Heredity (2017) 118, 31–41 & 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0018-067X/17 www.nature.com/hdy ORIGINAL ARTICLE Evolution of nickel hyperaccumulation and serpentine adaptation in the Alyssum serpyllifolium species complex MK Sobczyk1, JAC Smith1, AJ Pollard2 and DA Filatov1 Metal hyperaccumulation is an uncommon but highly distinctive adaptation found in certain plants that can grow on metalliferous soils. Here we review what is known about evolution of metal hyperaccumulation in plants and describe a population-genetic analysis of the Alyssum serpyllifolium (Brassicaceae) species complex that includes populations of nickel- hyperaccumulating as well as non-accumulating plants growing on serpentine (S) and non-serpentine (NS) soils, respectively. To test whether the S and NS populations belong to the same or separate closely related species, we analysed genetic variation within and between four S and four NS populations from across the Iberian peninsula. Based on microsatellites, genetic variation was similar in S and NS populations (average Ho = 0.48). The populations were significantly differentiated from each other (overall FST = 0.23), and the degree of differentiation between S and NS populations was similar to that within these two groups. However, high S versus NS differentiation was observed in DNA polymorphism of two genes putatively involved in adaptation to serpentine environments, IREG1 and NRAMP4, whereas no such differentiation was found in a gene (ASIL1) not expected to play a specific role in ecological adaptation in A. serpyllifolium. These results indicate that S and NS populations belong to the same species and that nickel hyperaccumulation in A. serpyllifolium appears to represent a case of adaptation to growth on serpentine soils. Further functional and evolutionary genetic work in this system has the potential to significantly advance our understanding of the evolution of metal hyperaccumulation in plants. Heredity (2017) 118, 31–41; doi:10.1038/hdy.2016.93; published online 26 October 2016 INTRODUCTION Of various hypotheses proposed to explain the possible adaptive Metal hyperaccumulation in plants advantage conferred by hyperaccumulation of metals (Boyd and One of the most extraordinary adaptations known in the plant Martens, 1992), only the herbivore/pathogen ‘elemental defence’ kingdom is the ability of certain plants to hyperaccumulate trace hypothesis has gathered plentiful supporting experimental evidence elements in their above-ground biomass. This trait is present in only (Boyd, 2004, 2007). There have been many reports of decreased ∼ 500 species, representing o0.2% of all angiosperm species (Reeves herbivory or reduced pathogen infection on plants hyperaccumulating and Baker, 2000; Verbruggen et al., 2009; Krämer, 2010; Van der Ent metals (Poschenrieder et al., 2006; Boyd, 2007; Fones et al.,2010; et al., 2013). In contrast to metal excluders whose strategy is to control Rascio and Navari-Izzo, 2011). However, in some studies no protective the uptake of metals into the root and prevent metal translocation to effect of metal hyperaccumulation against herbivory was observed aerial organs, hyperaccumulators accumulate metals in the shoot to (Noret et al., 2007), and designing trials to demonstrate such an effect levels toxic to most other plants (Baker, 1981; Baker and Brooks, 1989; in the field is particularly challenging, and hence further work is Baker et al., 2000; Pollard et al., 2002; Krämer, 2010; Rascio and required to substantiate the selective advantage offered by metal Navari-Izzo, 2011). This is remarkable as the photosynthetic apparatus hyperaccumulation. Nonetheless, it remains broadly true that hyper- is one of the major targets of metal phytotoxicity, typically resulting in accumulator species show a very high degree of metal tolerance severe symptoms such as chlorosis and necrosis, wilting, abnormal (otherwise the accumulation of such exceptional concentrations of development and reduced growth (Pandey and Sharma, 2002; metals in the shoot would be suicidal), and that the degree of metal Rahman et al., 2005; Marschner and Marschner, 2012). These toxic tolerance of different species or populations tends to be quite closely effects are a product of numerous harmful interactions at the cellular correlated with the metal content of the natural substrates on which level (Haydon and Cobbett, 2007), including nonspecific binding of they grow (Antonovics et al., 1971; Roosens et al., 2003; de la Fuente metals to enzyme functional groups and displacement of other metals et al., 2007; Pollard et al.,2014). from their binding sites, generation of reactive oxygen species by Two broad hypotheses have been considered in the literature redox-active metals that can lead to disruption of the electron- regarding the origin and spread of metallicolous populations, that is, transport chain (Qadir et al., 2004), lipid peroxidation and subsequent those adapted to soils containing an elevated concentration of a impairment of membrane integrity (Pandolfini et al.,1992;Roset al., particular metal (Pauwels et al., 2005). One is based on the observation 1992; Gonnelli et al., 2001; Haydon and Cobbett, 2007; Krämer, 2010; that populations inhabiting metalliferous outcrops are often separated Hanikenne and Nouet, 2011). by large geographic distances; this would limit dispersal and genetic 1Department of Plant Sciences, University of Oxford, Oxford, UK and 2Department of Biology, Furman University, Greenville, SC, USA Correspondence: DA Filatov, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK. E-mail: [email protected] Received 27 February 2016; revised 7 August 2016; accepted 12 August 2016; published online 26 October 2016 Evolution of metal hyperaccumulation in plants MK Sobczyk et al 32 exchange between metallicolous populations, and instead would populations of A. halleri (Meyer et al., 2009). Large variation in the favour evolution of locally adapted metallicolous populations from degree of zinc tolerance within non-metallicolous populations and nearby non-metalliferous sites (Schat et al., 1996) driven by ecological individuals can then be explained by either local gene flow from speciation (Rundle and Nosil, 2005). The other hypothesis proposes a metallicolous to non-metallicolous populations, or as a result of single origin of a genetic adaptation to metalliferous substrates, its ancestral standing genetic variation present within the non- spread across outlying metalliferous sites and subsequent differentia- metallicolous populations, that may have been exploited as the initial tion between more recently established metallicolous populations basis for metal tolerance in metallicolous populations. because of genetic drift. These alternative views resonate with earlier debates as to whether local populations of metal-tolerant plants Nickel hyperaccumulation occurring on metalliferous outcrops represent ‘neoendemics’ or Metal hyperaccumulator plants are known to accumulate zinc, ‘palaeoendemics’, respectively (equivalent to the ‘insular’ and cadmium, copper, cobalt, manganese and the metalloids arsenic, ‘depleted’ species of Stebbins (1942), as discussed by Kazakou et al. thallium and selenium, but the largest number (~80%) of hyper- (2008)). Correspondingly, non-metallicolous populations of such accumulator species described are known to accumulate nickel (Reeves species could be either ancestral, as in the first scenario, or locally and Baker, 2000; Verbruggen et al., 2009; Krämer, 2010), possibly derived from metallicolous populations, as in the second. because of its prevalence in ultramafic rocks across the continents (Van der Ent et al.,2013).Ultramafic substrates are derived from Evolution of metal tolerance in plants igneous rocks with a low silica content but rich in maficmineralssuch The question of whether metal tolerance is a constitutive trait across as magnesium, iron and nickel. Weathering of ultramafic bedrock all populations of a species, or the degree to which ecotypic creates serpentine soils with distinctive physical and chemical char- differentiation has occurred in response to different substrates, has acteristics, including particularly high contents of nickel, cobalt and been one of the most important issues underpinning hypotheses chromium, high Mg/Ca ratio and low levels of the essential macro- concerning the evolutionary origins of this trait. Two of the most nutrients nitrogen, phosphorus and potassium (Brooks, 1987; intensively studied species of hyperaccumulator plants, Arabidopsis Kazakou et al., 2008). Most types of soil show nickel concentrations ’ halleri (L.) O Kane & Al-Shehbaz (formerly Cardaminopsis halleri (L.) typically between 7 and 50 mg kg − 1, whereas in serpentine soils the Hayek) and Noccaea caerulescens (J Presl and C Presl) FK Mey. nickel content usually ranges from 700 to 8000 mg kg − 1 (Reeves, (formerly Thlaspi caerulescens J Presl and C Presl), display a basal, 1992; Reeves and Baker, 2000). This characteristic geochemistry, constitutive level of zinc tolerance and hyperaccumulation in both combined with a characteristically thin soil cover and granular texture, metallicolous and non-metallicolous populations (Meerts and Van poor water-holding capacity, ready erosion and exposure to high light Isacker, 1997; Bert et al., 2000, 2002; Escarré et al., 2000; Frérot et al., intensity, makes serpentine soils a notoriously difficult environment 2003; Pauwels et al.,