins in photosynthetic reactions. For example, complete sequencing of rbcL in the chloroplast genome is a prime target for genetic engineering to improve photosynthetic efficiency (Parry et al., 2007). With NGS, such as Restriction-site Associated DNA Sequencing (RADSeq), genetic diversity, gene flow and population structure for both the chloroplast and the nuclear genome can be determined. In several locations in the world, a significant positive relationship was reported between the occurrence of albino mangrove propagules and high concentrations of hydrocarbons in the sediment (Klekowski et al., 1994a, b; Duke and Watkinson, 2002; Paul and Young, 2005). In this study, trees of A. marina, bearing albino propagules occurred in sites with high levels of both total hydrocarbon and petroleum-based PAHs (Duke and Watkinson, 2002; Paul and Young, 2005). In oil contaminated A. marina, Bruguiera gymnorrhiza and R. mucronata, most PAHs were retained within roots (96–99%) with little translocation to shoots (1–4%). Dominant PAHs detected in leaves of the three species included phenanthrene, naphthalene, fluorene, acenaphthene, chrysene, benzo[k+b]fluoranthene and anthracene (Naidoo and Naidoo, 2016, 2017, 2018). These PAHs were also present in mangrove sediments in Australia that supported A. marina trees with albino propagules (Duke and Watkinson, 2002). In other studies where oil concentration in the soil was not measured, the presence of albino propagules was associated with prior oil pollution of the habitat (Perry, 1998; Paul and Young, 2005). The albino mutation causative agent occurs over timespans of several decades after oil spills so it is difficult to pinpoint the cause. Moreover, chlorophyll deficient mutants have been reported across a diverse range of mangrove species including Avicennia, Rhizophora, Kandelia, Ceriops and Pelliciera (Table 1, Fig. 1), demonstrating that this phenomenon is widespread. These results have potentially serious implications for coastal management as high mutant densities may be indicative of long term genetic deterioration of mangroves following oil pollution. Additionally, there are only about 70 mangrove species and they occupy a narrow ecological range, constrained mostly between mean sea level and high spring tide (Duke and Watkinson, 2002; Lewis et al., 2011). Mangrove ecosystems are most vulnerable when conditions change rapidly during coastal
5. Conclusions This study demonstrated that chlorophyll deficient propagules of A. marina are caused by a lethal genetic mutation in both nuclear (ITS) and coding chloroplast (matK) genes. There were greater mutations in the chloroplast dataset compared to the nuclear dataset. These data suggest that PAHs have a greater effect on the chloroplast genome compared to that of the nucleus. Albinism will reduce regenerative capacity and represents an additional and unquantified aspect of general deterioration in mangrove habitat and fitness. Our data, together with others, provide compelling evidence that petroleum hydrocarbons are responsible for lethal mutations in mangroves. Several significant correlations in different parts of the world and in different mangrove species are compatible with a cause-effect relationship. Our findings raise environmental management issues concerning the persistence of petroleum hydrocarbons in intertidal environments and their harmful impacts. Improving the understanding of the molecular basis of oil pollution will provide tools for biomonitoring and bioremediation after spills. Author contributions DV performed the genetic analyses, SP provided the albino propagules and GN initiated the study. DV and GN wrote the manuscript while all contributed to the interpretation and discussion of the results. Acknowledgements This research was supported by the National Research Foundation (grant number 93560 to G Naidoo) and the University of KwaZuluNatal. The authors are grateful to A Rajh for technical assistance. References Barbier, E.B., 2016. The protective service of mangrove ecosystems: a review of valuation methods. Mar. Pollut. Bull. 109, 676–681. Chen, X.Y., Lin, P., Lin, Y.M., 1996. Mating systems and spontaneous mutation rates for chlorophyll-deficiency in populations of the mangrove Kandelia candel. Hereditas 125, 47–52. Corredor, J.E., Morell, J.M., Klekowski Jr., E.J., Lowenfeld, R., 1995. Mangrove genetics. III. Pigment fingerprints of chlorophyll deficient mutants. Int. J. Plant Sci. 156, 55–60. Cuénoud, P., Savolainen, V., Chatrou, L.W., Powell, M., Grayer, R.J., Chase, M.W., 2002. Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. Am. J. Bot. 89 (1), 132–144. Da Silva, E.M., Peso-Aguiar, M.C., Navarro, M de F.T., Chastinet, C.de B.E.A., 1997. Impact of petroleum pollution on aquatic coastal ecosystems in Brazil. Environ. Toxicol. Chem. 16 (1), 112–118. Duke, N.C., Watkinson, A.J., 2002. Chlorophyll-deficient propagules of Avicennia marina and apparent longer term deterioration of mangrove fitness in oil-polluted sediments. Mar. Pollut. Bull. 44, 1269–1276. Friess, D.A., 2016. Ecosystem Services and Disservices of Mangrove Forests: Insights from Historical Colonial Observations, vol. 183 Forests. Giska, I., Babik, W., van Gestel, C.A., van Straalen, N.M., Laskowski, R., 2015. Genomewide genetic diversity of rove beetle populations along a metal pollution gradient. Ecotoxicol. Environ. Saf. 119, 98–105.
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Gomes, N.C.M., Borges, L.R., Paranhos, R., Pinto, F.N., Krögerrecklenfort, E., MendonçaHagler, L.C., Smalla, K., 2007. Diversity of ndo genes in mangrove sediments exposed to different sources of polycyclic aromatic hydrocarbon pollution. Appl. Environ. Microbiol. 73 (22), 7392–7399. Guedes, F.A. de F., Rossetto, P. de B., Guimarães, F., Wilwerth, M.W., Paes, J.E.S., Nicolás, M.F., Reinert, F.R., Peixoto, S., Alves-Ferreira, M., 2018. Characterization of Laguncularia racemosa transcriptome and molecular response to oil pollution. Aquat. Toxicol. 205, 36–50. Handler, S.H., Teas, H.J., 1983. Inheritance of albinism in the red mangrove, Rhizophora mangle L. In: Teas, H.J. (Ed.), Tasks for Vegetation Science. W. Junk, The Hague, pp. 117–121. Hobbs, J.P., van Herwerden, L., Jerry, D., Jones, G., Munday, P., 2013. High genetic diversity in geographically remote populations of endemic and widespread coral reef angelfishes (genus: Centropyge). Diversity 5 (1), 39–50. Hofker, M.H., Fu, J., Wijmenga, C., 2014. The genome revolution and its role in understanding complex diseases. Biochim. Biophys. Acta (BBA) - Mol. Basis Dis. 1842 (10), 1889–1895. Huang, J., Lu, X., Zhang, W., Huang, R., Chen, S., Zheng, Y., 2014. Transcriptome sequencing and analysis of leaf tissue of Avicennia marina using the Illumina platform. PLoS One 9, e108785. https://doi.org/10.1371/journal.pone.0108785. Klekowski Jr., E.J., 1988. Mutation Developmental Selection, and Plant Evolution. Columbia University Press, New York. Klekowski Jr., E.J., 1992. Mutation rates in diploid annuals. Are they immutable? Int. J. Plant Sci. 153, 462–465. Klekowski Jr., E.J., Corredor, J.E., Lowenfeld, R., Klekowski, E.H., Morell, J.M., 1994a. Using mangroves to screen for mutagens in tropical marine environments. Mar. Pollut. Bull. 28, 346–350. Klekowski Jr., E.J., Corredor, J.E., Morell, J.M., Del-Castillo, C.A., 1994b. Petroleum pollution and mutation in mangroves. Mar. Pollut. Bull. 28, 166–169. Klekowski Jr., E.J., Godfrey, P.J., 1989. Aging and mutation in plants. Nature 340, 389–391. Klekowski Jr., E.J., Lowenfeld, R., Hepler, P.K., 1994c. Mangrove genetics. II. Outcrossing and lower spontaneous mutation rates in Puerto Rican Rhizophora. Int. J. Plant Sci. 155, 373–381. Lewis, M., Pryor, R., Wilking, L., 2011. Fate and effects of anthropogenic chemicals in mangrove ecosystems: a review. Environ. Pollut. 180, 345–367. Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25 (11), 1451–1452. Lowenfeld, R., Klekowski, E.J., 1992. Mangrove genetics. I. Mating system and mutation rates of Rhizophora mangle in Florida and San Salvador Island, Bahamas. Int. J. Plant Sci. 153, 384–400. Naidoo, G., Naidoo, K., 2016. Uptake of polycyclic aromatic hydrocarbons and their cellular effects in the mangrove Bruguiera gymnorrhiza. Mar. Pollut. Bull. 113, 193–199. Naidoo, G., Naidoo, K., 2017. Ultrastructural effects of polycyclic aromatic hydrocarbons in the mangroves Avicennia marina and Rhizophora mucronata. Flora 235, 1–9. Naidoo, G., Naidoo, K., 2018. Uptake and translocation of polycyclic hydrocarbons in the mangroves Avicennia marina and Rhizophora mucronata. Environ. Sci. Pollut. Res. 25, 28875–28883.
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Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Oil induces chlorophyll deficient propagules in mangroves a
a
b
Dimitri Veldkornet , Anusha Rajkaran , Swapan Paul , Gonasageran Naidoo a b c
T c,∗
Department of Biodiversity and Conservation Biology, University of the Western Cape, South Africa Sydney Olympic Park Authority, Sydney, Australia University of KwaZulu-Natal, School of Life Sciences, Westville, South Africa
A R T I C LE I N FO
A B S T R A C T
Keywords: Albino Avicennia marina Chlorophyll deficiency Gene mutation Oil pollution Polycyclic aromatic hydrocarbons (PAHs)
In Australia, some trees of the mangrove, Avicennia marina, growing in a chronic oil polluted site, produce chlorophyll deficient (albino) propagules. We tested the hypothesis that albinism was due to an oil-induced mutant allele that controls photosynthesis. We determined whether there are genetic differences between normal and chlorophyll deficient propagules. Four gene regions (nuclear 18S–26S cistron; chloroplast - trnH-psbA, rsp16 and matK) were sequenced and analysed for normal and albino propagules. Mutations occurred in both nuclear (ITS) and coding chloroplast (matK) genes of albino propagules. There were 10 mutational differences between normal and albino propagules in the matK samples. Analysis of molecular variation (AMOVA) of the matK dataset indicated highly significant genetic differentiation between normal and albino propagules. Our study suggests for the first time that PAHs from a chronic oil polluted site resulted in mutations in both nuclear and chloroplast genes, resulting in the production of albino propagules.
1. Introduction Mangroves occur in low wave energy sheltered locations of the tropics and subtropics and have high ecological and conservation value due to their high productivity and multiple ecosystem services they provide (Barbier, 2016; Friess, 2016). Mangrove ecosystems occur in close proximity to urban and industrial areas and are therefore highly vulnerable to oil pollution (Lewis et al., 2011). Oil is composed of a variety of hydrocarbons (USEPA, 2008) such as alkanes, cyclohexenes and aromatics (polycyclic aromatic hydrocarbons, PAHs). PAHs degrade very slowly and persist in the environment for prolonged periods due to their insolubility in water and low rates of degradation (Lewis et al., 2011). PAHs are phytotoxic and affect plants at all stages of growth (Gomes et al., 2007; Naidoo et al., 2010). Oil contamination of mangroves reduces germination and growth, induces leaf abscission, decreases ion and water uptake, reduces electron transport rate (ETR) and quantum yield through Photosystem II (ΦPSII), disrupts membranes and organelles and increases mortality (Zhang et al., 2007; Naidoo et al., 2010; Naidoo and Naidoo, 2016, 2017, 2018). In several parts of the world, there have been reports of mangroves producing propagules which lack their normal green coloration and are either yellow, pink or red (Fig. 1). These mutants are chlorophyll deficient and are referred to as albinos. Albino propagules have been reported in Central America, South East Asia, Florida, Puerto Rico and
∗
Corresponding author. E-mail address: [email protected] (G. Naidoo).
https://doi.org/10.1016/j.marpolbul.2019.110667 Received 25 June 2019; Accepted 11 October 2019 Available online 02 November 2019 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.
Australia. Mutants have been observed in several genera of mangroves including Avicennia, Rhizophora, Kandelia, Ceriops and Pelliciera (Table 1). Mutants of Rhizophora, Ceriops and Kandelia can be observed whilst attached to the parent tree as they are large and contrast with the green foliage and normal sibling propagules. This allows for easy visual recognition of potential genotypic differences in the trees. Mutants of Avicennia propagules are more difficult to locate because of their small size (Fig. 1). Albino bearing parent trees appear normal but are heterozygous and possess a mutant recessive gene. Albino-bearing trees produce normal and albino propagules in some measurable ratio of frequencies and have been scored for the mutant trait (Klekowski and Godfrey, 1989; Lowenfeld and Klekowski, 1992; Klekowski et al., 1994 a,b,c; Chen et al., 1996). The chlorophyll deficient genotype is presumably due to a mutant allele in the nuclear genes that controls photosynthesis in the chloroplast. In fiowering plants, approximately 300 nuclear gene loci control the presence of chlorophyll in the chloroplasts (Klekowski, 1992; Corredor et al., 1995). Chlorophyll deficient mutants are recessive, with homozygotes germinating as white or yellow offspring. Albino propagules produce at least a pair of leaves before dying, usually within 3 months, after depletion of maternal reserves, (Paul and Young, 2005). Environmental conditions such as petroleum contaminated sediments may enhance the intraspecific mutation rates for R. mangle populations (Klekowski et al., 1994c). In many other localities where
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polluted site were identified as producing albino propagules.Yellow and red albino propagules survive for about 3 months and their growth is stunted with abnormal leaves and branches (Paul and Young, 2005). Most of the trees that bear albino propagules are over 30 years old and also exhibit abnormal adventitious roots that arise from the tree trunks (Fig. 1). These abnormal roots were induced experimentally by oil in field and laboratory studies (Naidoo et al., 2010). 2.2. DNA extraction and analysis DNA was extracted from freeze-dried propagules using the Qiagen DNeasy Plant Mini Kit (www.qiagen.com), following the kit's protocol and visualised on 0.8% agarose gels. Four gene regions were selected in this study because they readily amplify across a number of taxa. The nuclear 18S–26S cistron was sequenced using primers ITS4 (forward: TCC TCC GCT TAT TGA TAT GC) and ITS5m (reverse: GGA AGG AG AAG TCG TAA CAA GG) (Sang et al., 1995). The two non-coding chloroplast gene regions were trnH-psbA (trnH forward primer: CGC GCA TGG TGG ATT CAC AAT CC) (Tate and Simpson, 2003), and psbA3 (reverse primer: GTT ATG CAT GAA CGT AAT GCT C) (Sang et al., 1995), and rsp16 (using primers rps16F: AAA CGA TGT GGT ARA AAG CAA C and rps16R: AAC ATC WAT TGC AAS GAT TCG ATA), (Shaw et al., 2005); and the coding region matK using primers KIM 3F (CGT ACA GTA CTT TTG TGT TTA CGA G) -KIM 1R (ACC CAG TCC ATC TGG AAA TCT TGG TTC), (Cuénoud et al., 2002). PCR was performed using the EmeraldAmp PCR Master Mix (Takara Bio Inc., Shiga, Japan) with 0.5 mM of each primer. The PCRs were performed in volumes of 25 μl containing 1 μl of the DNA template of unknown concentration, 12.5 μl 2X EmeraldAmp® PCR Master Mix, 0.5 μl BSA, 0.5 μl of each primer and sterile distilled water. The PCR protocol consisted of an initial 2 min denaturing step at 94 °C; 40 cycles, each comprising 94 °C for 1 min 45 s, 55 °C for 30 s, 72 °C for 2 min; and a final 6 min extension step at 72 °C. All PCRs were performed on a GeneAmp 2700 PCR System (Applied Biosystems, Foster City, CA, USA). Sequencing was conducted by Inqaba Biotechnical Industries. Sequences generated were assembled with CODONCODE ALIGNER v5.4 (Codon Code Corp, Barnstable, MA, USA, http://www.codoncode.com). Each of the four gene regions were handled differently in alignment. Alignment of trnH-psbA and rps16 was unambiguous, because of the absence of indel variation, and all sequences were readily assembled. The MUSCLE function was used for ITS and matK, then checked manually and adjusted where necessary, with gaps positioned to minimize nucleotide mismatches. Gap penalties and mismatch scores were based on the default setting in Codoncode Aligner. Haplotype diversity (h, the probability that two haplotypes chosen at random in a sample are different) and genetic diversity (π, the mean number of differences between all pairs of haplotypes) (Nei and Li, 1979; Nei and Tajima, 1981) for all samples were calculated in DnaSP v5 (Librado and Rozas, 2009). Population genetic structure was investigated for the nuclear and chloroplast data using analyses of molecular variance (AMOVA) in Arlequin 3.5.2.2 to test for significance under the null hypothesis of panmixia with 10 000 permutations. Pairwise calculations of ФST, an analogue of FST, consider haplotype frequency and the extent of differentiation among haplotypes and assume that the mutation rate is negligible compared to the migration rate.
Fig. 1. Top left: Albino propagules of A. marina in Sydney; Top right: Albino propagules of Kandelia obovata in southern China; Bottom left: Albino propagules of Rhizophora apiculata in Palau; Bottom right: adventitious roots on stems of old A. marina trees in an oil-polluted site, Sydney, Australia. Pictures of Kandelia and Rhiziphora are courtesy of K. Krauss.
albino propagules were reported (Table 1), the soil had significant amounts of biogenic hydrocarbons, including PAHs, or a history of chronic oil contamination (Klekowski et al., 1994a,b; Duke and Watkinson, 2002; Paul and Young, 2005). These observations suggest that petroleum hydrocarbons may exacerbate lethal mutations in mangroves, suggesting a cause-effect relationship. Molecular responses to oil stress and effects of oil pollution on mutation in mangroves, however, have not been adequately investigated and are poorly understood (Huang et al., 2014; Guedes et al., 2018). Despite their ecological and economic relevance, mangrove plant sequences are poorly represented in biological databases. The objective of this study was to determine whether there were genetic differences between normal and chlorophyll deficient propagules of A. marina. We employed standard Sanger sequencing and statistical analyses and report mutational differences in nuclear and coding chloroplast DNA between normal and albino propagules of this pioneer mangrove species. As far as we are aware, this is one of a few studies to investigate the genetic effects of oil and provides novel insights on the long term dynamics of oil pollution on mangroves. 2. Materials and methods 2.1. Study site Albino and normal propagules of A. marina, were collected from Badu Mangroves, Sydney Olympic Park, Australia (33° 15′ 24.3″ S; 151° 04′ 48.1” E). This area has a long history of industrial pollution for about 30 years. The entire area, situated on the former mudflat of Homebush Bay, was infilled with industrial waste and refuse and continues to receive road and catchment runoff and is a highly polluted site. The mangrove sediment in this bay is highly contaminated with petroleum hydrocarbons. The concentration of total petroleum hydrocarbons in the sediment ranged from 385 to 554 pg g−1 (Paul and Young, 2005), which is more than 50-times that found in another polluted study site in Queensland, Australia, with albino propagules (Duke and Watkinson, 2002). Propagules of A. marina with green cotyledons are considered ‘normal’. Albino propagules (Fig. 1) were of two types: yellow-orange (with bright yellow cotyledons) and reddishgreen (with reddish-green cotyledons). In total, 30 trees in this oil
3. Results The non-coding chloroplast gene regions (rps16 and trnH-psbA) showed no differences between normal and albino samples and were consequently removed from further analysis. This was probably due to PAHs not affecting gene regions that do not code for any amino acids and are not under selection pressure (Shaw et al., 2005). Results from the genetic analysis indicated mutations in both nuclear (ITS) and coding chloroplast (matK) genes (Tables 2 and 3). 2
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Table 1 Reports of chlorophyll deficient mangrove propagules in different species and various localities. Mangrove species
Location
Pollutant
Reference
Rhizophora mangle
Florida Puerto Rico
oil oil oil oil oil oil oil oil oil oil, pesticides ? oil ? ? ?
Handler and Teas (1983) Handler and Teas (1983) Lowenfeld and Klekowski (1992) Klekowski (1988) Klekowski et al. (1994a),b,c Perry (1998) Duke and Watkinson (2002) Duke and Watkinson (2002) Duke and Watkinson (2002) Paul and Young (2005) Da Silva et al. (1997) Duke and Watkinson (2002) Duke and Watkinson (2002) Duke and Watkinson (2002) Chen et al. (1996)
Hawaii Central America Australia Australia
Rhizophora stylosa Avicennia marina Avicennia germinans
Brazil Australia Australia Central America China
Ceriops Pelliciera rhizophorae Kandelia candel
Nucleotide diversity was higher compared to haplotype diversity for ITS and matK datasets. In the ITS dataset, nucleotide diversity ranged from 0.0007 (normal) to 0.0020 (albino) for the nuclear dataset, whereas the haplotype diversity was higher for albino samples (0.667) compared to normal samples (0.500). There were two unique haplotypes with one base mutation for normal samples and two mutations for albino samples. AMOVA suggested no significant genetic differentiation between normal and albino samples (ФST = 0.26667; p > 0.05); but a high degree of variation within populations (73.33%) compared to variation among populations (26.67%). Sequences were more divergent in the matK (π = 0.0090; h = 0.571) compared to the ITS dataset and separated normal and albino samples into two distinct haplotypes. There were 10 mutational differences between normal and albino samples. AMOVA of the matK dataset suggests highly significant, genetic differentiation between normal and albino samples (ФST = 1.000; p < 0.05) with a high degree of variation among populations (100%).
Table 3 Summary statistics for 18S–26S cistron intergenic spacer (ITS) coding chloroplast DNA (matK) between normal and albino samples of Avicennia marina. π = nucleotide diversity, h = haplotype diversity. Gene region
Type
No. Base pairs
π
h
No. Haplotypes
Mutations
ITS
Normal Albino Combined Normal Albino Combined
670 670 670 633 633 633
0.0007 0.0020 0.0020 0.0000 0.0000 0.0090
0.500 0.667 0.607 0.000 0.000 0.571
2 2 3 1 1 2
1 2 3 0 0 10
MatK
populations and lower their evolutionary potential (Giska et al., 2015). Exposure to pollution may also result in reduced individual fitness and decreased size of wild populations. In addition, when populations have become small, genetic drift may remove variation faster. The genetic diversity is predicted to be low because genetic variation is increasingly lost through genetic drift (Simonsen and Klok, 2010; Hobbs et al., 2013; Giska et al., 2015). We speculate that the low genetic diversity and high haplotype diversity in albino mangrove propagules resulted in genetic drift which supports the genetic erosion hypothesis of van Straalen and Timmermans (2002). We suggest that exposure to chonic contaminants, such as PAHs, may lead to reduced genetic diversity. In a recent study, Arabidopsis thaliana seedlings exposed to the water-soluble fraction of marine fuel MF380 (WSF-MF380) exhibited responses similar to those of heat, hypoxia, osmotic and oxidative stresses (Nardeli et al., 2016). In the mangrove, L. racemosa, oil exposure modulated the expression of genes related to protein homeostasis, photosynthesis, hypoxia and ethylene response (Guedes et al., 2018). In other studies, adverse effects of PAHs on chloroplasts and on photosynthesis have been reported in mangroves (Naidoo et al., 2010; Naidoo and Naidoo, 2016, 2017; 2018). In A. thaliana, PAHs were
4. Discussion Data from this study suggest that PAHs from a chronic oil polluted site resulted in mutations in the mangrove A. marina. Chlorophyll-deficiency in mangrove propagules may be caused either by mutation of genes in the nuclear or chloroplast genome (Klekowski, 1992, Klekowski and Godfrey, 1989). Our data indicated that mutations occurred in both nuclear (ITS) and coding chloroplast (matK) genes. Previously, mutant alleles in albino propagules of Rhizophora were determined by their effects on chloroplast ultrastructure (Klekowski et al., 1994b). Genetic diversity can be influenced by a range of factors including population size, natural selection, mutation rates, gene flow between populations, introgression from hybridisation and historical effects on these factors such as population bottlenecks (Hobbs et al., 2013). Chronic exposure to pollution may decrease genetic diversity of
Table 2 Segregating sites in 18S–26S cistron intergenic spacer (ITS) and coding chloroplast DNA (matK) between normal and albino. samples of Avicennia marina. MatK Type Normal
Albino
Position Consensus N1 N2 N3 N4 A1 A2 A3 A4
ITS 81 C . . . . G . . .
311 G . . . . T . . .
355 A . . . . C . . .
547 A . . . . T . . .
562 A . . . . G . . .
575 A . . . . T T T T
3
579 A . . . . G . . .
581 T . . . . A . . .
590 T . . . . G . . .
597 C . . . . T . . .
191 C . . . . . . T T
525 G . . . . . . A A
625 T C . . . . . . .
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development, severe storms and chronic oil pollution. The more sensitive mangrove species might change in genetic makeup by direct mutation such as the production of albino propagules, while others may change by genetic selection over many generations for greater tolerance to chronic pollution (Duke and Watkinson, 2002). Mangroves subject to chronic oil pollution have relatively lower biodiversity and plants generally exhibit genetic abnormalities (Duke and Watkinson, 2002; Paul and Young, 2005; Naidoo et al., 2010; Naidoo and Naidoo, 2017). Chronic oil pollution leads to decline in ecosystem function and eventual collapse as there is little or no seedling recruitment. In such cases, the benefits and services of mangrove ecosystems will be lost. Shorelines would be subjected to greater erosion and there will be a reduction in estuarine and nearshore fisheries (Barbier, 2016; Friess, 2016).
reported to have caused the modulation of photosynthesis-related genes (Weisman et al., 2010). In A. marina, our results showed greater mutations in the chloroplast dataset compared to the nuclear dataset. This suggests that PAHs may have a greater effect on the chloroplast genome, which ultimately results in chlorophyll-deficiency in mangroves. Photosynthesis is a complex process that includes many potential target reactions, such as light harvesting, carbon fixation, photophosphorylation and electron transport (Wang et al., 2014). The nucleus plays a critical role in maintaining normal function of the chloroplast (Richly and Leister, 2004). The chloroplast, as the site of photosynthesis, requires adaptive changes in the expression levels of nuclear genes to respond to endogenous and environmental stimuli. These changes in gene expression are regulated by a complex signalling network (Wang et al., 2014). Richly and Leister (2004) reported that the nuclear genome encodes about 2090 predicted chloroplast proteins suggesting that the nucleus plays a critical role in the regulation of photosynthesis and environmental perturbations via gene expression. Conclusions about the identity of specific genes mutated in each of the genomes, however, require additional studies with greater sample size and advanced sequencing techniques. Detecting genetic responses to environmental change is difficult in the absence of a priori candidate loci with a large effect on the phenotype of interest.This suggests that genome-wide approaches allow for better insight into population genetic processes occurring across a whole genome (Giska et al., 2015). Next Generation Sequencing (NGS) techniques (Hofker et al., 2014) may be more appropriate in the identification of genes that are directly associated with chlorophyll-deficiency. Whole genome sequencing may be used to determine the mutation frequency for genomes and the genes responsible for coding proteins in photosynthetic reactions. For example, complete sequencing of rbcL in the chloroplast genome is a prime target for genetic engineering to improve photosynthetic efficiency (Parry et al., 2007). With NGS, such as Restriction-site Associated DNA Sequencing (RADSeq), genetic diversity, gene flow and population structure for both the chloroplast and the nuclear genome can be determined. In several locations in the world, a significant positive relationship was reported between the occurrence of albino mangrove propagules and high concentrations of hydrocarbons in the sediment (Klekowski et al., 1994a, b; Duke and Watkinson, 2002; Paul and Young, 2005). In this study, trees of A. marina, bearing albino propagules occurred in sites with high levels of both total hydrocarbon and petroleum-based PAHs (Duke and Watkinson, 2002; Paul and Young, 2005). In oil contaminated A. marina, Bruguiera gymnorrhiza and R. mucronata, most PAHs were retained within roots (96–99%) with little translocation to shoots (1–4%). Dominant PAHs detected in leaves of the three species included phenanthrene, naphthalene, fluorene, acenaphthene, chrysene, benzo[k+b]fluoranthene and anthracene (Naidoo and Naidoo, 2016, 2017, 2018). These PAHs were also present in mangrove sediments in Australia that supported A. marina trees with albino propagules (Duke and Watkinson, 2002). In other studies where oil concentration in the soil was not measured, the presence of albino propagules was associated with prior oil pollution of the habitat (Perry, 1998; Paul and Young, 2005). The albino mutation causative agent occurs over timespans of several decades after oil spills so it is difficult to pinpoint the cause. Moreover, chlorophyll deficient mutants have been reported across a diverse range of mangrove species including Avicennia, Rhizophora, Kandelia, Ceriops and Pelliciera (Table 1, Fig. 1), demonstrating that this phenomenon is widespread. These results have potentially serious implications for coastal management as high mutant densities may be indicative of long term genetic deterioration of mangroves following oil pollution. Additionally, there are only about 70 mangrove species and they occupy a narrow ecological range, constrained mostly between mean sea level and high spring tide (Duke and Watkinson, 2002; Lewis et al., 2011). Mangrove ecosystems are most vulnerable when conditions change rapidly during coastal
5. Conclusions This study demonstrated that chlorophyll deficient propagules of A. marina are caused by a lethal genetic mutation in both nuclear (ITS) and coding chloroplast (matK) genes. There were greater mutations in the chloroplast dataset compared to the nuclear dataset. These data suggest that PAHs have a greater effect on the chloroplast genome compared to that of the nucleus. Albinism will reduce regenerative capacity and represents an additional and unquantified aspect of general deterioration in mangrove habitat and fitness. Our data, together with others, provide compelling evidence that petroleum hydrocarbons are responsible for lethal mutations in mangroves. Several significant correlations in different parts of the world and in different mangrove species are compatible with a cause-effect relationship. Our findings raise environmental management issues concerning the persistence of petroleum hydrocarbons in intertidal environments and their harmful impacts. Improving the understanding of the molecular basis of oil pollution will provide tools for biomonitoring and bioremediation after spills. Author contributions DV performed the genetic analyses, SP provided the albino propagules and GN initiated the study. DV and GN wrote the manuscript while all contributed to the interpretation and discussion of the results. Acknowledgements This research was supported by the National Research Foundation (grant number 93560 to G Naidoo) and the University of KwaZuluNatal. The authors are grateful to A Rajh for technical assistance. References Barbier, E.B., 2016. The protective service of mangrove ecosystems: a review of valuation methods. Mar. Pollut. Bull. 109, 676–681. Chen, X.Y., Lin, P., Lin, Y.M., 1996. Mating systems and spontaneous mutation rates for chlorophyll-deficiency in populations of the mangrove Kandelia candel. Hereditas 125, 47–52. Corredor, J.E., Morell, J.M., Klekowski Jr., E.J., Lowenfeld, R., 1995. Mangrove genetics. III. Pigment fingerprints of chlorophyll deficient mutants. Int. J. Plant Sci. 156, 55–60. Cuénoud, P., Savolainen, V., Chatrou, L.W., Powell, M., Grayer, R.J., Chase, M.W., 2002. Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. Am. J. Bot. 89 (1), 132–144. Da Silva, E.M., Peso-Aguiar, M.C., Navarro, M de F.T., Chastinet, C.de B.E.A., 1997. Impact of petroleum pollution on aquatic coastal ecosystems in Brazil. Environ. Toxicol. Chem. 16 (1), 112–118. Duke, N.C., Watkinson, A.J., 2002. Chlorophyll-deficient propagules of Avicennia marina and apparent longer term deterioration of mangrove fitness in oil-polluted sediments. Mar. Pollut. Bull. 44, 1269–1276. Friess, D.A., 2016. Ecosystem Services and Disservices of Mangrove Forests: Insights from Historical Colonial Observations, vol. 183 Forests. Giska, I., Babik, W., van Gestel, C.A., van Straalen, N.M., Laskowski, R., 2015. Genomewide genetic diversity of rove beetle populations along a metal pollution gradient. Ecotoxicol. Environ. Saf. 119, 98–105.
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