Herrera et al. – 1 1 Transgenerational epigenetics: inheritance of global cytosine methylation and 2 methylation-related epigenetic markers in the shrub Lavandula latifolia 1 3 4 Carlos M. Herrera2,4, Conchita Alonso2, Mónica Medrano2, Ricardo Pérez3 and Pilar 5 Bazaga2 6 7 8 1 Manuscript received _______; revision accepted _______. 9 2 Estación Biológica de Doñana, Consejo Superior de Investigaciones Científicas 10 (CSIC), Avenida Américo Vespucio 26, 41092 Sevilla, Spain 11 3 Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de La 12 Cartuja, Consejo Superior de Investigaciones Científicas (CSIC)–Universidad de 13 Sevilla, Sevilla, Spain 14 4 Author for correspondence (e-mail: [email protected]) 15 16 17 18 Running head: Transgenerational epigenetics in Lavandula Herrera et al. – 2 19 Abstract 20 PREMISE OF THE STUDY: The ecological and evolutionary significance of natural 21 epigenetic variation (i.e., not based on DNA sequence variants) variation will depend 22 critically on whether epigenetic states are transmitted from parents to offspring, but 23 little is known on epigenetic inheritance in non-model plants. 24 METHODS: We present a quantitative analysis of transgenerational transmission of 25 global DNA cytosine methylation (= proportion of all genomic cytosines that are 26 methylated) and individual epigenetic markers (= methylation status of anonymous 27 MSAP markers) in the shrub Lavandula latifolia. Methods based on parent-offspring 28 correlations and parental variance component estimation were applied to epigenetic 29 features of field-growing plants (‘maternal parents’) and greenhouse-grown progenies. 30 Transmission of genetic markers (AFLP) was also assessed for reference. 31 KEY RESULTS: Maternal parents differed significantly in global DNA cytosine 32 methylation (range = 21.7-36.7%). Greenhouse-grown maternal families differed 33 significantly in global methylation, and their differences were significantly related to 34 maternal origin. MSAP markers exhibited significant transgenerational transmission, as 35 denoted by significant maternal variance component of marker scores in greenhouse 36 families and significant mother-offspring correlations of marker scores. 37 CONCLUSIONS: Although transmission-related measurements for global methylation and 38 MSAP markers were quantitatively lower than those for AFLP markers taken as 39 reference, this study has revealed extensive transgenerational transmission of genome- 40 wide global cytosine methylation and anonymous epigenetic markers in L. latifolia. 41 Similarity of results for global cytosine methylation and epigenetic markers lends 42 robustness to this conclusion, and stresses the value of considering both types of 43 information in epigenetic studies of non-model plants. Herrera et al. – 3 44 KEY WORDS: amplified fragment length polymorphism (AFLP); epigenetic variation; 45 global cytosine methylation; inheritance; Lamiaceae; methylation-sensitive amplified 46 polymorphism (MSAP). 47 48 Natural epigenetic variation (i.e., not based on DNA sequence variants) has been 49 recently related to phenotypic and functional diversity in plants (Medrano et al., 2014; Ci 50 et al., 2015; Hu et al., 2015; Kooke et al., 2015; Wilschut et al., 2016). The ecological and 51 evolutionary significance of such epigenetic variation will depend critically on whether 52 epigenetic states that are related to organismal features are transmitted from parents to 53 offspring (i.e., epigenetic states are not completely reset between generations; Jablonka 54 and Raz, 2009; Jablonka and Lamb, 2015; Uller et al., 2015; Kronholm and Collins, 55 2016), a situation that has been reported frequently by recent investigations (Cortijo et al., 56 2014; Miska and Ferguson-Smith, 2016; Quadrana and Colot, 2016; Zheng et al., 2017). 57 In contrast to developmental epigenetics, transgenerational epigenetics involves heritable 58 changes in DNA methylation (see Quadrana and Colot, 2016, for review). Although it has 59 been sometimes advocated that the concept of transgenerational epigenetic inheritance 60 should be restricted to cases where epigenetic changes remain unaltered for a number of 61 successive generations (e.g., Pecinka and Mittelsten Scheid, 2012), recent usage tends to 62 be less restrictive and the concept can also be applied in situations where epigenetic states 63 are not reset between two consecutive generations (Quadrana and Colot, 2016). We adhere 64 to this more liberal usage in this paper. 65 Available evidence of transgenerational epigenetic transmission in plants refers nearly 66 exclusively to a handful of crop and model species with detailed genomic information 67 available (see reviews in Hauser et al., 2011; Quadrana and Colot, 2016). Remarkably few 68 attempts have been done so far at quantitatively assessing epigenetic inheritance in non- Herrera et al. – 4 69 model species without a reference genome (but see Herrera et al., 2013; Avramidou et al., 70 2015). Broadening the phylogenetic scope of studies on transgenerational epigenetics 71 (sensu Quadrana and Colot, 2016) beyond the narrow domain of model plants will help to 72 evaluate the generality of the phenomenon and contribute to a better understanding of the 73 importance of epigenetic mechanisms in plants, as recently emphasized by Rensing (2017) 74 in a related context. DNA cytosine methylation is a key mechanism for epigenetic 75 regulation in plants (Grant-Downton and Dickinson, 2005, 2006; Gehring and Henikoff, 76 2007) whose magnitude, genome-wide patterns and functional aspects have a significant 77 phylogenetic component and vary across plant lineages (Zemach et al., 2010; Alonso et 78 al., 2016; Vidalis et al., 2016). Phylogenetically restricted and/or biased species sampling 79 might therefore lead to distorted or biased views on the prevalence of epigenetic 80 inheritance in plants as a whole. 81 We present in this paper a quantitative analysis of transgenerational transmission of 82 global DNA cytosine methylation (= proportion of all genomic cytosines that are 83 methylated; also sometimes termed “bulk methylation” or “genome-wide methylation”) 84 and individual epigenetic markers (= methylation status of anonymous genomic markers) 85 in the shrub Lavandula latifolia. Although data on global cytosine methylation are not 86 informative on the genomic positions where methylation occurs, its analysis is helpful to 87 evaluate the overall importance of this epigenetic mark in non-model organisms without 88 detailed genomic information (Rozhon et al., 2008; Alonso et al., 2016). Furthermore, 89 variation in global cytosine methylation is correlated with changes in the methylation 90 status of specific genic and intergenic regions, and is consequential for gene expression, 91 genomic instability (Steward et al., 2002; Baubec et al., 2009; Bonchev and Parisod, 2013; 92 Vidalis et al., 2016) and organismal features (Sano et al., 1990; Tatra et al., 2000; Kondo 93 et al., 2006). Anonymous epigenetic markers, on the other hand, provide information on Herrera et al. – 5 94 the genome-wide patterns of cytosine methylation, and their variation across individuals or 95 populations is often related to differences in functional traits (Medrano et al., 2014; 96 Herrera et al., 2017). Taken together, global cytosine methylation and anonymous 97 epigenetic markers thus provide complementary views of genome-wide epigenetic 98 characteristics of individuals, and have been often applied in ecological epigenetics studies 99 of non-model plants (Schulz et al., 2013; Alonso et al., 2014, 2016, 2017; Wilschut et al., 100 2016). 101 Epigenetic characteristics will be treated here as individual phenotypic traits, and 102 standard methods commonly used in quantitative genetics studies of trait heritability, 103 namely parent-offspring correlations and parental variance component estimation in the 104 progeny (Roff, 1997; Lynch and Walsh, 1998), will be applied. Some peculiarities of 105 epigenetic variation, notably its potential lability in response to the environment, call for 106 extended quantitative genetic models and more complex experimental and analytical 107 designs (Gorelick, 2005; Gorelick and Laubichler, 2008). For this reason, we will not 108 attempt here to relate formally our quantitative results to the notion of ‘heritability’. In 109 addition to documenting extensive inheritance of the epigenetic features considered in 110 Lavandula latifolia, the present study also aims to illustrate a simple analytical approach 111 that can be easily implemented experimentally to test and quantify the transgenerational 112 transmission of anonymous epigenetic markers in non-model plants. In order to obtain a 113 reference level against which to compare the transmission of molecular epigenetic 114 characteristics, we will also concurrently assess transmission of anonymous genetic 115 markers using the same experimental and analytical methods. 116 MATERIALS AND METHODS 117 Study species—Lavandula latifolia (Lamiaceae) is a long-lived, low evergreen shrub 118 characteristic of clearings and well-lit undergrowth in open woodlands of the eastern Herrera et al. – 6 119 Iberian Peninsula at 1000–1600 m a.s.l. Flowering takes place during July–October. 120 Flowers are hermaphrodite, self-compatible, and insect pollinated. The species reproduces 121 exclusively by seeds, which are small (1 mg), lack special mechanisms for dispersal, and 122 ripen by the end of summer (see, e.g., Herrera and