Identification of exotic genetic components and DNA methylation pattern analysis of three cotton introgression lines from <Emphasis Type="Italic">Gossypium bickii</Emphasis>

The impact of alien DNA fragments on plant genome has been studied in many species. However, little is known about the introgression lines of Gossypium. To study the consequences of introgression in Gossypium, we investigated ∼2000 genomic and ∼800 epigenetic sites in three typical cotton introgression lines, as well as their cultivar (Gossypium hirsutum) and wild parents (Gossypium bickii), by amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP). The results demonstrate that an average of 0.5% of exotic DNA segments from wild cotton is transmitted into the genome of each introgression line, with the addition of other forms of genetic variation. In total, an average of 0.7% of genetic variation sites is identified in introgression lines. Simultaneously, the overall cytosine methylation level in each introgression line is very close to that of the upland cotton parent (an average of 22.6%). Further dividing patterns reveal that both hypomethylation and hypermethylation occurred in introgression lines in comparison with the upland cotton parent. Sequencing of nine methylation polymorphism fragments showed that most (7 of 9) of the methylation alternations occurred in the noncoding sequences. The molecular evidence of introgression from wild cotton into introgression lines in our study is identified by AFLP. Moreover, the causes of petal variation in introgression lines are discussed.     

Genetic and epigenetic status of triple exotic consanguinity cotton introgression lines

Introgression lines are some of the most important germplasm for breeding applications and other research conducted on cotton crops. The DNA methylation level among 10 introgression lines of cotton (Gossypium hirsutum) and three exotic parental species (G. arboreum, G. thurberi and G. barbadense) were assessed by methylation-sensitive amplified polymorphism (MSAP) technology. The methylation level in the introgression lines ranged from 33.3 to 51.5%. However, the lines PD0111 and PD0113 had the lowest methylation level (34.6 and 33.3%, respectively) due to demethylation of most non-coding sequences. Amplified fragment length polymorphism (AFLP) was used to evaluate the genetic polymorphism in the cotton introgression lines. A high degree of polymorphism was observed in all introgression lines (mean 47.2%) based on AFLP and MSAP analyses. This confirmed the effects of genetic improvement on cotton introgression lines. The low methylation varieties, PD0111 and PD0113 (introgression lines), clustered outside of the introgression lines based on MSAP data, which was incongruent with an AFLP-based dendrogram. This phenomenon could be caused by environmental changes or introgression of exotic DNA fragments.     

Extent and pattern of DNA methylation alteration in rice lines derived from introgressive hybridization of rice and Zizania latifolia Griseb

We have reported previously that introgression by Zizania latifolia resulted in extensive DNA methylation changes in the recipient rice genome, as detected by a set of pre-selected DNA segments. In this study, using the methylation-sensitive amplified polymorphism (MSAP) method, we globally assessed the extent and pattern of cytosine methylation alterations in three typical introgression lines relative to their rice parent at ∼2,700 unbiased genomic loci each representing a recognition site cleaved by one or both of the isoschizomers, HpaII/MspI. Based on differential digestion by the isoschizomers, it is estimated that 15.9% of CCGG sites are either fully methylated at the internal Cs and/or hemi-methylated at the external Cs in the rice parental cultivar Matsumae. In comparison, a statistically significant increase in the overall level of both methylation types was detected in all three studied introgression lines (19.2, 18.6, 19.6%, respectively). Based on comparisons of MSAP profiles between the isoschizomers within the rice parent and between parent and the introgression lines, four major groups of MSAP banding patterns are recognized, which can be further divided into various subgroups as a result of inheritance of, or variation in, parental methylation patterns. The altered methylation patterns include hyper- and hypomethylation changes, as well as inter-conversion of hemi- to full-methylation, or vice versa, at the relevant CCGG site(s). Most alterations revealed by MSAP in low-copy loci can be validated by DNA gel blot analysis. The changed methylation patterns are uniform among randomly selected individuals for a given introgression line within or among selfed generations. Sequencing on 31 isolated fragments that showed different changing patterns in the introgression line(s) allowed their mapping onto variable regions on one or more of the 12 rice chromosomes. These segments include protein-coding genes, transposon/retrotransposons and sequences with no homology. Possible causes for the introgression-induced methylation changes and their implications for genome evolution and crop breeding are discussed.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Z. Y. Dong and Y. M. Wang have equally contributed to the work.     

红豆杉脱分化过程中的遗传和表观遗传变异

采用扩增片段长度多态性（AFLP）和甲基化敏感扩增多态性（MSAP）技术分析红豆杉脱分化前后基因组DNA和DNA甲基化状态的变化。选用32个AFLP引物组合从红豆杉植株及其愈伤组织分别扩增出1834个片段,无多态性片段产生。这说明红豆杉植株在诱导形成愈伤组织的过程中基因组DNA保持高度的遗传稳定性。另用32个MSAP引物组合从红豆杉植株及其愈伤组织分别扩增出1197个片段,总扩增位点的甲基化水平由脱分化前的12．4％上升为16．2％,表明红豆杉在脱分化过程中的某些位点发生了甲基化。红豆杉脱分化前后的DNA甲基化模式也存在较大差异,说明DNA甲基化对愈伤组织形成有调控作用。     

Genomic Change,Retrotransposon Mobilization and Extensive Cytosine Methylation Alteration in Brassica napus Introgressions from Two Intertribal Hybridizations

Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP), sequence-specific amplification polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ∼10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4–39.8%) and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed.     

Analysis of DNA methylation polymorphism in a set of stable chromosome translocation lines

Interspecific hybridization is associated with the origin of novel traits and confers increased vigor compared with the parent lines, although its molecular basis is poorly understood. We report here the identification of genetic and epigenetic changes in a set of wheat–rye translocation lines (R59, R57, and R25) which exhibited novel heritable morphological characteristics compared with the parent lines (MY11 and L155). Genome in situ hybridization and amplified fragment length polymorphism analyses revealed no obvious variations in the primary structure of the genome in different translocation lines, with the exception of the same 1RS chromosome translocation. Global assessment of the extent and pattern of cytosine methylation alterations by methylation-sensitive amplified polymorphism (MSAP) analyses revealed differences in the extent of genomic DNA methylation between the rye and wheat parent lines. Fully-methylated sites were significantly increased and hemi-methylated sites were markedly decreased in the genome of translocation lines compared with the wheat parental cultivar MY11. Comparisons of different MSAP patterns revealed both monomorphic and polymorphic sites between translocation lines and wheat parents. Sequencing of 44 isolated fragments that showed methylation alterations indicated that cellular genes and especially transposable elements were targets for methylation alterations in translocation lines. The present study provides further understanding of the rules governing the distribution and existence of DNA methylation variations induced in the wheat genome during alien germplasm introduction. Furthermore, our study provides insights into the relationship between DNA methylation and hybrid vigor as well as a theoretical basis for further fundamental research and breeding application.     

Extensive de Novo genomic variation in rice induced by introgression from wild rice (Zizania latifolia Griseb.)

To study the possible impact of alien introgression on a recipient plant genome, we examined >6000 unbiased genomic loci of three stable rice recombinant inbred lines (RILs) derived from intergeneric hybridization between rice (cv. Matsumae) and a wild relative (Zizania latifolia Griseb.) followed by successive selfing. Results from amplified fragment length polymorphism (AFLP) analysis showed that, whereas the introgressed Zizania DNA comprised <0.1% of the genome content in the RILs, extensive and genome-wide de novo variations occurred in up to 30% of the analyzed loci for all three lines studied. The AFLP-detected changes were validated by DNA gel-blot hybridization and/or sequence analysis of genomic loci corresponding to a subset of the differentiating AFLP fragments. A BLAST analysis revealed that the genomic variations occurred in diverse sequences, including protein-coding genes, transposable elements, and sequences of unknown functions. Pairwise sequence comparison of selected loci between a RIL and its rice parent showed that the variations represented either base substitutions or small insertion/deletions. Genome variations were detected in all 12 rice chromosomes, although their distribution was uneven both among and within chromosomes. Taken together, our results imply that even cryptic alien introgression can be highly mutagenic to a recipient plant genome.     

Comparative analyses of genetic/epigenetic diversities and structures in a wild barley species (Hordeum brevisubulatum) using MSAP, SSAP and AFLP

We analyzed genetic diversity and population genetic structure of four artificial populations of wild barley (Hordeum brevisubulatum); 96 plants collected from the Songnen Prairie in northeastern China were analyzed using amplified fragment length polymorphism (AFLP), specific-sequence amplified polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) markers. Indices of (epi-)genetic diversity, (epi-)genetic distance, gene flow, genotype frequency, cluster analysis, PCA analysis and AMOVA analysis generated from MSAP, AFLP and SSAP markers had the same trend. We found a high level of correlation in the artificial populations between MSAP, SSAP and AFLP markers by the Mantel test (r > 0.8). This is incongruent with previous findings showing that there is virtually no correlation between DNA methylation polymorphism and classical genetic variation; the high level of genetic polymorphism could be a result of epigenetic regulation. We compared our results with data from natural populations. The population diversity of the artificial populations was lower. However, different from what was found using AFLP and SSAP, based on MSAP results the methylation polymorphism of the artificial populations was not significantly reduced. This leads us to suggest that the DNA methylation pattern change in H. brevisubulatum populations is not only related to DNA sequence variation, but is also regulated by other controlling systems.     

Molecular diversity, genomic constitution, and QTL mapping of fiber quality by mapped SSRs in introgression lines derived from Gossypium hirsutum?×?G. darwinii Watt

Because the genetic basis of current upland cotton cultivars is narrow, exploring new germplasm resources and discovering novel alleles relevant to important agronomic traits have become two of the most important themes in the field of cotton research. In this study, G. darwinii Watt, a wild cotton species, was crossed with four upland cotton cultivars with desirable traits. A total of 105 introgression lines (ILs) were successfully obtained. By using 310 mapped SSRs evenly distributed across the interspecific linkage map of G. hirsutum?×?G. barbadense, these 105 ILs and their corresponding parents were analyzed. A total of 278 polymorphic loci were detected among the 105 ILs, and the average length of introgression segments accumulated to 333.5?cM, accounting for 6.7?% of the whole genome. These lines included many variations. However, high similarity coefficients existed between lines, even between those derived from different parents. Finally, all the ILs and their upland cotton parents were used for association mapping of fiber quality in three environments. A total of 40 SSRs were found to be associated with five fiber quality indexes (P?相似文献   

Cleaved AFLP (cAFLP), a modified amplified fragment length polymorphism analysis for cotton

In certain plant species including cotton (Gossypium hirsutum L. or Gossypium barbadense L.), the level of amplified fragment length polymorphism (AFLP) is relatively low, limiting its utilization in the development of genome-wide linkage maps. We propose the use of frequent restriction enzymes in combination with AFLP to cleave the AFLP fragments, called cleaved AFLP analysis (cAFLP). Using four Upland cotton genotypes (G. hirsutum) and three Pima cotton (G. barbadense), we demonstrated that cAFLP generated 67% and 132% more polymorphic markers than AFLP in Upland and Pima cotton, respectively. This resulted in 15.5 and 25.5 polymorphic cAFLP markers per AFLP primer combination, as compared to 9.1 and 11.0 polymorphic AFLP. The cAFLP-based genetic similarity (GS) is generally lower than the AFLP-based GS, even though both marker systems are overall congruent. In some cases, cAFLP can better resolve genetic relationships between genotypes, rendering a higher discriminatory power. Given the high-resolution power of capillary-based DNA sequencing system, we further propose that AFLP and cAFLP amplicons from the same primer combination can be pooled as one sample before electrophoresis. The combination produced an average of 18.5 and 31.0 polymorphic markers per primer pair in Upland and Pima cotton, respectively. Using several restriction enzyme combinations before pre-selective amplification in combination with various frequent 4 bp-cutters or 6 bp-cutters after selective amplification, the pooled AFLP and cAFLP will provide unlimited number of polymorphic markers for genome-wide mapping and fingerprinting.     

A genetic map of an Australian wild Gossypium C genome and assignment of homoeologies with tetraploid cultivated cotton

Genetic diversity for traits such as fibre quality or disease resistance to microorganisms is limited in the elite cotton germplasm; consequently, cotton breeders are looking for novel alleles in the secondary or even in the tertiary gene pools. The wild Australian Gossypium species (tertiary gene pool) represent an alternative source of novel alleles. However, to use these species efficiently, enabling tools are required. Chromosome-specific molecular markers are particularly useful tools to track the transmission of this exotic genetic material into the cultivated cotton during introgression. In this study, we report the construction of a genetic linkage map of the Australian wild C-genome species Gossypium sturtianum. The map, based on an F(2) population of 114 individuals, contains 291 AFLP loci. The map spans 1697 cM with an average distance of 5.8 cM between markers. To associate C-genome chromosomes with the A and D subgenomes of cultivated cotton, 29 SSR and RFLP-STS markers were assigned to chromosomes using cultivated cotton mapped marker information. Polymorphisms were revealed by 51 AFLP primer combinations and 38 RFLP-STS and 115 SSR cotton mapped markers. The utility of transferring RFLP-STS and SSR cotton mapped markers to other Gossypium species shows the usefulness of a comparative approach as a source of markers and for aligning the genetic map of G. sturtianum with the cultivated species in the future. This also indicates that the overall structure of the G. sturtianum linkage groups is similar to that of the A and D subgenomes of cotton at the gross structural level. Applications of the map for the Australia wild C-genome species and cotton breeding are discussed.     

Genetic diversity and methylation polymorphism analysis of Chrysanthemum nankingense

The diploid species Chrysanthemum nankingense (Anthemideae, Asteraceae) is closely related to the commercially important hexaploid ornamental species Chrysanthemum morifolium and is well adapted to poor environments. In this study, phenotypic variants of C. nankingense were first identified by morphological traits. Using EST-SSR (simple sequence repeat) analysis, we detected some absent EST-SSRs. The percentage of AFLP (amplified fragment length polymorphism) polymorphic fragments was 78.2%, indicating high genetic diversity. To evaluate the genome methylation level and methylation polymorphism, we used the MSAP (methylation-sensitive amplification polymorphism) technique to analyze the 30 C. nankingense lines. The total DNA methylation level ranged from 54.6% to 62.6%. Most of the MSAP-methylated fragments (97%) were polymorphic in the lines. The U-values associated with hemi-methylation were larger than those associated with full methylation in four of the 30 lines, and six individual values were statistically significant (U > 1.96). The high genomic diversity as well as the high methylation polymorphism may be responsible for the morphological polymorphism. There was no significant correlation between the phenotypic and genetic diversity among the lines.     

Genetic structure in cultivated and wild carrots (Daucus carota L.) revealed by AFLP analysis

Genetic variation within and among five Danish populations of wild carrot and five cultivated varieties was investigated using amplified fragment length polymorphism (AFLP). Ten AFLP primer combinations produced 116 polymorphic bands. Based on the marker data an UPGMA-cluster analysis and principal component analysis (PCA) separated the Daucus collections into three groups, consisting of the wild populations, the old varieties, and the recently bred varieties. The genetic distance between the wild populations reflected the physical distance between collection sites. Analysis of genetic diversity showed that the old varieties released between 1974 and 1976 were more heterogeneous than the newly developed F₁ hybrid varieties. The analysis of molecular variation (AMOVA) showed that the major part of the genetic variation in the plant material was found within populations/varieties. The presence of markers specific to the cultivated carrot makes it possible to detect introgression from cultivated to wild types. Received: 6 October 1999 / Accepted: 4 November 1999     

The use of multiple alien chromosome addition aneuploids facilitates genetic linkage mapping of the Gossypium G genome.

Primary germplasm pools represent the most accessible source of new alleles for crop improvement, but not all effective alleles are available in the primary germplasm pool, and breeders must sometimes confront the difficulties of introgressing genes from the secondary and tertiary germplasm pools in cotton by using synthetic polyploids as introgression bridges. Two parental Gossypium nelsonii x Gossypium australe AFLP genetic linkage maps were used to identify G genome chromosome-specific molecular markers, which in turn were used to track the fidelity and frequency of G. australe chromosome transmission in a Gossypium hirsutum x G. australe hexaploid bridging family. Conversely, when homoeologous recombination is low, first generation aneuploids are useful adjuncts to genetic linkage mapping. Although locus ordering was not possible, the distribution of AFLP markers among 18 multiple chromosome addition aneuploids identified mapping errors among the G. australe and G. nelsonii linkage groups and assigned non-segregating G. australe AFLPs to linkage groups. Four putatively recombined G. australe chromosomes were identified in 5 of the 18 aneuploids. The G. australe and G. nelsonii genetic linkage maps presented here represent the first AFLP genetic linkage maps for the Gossypium G genome.     

Development of Oryza rufipogon and O. sativa Introgression Lines and Assessment for Yield-related Quantitative Trait Loci

Introgression lines population was effectively used in mapping quantitative trait loci （QTLs）, identifying favorable genes, discovering hidden genetic variation, evaluating the action or interaction of QTLs in multiple conditions and providing the favorable experimental materials for plant breeding and genetic research. In this study, an advanced backcross and consecutive selfing strategy was used to develop introgression lines （ILs）, which derived from an accession of Oryza rufipogon Griff. collected from Yuanjiang County, Yunnan Province of China, as the donor, and an elite indica cultivar Teqing （O. sativa L.）, as the recipient. Introgression segments from O. rufipogon were screened using 179 polymorphic simple sequence repeats （SSR） markers in the genome of each IL. Introgressed segments carried by the introgression lines population contained 120 ILs covering the whole O. rufipogon genome. The mean number of homozygous O. rufipogon segments per introgression line was about 3.88. The average length of introgressed segments was approximate 25.5 cM, and about 20.8% of these segments had sizes less than 10 cM. The genome of each IL harbored the chromosomal fragments of O. rufipogon ranging from 0.54% to 23.7%, with an overall average of 5.79%. At each locus, the ratio of substitution of O. rufipogon alleles had a range of 1.67-9.33, with an average of 5.50. A wide range of alterations in morphological and yield-related traits were also found in the introgression lines population. Using single-point analysis, a total of 37 putative QTLs for yield and yield components were detected at two sites with 7%-20% explaining the phenotypic variance. Nineteen QTLs （51.4%） were detected at both sites, and the alleles from O. rufipogon at fifteen loci （40.5%） improved the yield and yield components in the Teqing background. These O. rufipogon-O, sativa introgression lines will serve as genetic materials for identifying and using favorable genes from common wild rice.     

Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation in the rice genome, using the technique of methylation-sensitive amplified polymorphism (MSAP), which is a modification of the amplified fragment length polymorphism (AFLP) method that makes use of the differential sensitivity of a pair of isoschizomers to cytosine methylation. The tissues assayed included seedlings and flag leaves of an elite rice hybrid, Shanyou 63, and the parental lines Zhenshan 97 and Minghui 63. In all, 1076 fragments, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified using 16 pairs of selective primers. A total of 195 sites were found to be methylated at cytosines in one or both parents, and the two parents showed approximately the same overall degree of methylation (16.3%), as revealed by the incidence of differential digestion by the isoschizomers. Four classes of patterns were identified in a comparative assay of cytosine methylation in the parents and hybrid; increased methylation was detected in the hybrid compared to the parents at some of the recognition sites, while decreased methylation in the hybrid was detected at other sites. A small proportion of the sites was found to be differentially methylated in seedlings and flag leaves; DNA from young seedlings was methylated to a greater extent than that from flag leaves. Almost all of the methylation patterns detected by MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrate that the MSAP technique is highly efficient for large-scale detection of cytosine methylation in the rice genome. We believe that the technique can be adapted for use in other plant species. Received: 23 October 1998 / Accepted: 11 January 1999     

Citrus somatic hybrid: an alternative system to study rapid structural and epigenetic reorganization in allotetraploid genomes

Citrus somatic hybrids produced in the past years provide a novel opportunity to study the immediate effects of allopolyploidization on genome structure and methylation. Here, we present a first attempt to investigate the alterations in genome structure and methylation in three sets of citrus somatic allotetraploids and their diploid parents using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques. Our results indicate that all the allotetraploids mainly have the AFLP and MSAP banding patterns containing specific bands from both parents plus some alterations. The incidences of the AFLP polymorphic bands in allotetraploids show a range from 4.61 to 7.88 %, while from 12.50 to 15.67 % of the sites are methylated. In addition, the proportions of callus-parent-specific DNA structure and methylation alterations are much greater than those of leaf-parent-specific alterations in the somatic hybrids. Furthermore, we find that the somatic hybrids take on a greater divergence from the callus parent and a closer relationship to leaf parent in all groups of plants by dendrogram analysis based on AFLP or MSAP data. Taken together, our results suggest that somatic hybrids are very useful in elucidating the immediate changes that occur in newly synthesized allotetraploid.     

茄子空间诱变效应及其AFLP分析

为了研究茄子空间诱变效果,比较空间诱变系与原始对照间的分子水平差异,揭示空间诱变的分子效应,对3个不同茄子原始自交系（K_1、L_1、M_1）及其对应空间诱变系（K_2、L_2、M_2）生物学性状进行了比较研究,并利用AFLP分子标记技术进行了DNA指纹分析。结果表明,与原始自交系相比,3个空间诱变系的生育期、株型、果形均未发生明显变化,发生变化的性状是平均单果重和种子千粒重。诱变系M_2的平均单果重比其对照增加16.82%,差异极显著;种子千粒重有降低趋势,其中L_2比L_2显著降低了10.24%。48对AFLP选择性扩增引物在6个自交系和诱变系间扩增得到40条可以揭示诱变系与原始自交系间多态性的条带;部分目标片段测序结果经BLAST分析表明与已报道的高频突变相关DNA区域或蛋白同源性很高。可见,空间诱变技术能够引起茄子的遗传变异,而且这种变异是以DNA水平的变异为基础的。     

Epigenetic variation predicts regional and local intraspecific functional diversity in a perennial herb

The ecological significance of epigenetic variation has been generally inferred from studies on model plants under artificial conditions, but the importance of epigenetic differences between individuals as a source of intraspecific diversity in natural plant populations remains essentially unknown. This study investigates the relationship between epigenetic variation and functional plant diversity by conducting epigenetic (methylation‐sensitive amplified fragment length polymorphisms, MSAP) and genetic (amplified fragment length polymorphisms, AFLP) marker–trait association analyses for 20 whole‐plant, leaf and regenerative functional traits in a large sample of wild‐growing plants of the perennial herb Helleborus foetidus from ten sampling sites in south‐eastern Spain. Plants differed widely in functional characteristics, and exhibited greater epigenetic than genetic diversity, as shown by per cent polymorphism of MSAP fragments (92%) or markers (69%) greatly exceeding that for AFLP ones (41%). After controlling for genetic structuring and possible cryptic relatedness, every functional trait considered exhibited a significant association with at least one AFLP or MSAP marker. A total of 27 MSAP (13.0% of total) and 12 AFLP (4.4%) markers were involved in significant associations, which explained on average 8.2% and 8.0% of trait variance, respectively. Individual MSAP markers were more likely to be associated with functional traits than AFLP markers. Between‐site differences in multivariate functional diversity were directly related to variation in multilocus epigenetic diversity after multilocus genetic diversity was statistically accounted for. Results suggest that epigenetic variation can be an important source of intraspecific functional diversity in H. foetidus, possibly endowing this species with the capacity to exploit a broad range of ecological conditions despite its modest genetic diversity.