Monitoring genetic diversity in tropical trees with multilocus dominant markers

Since no universal codominant markers are currently available, dominant genetic markers, such as amplified fragment length polymorphism (AFLP), are valuable tools for assessing genetic diversity in tropical trees. However, the measurement of genetic diversity (H) with dominant markers depends on the frequency of null homozygotes (Q) and the fixation index (F) of populations. While Q can be estimated for AFLP loci, F is less accessible. Through a modelling approach, we show that the monolocus estimation of genetic diversity is strongly dependent on the value of F, but that the multilocus diversity estimate is surprisingly robust to variations in F. The robustness of the estimate is due to a mechanistic effect of compensation between negative and positive biases of H by different AFLP loci exhibiting contrasting frequency profiles of Q. The robustness was tested across contrasting theoretical frequency profiles of Q and verified for 10 neotropical species. Practical recommendations for the implementation of this analytical method are given for genetic surveys in tropical trees, where such markers are widely applied.     

A model-based method for identifying species hybrids using multilocus genetic data

We present a statistical method for identifying species hybrids using data on multiple, unlinked markers. The method does not require that allele frequencies be known in the parental species nor that separate, pure samples of the parental species be available. The method is suitable for both markers with fixed allelic differences between the species and markers without fixed differences. The probability model used is one in which parentals and various classes of hybrids (F(1)'s, F(2)'s, and various backcrosses) form a mixture from which the sample is drawn. Using the framework of Bayesian model-based clustering allows us to compute, by Markov chain Monte Carlo, the posterior probability that each individual belongs to each of the distinct hybrid classes. We demonstrate the method on allozyme data from two species of hybridizing trout, as well as on two simulated data sets.     

Genetic characterization of hybridization and introgression between anadromous rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki)

Interspecific hybridization represents a dynamic evolutionary phenomenon and major conservation problem in salmonid fishes. In this study we used amplified fragment length polymorphisms (AFLP) and mitochondrial DNA (mtDNA) markers to describe the extent and characterize the pattern of hybridization and introgression between coastal rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki). Hybrid individuals were initially identified using principle coordinate analysis of 133 polymorphic AFLP markers. Subsequent analysis using 23 diagnostic AFLP markers revealed the presence of F1, rainbow trout backcross, cutthroat trout backcross and later-generation hybrids. mtDNA analysis demonstrated equal numbers of F1 hybrids with rainbow and cutthroat trout mtDNA indicating reciprocal mating of the parental types. In contrast, rainbow and cutthroat trout backcross hybrids always exhibited the mtDNA from the recurrent parent, indicating a male hybrid mating with a pure female. This study illustrates the usefulness of the AFLP technique for generating large numbers of species diagnostic markers. The pattern of hybridization raises many questions concerning the existence and action of reproductive isolating mechanisms between these two species. Our findings are consistent with the hypothesis that introgression between anadromous populations of coastal rainbow and coastal cutthroat trout is limited by an environment-dependent reduction in hybrid fitness.     

Genetic diversity analysis using lowly polymorphic dominant markers: the example of AFLP in pigs

DNA markers are commonly used for large-scale evaluation of genetic diversity in farm animals, as a component of the management of animal genetic resources. AFLP markers are useful for such studies as they can be generated relatively simply; however, challenges in analysis arise from their dominant scoring and the low level of polymorphism of some markers. This paper describes the results obtained with a set of AFLP markers in a study of 59 pig breeds. AFLP fingerprints were generated using four primer combinations (PC), yielding a total of 148 marker loci, and average harmonic mean of breed sample size was 37.3. The average proportion of monomorphic populations was 63% (range across loci: 3%-98%). The moment-based method of Hill and Weir (2004, Mol Ecol 13:895-908) was applied to estimate gene frequencies, gene diversity (F(ST)), and Reynolds genetic distances. A highly significant average F(ST) of 0.11 was estimated, together with highly significant PC effects on gene diversity. The variance of F(ST) across loci also significantly exceeded the variance expected under the hypothesis of AFLP neutrality, strongly suggesting the sensitivity of AFLP to selection or other forces. Moment estimates were compared to estimates derived from the square root estimation of gene frequency, as currently applied for dominant markers, and the biases incurred in the latter method were evaluated. The paper discusses the hypotheses underlying the moment estimations and various issues relating to the biallelic, dominant, and lowly polymorphic nature of this set of AFLP markers and to their use as compared to microsatellites for measuring genetic diversity.     

Molecular linkage maps of the Populus genome.

We report molecular genetic linkage maps for an interspecific hybrid population of Populus, a model system in forest-tree biology. The hybrids were produced by crosses between P. deltoides (mother) and P. euramericana (father), which is a natural hybrid of P. deltoides (grandmother) and P. nigra (grandfather). Linkage analysis from 93 of the 450 backcross progeny grown in the field for 15 years was performed using random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), and inter-simple sequence repeats (ISSRs). Of a total of 839 polymorphic markers identified, 560 (67%) were testcross markers heterozygous in one parent but null in the other (segregating 1:1), 206 (25%) were intercross dominant markers heterozygous in both parents (segregating 3:1), and the remaining 73 (9%) were 19 non-parental RAPD markers (segregating 1:1) and 54 codominant AFLP markers (segregating 1:1:1:1). A mixed set of the testcross markers, non-parental RAPD markers, and codominant AFLP markers was used to construct two linkage maps, one based on the P. deltoides (D) genome and the other based on P. euramericana (E). The two maps showed nearly complete coverage of the genome, spanning 3801 and 3452 cM, respectively. The availability of non-parental RAPD and codominant AFLP markers as orthologous genes allowed for a direct comparison of the rate of meiotic recombination between the two different parental species. Generally, the rate of meiotic recombination was greater for males than females in our interspecific poplar hybrids. The confounded effect of sexes and species causes the mean recombination distance of orthologous markers to be 11% longer for the father (P. euramericana; interspecific hybrid) than for the mother (P. deltoides; pure species). The linkage maps constructed and the interspecific poplar hybrid population in which clonal replicates for individual genotypes are available present a comprehensive foundation for future genomic studies and quantitative trait locus (QTL) identification.     

Relationships between four measures of genetic distance and breeding behavior in spring wheat

We estimated the genetic distances among 10 spring wheat genotypes based on pedigree data, morphological traits and AFLP markers, used individually and combined with morphological traits, to find the best predictors of general- and specific-combining abilities among parental genotypes. Ten wheat parents were crossed in a diallel form, disregarding reciprocal hybrids, totaling 45 combinations. The F(1) hybrids, F(2) populations and parents were evaluated in the field in 2007. The experimental plots consisted of 20 plants for F(1) hybrids and 40 plants for parental and F(2) populations. All methods (pedigree data, AFLP markers and morphological traits, used individually and combined) were found to be useful for the assessment of genetic diversity. The significant coefficient correlations ranged from low (0.45) to moderate (0.67) between the distance measures and hybrid performance. There was significant agreement between the distance measures based on AFLP markers vs morphological traits + AFLP markers (r = 0.47) and between pedigree data vs morphological traits + AFLP markers (r = 0.43). The pedigree distance was positively associated with traits 100-kernel weight and grain yield per plant in F(1) (correlations of 0.67 and 0.62, respectively) and F(2) (correlations of 0.62 and 0.59, respectively) generations. These correlation values indicate that the genetic distance, based on pedigree data, could replace diallel crosses for the selection of parents with higher combining ability and with moderate reliability.     

Multicolour fluorescent detection and mapping of AFLP markers in chicken (Gallus domesticus)

We describe the mapping of amplified restriction fragment polymorphism (AFLP) markers in chicken (Gallus domesticus) using a multi-colour fluorescent detection system. DNA was used from a population consisting of four families with a total of 183 F2 individuals. The enzyme combination EcoRI/TaqI was used for double digestion, and fluorescently labelled fragments were analysed on an ABI PRISM 377 DNA sequencer. Polymorphic signals in the range of 50-500 bp were genotyped with the ABI PRISM Genotyper 2.0 software, which enabled the analysis of both dominant and incomplete dominant markers (with respect to AFLP, often referred to as codominant). In 19 sets consisting of 3 EcoRI/TaqI primer pair combinations each, a total of 475 polymorphic markers was detected. From these polymorphisms 344 markers could be mapped on the Wageningen linkage map. Fourteen markers were length polymorphisms of the same fragment and 28 markers Z-linked and uniformative; 64 AFLP markers appeared to be unlinked and 25 AFLP markers could not be accurately mapped on the basis of the genotyping results. The resulting AFLP/microsatellite linkage map is comprised of 33 linkage groups with a total of 835 loci.     

Genetic relationships between diploid and allotetraploid cherry species (Prunus avium, Prunus x gondouinii and Prunus cerasus)

Prunus avium L. (diploid, AA, 2n=2x=16), Prunus cerasus L. (allotetraploid, AAFF, 2n=4x=32) species, and their hybrid Prunus x gondouinii Rehd., constitute the most widely cultivated cherry tree species. P. cerasus is supposed to be an hybrid species produced by the union of unreduced P. avium gametes and normal P. fruticosa gametes. A continuum of morphological traits between these three species makes their assignation difficult. The aim of this paper is to study the genetic relationships between tetraploid and diploid cherry species. In all, 114 genotypes belonging to these species were analyzed using 75 AFLP markers. The coordinates of these genotypes on the first axis of a correspondence analysis allowed us to clearly distinguish each species, to identify misclassifications and to assign unknown genotypes to one species. We showed that there are specific alleles in P. cerasus, which are not present in the A genome of P. avium and which probably come from the F genome of P. cerasus. The frequencies of each marker in the A and the F genomes were estimated in order to identify A and F specific markers. We discuss the utility of these specific markers for finding the origin of the A and F genomes in the allopolyploid species.     

Species distinction in Irish populations of Quercus petraea and Q. robur: morphological versus molecular analyses

BACKGROUND AND AIMS: Populations of oak (Quercus petraea and Q. robur) were investigated using morphological and molecular (AFLP) analyses to assess species distinction. The study aimed to describe species distinction in Irish oak populations and to situate this in a European context. METHODS: Populations were sampled from across the range of the island of Ireland. Leaf morphological characters were analysed through clustering and ordination methods. Putative neutral molecular markers (AFLPs) were used to analyse the molecular variation. Cluster and ordination analyses were also performed on the AFLP markers in addition to calculations of genetic diversity and F-statisitcs. KEY RESULTS: A notable divergence was uncovered between the morphological and molecular analyses. The morphological analysis clearly differentiated individuals into their respective species, whereas the molecular analysis did not. Twenty species-specific AFLP markers were observed from 123 plants in 24 populations but none of these was species-diagnostic. Principal Coordinate Analysis of the AFLP data revealed a clustering, across the first two axes, of individuals according to population rather than according to species. High F(ST) values calculated from AFLP markers also indicated population differentiation (F(ST) = 0.271). Species differentiation accounted for only 13 % of the variation in diversity compared with population differentiation, which accounted for 27 %. CONCLUSIONS: The results show that neutral molecular variation is partitioned more strongly between populations than between species. Although this could indicate that the populations of Q. petraea and Q. robur studied may not be distinct species at a molecular level, it is proposed that the difficulty in distinguishing the species in Irish oak populations using AFLP markers is due to population differentiation masking species differences. This could result from non-random mating in small, fragmented woodland populations. Hybridization and introgression between the species could also have a significant role.     

Adaptation of the AFLP technique as a new tool to detect genetic instability and transposition in interspecific hybrids

An adapted amplified fragment length polymorphism (AFLP) protocol is presented for detection of hybrid instability in the genome of interspecific hybrids between Drosophila buzzatii and D. koepferae species. Analyses of 15 AFLP instability markers (new bands detected in hybrids) show that up to 81% are the result of transposable element (TE) activity. Twenty TEs associated with AFLP instability markers have been detected by this method in backcross hybrids and segmental hybrids, demonstrating its validity in detecting transposition events occurring during the hybridization process. New insertions of Helena TE have been observed in the hybrid genome after hybridization of the TGTCG22 instability marker by FISH. The AFLP marker technique proved to be an efficient method that improves upon traditional and bioinformatic tools previously used to detect TE mobilization. This newly adapted AFLP protocol may also be applied to a large number of organisms outside the Drosophila genus, making it of interest to evolutionary and population genetic researchers working with species where the knowledge of the genome is scarce.     

Efficiency of model-based Bayesian methods for detecting hybrid individuals under different hybridization scenarios and with different numbers of loci

Accurate detection of offspring resulting from hybridization between individuals of distinct populations has a range of applications in conservation and population genetics. We assessed the hybrid identification efficiency of two methods (implemented in the STRUCTURE and NEWHYBRIDS programs) which are tailored to identifying hybrid individuals but use different approaches. Simulated first- and second-generation hybrids were used to assess the performance of these two methods in detecting recent hybridization under scenarios with different levels of genetic divergence and varying numbers of loci. Despite the different approaches of the methods, the hybrid detection efficiency was generally similar and neither of the two methods outperformed the other in all scenarios assessed. Interestingly, hybrid detection efficiency was only minimally affected by whether reference population allele frequency information was included or not. In terms of genotyping effort, efficient detection of F1 hybrid individuals requires the use of 12 or 24 loci with pairwise F(ST) between hybridizing parental populations of 0.21 or 0.12, respectively. While achievable, these locus numbers are nevertheless higher than the number of loci currently commonly applied in population genetic studies. The method of STRUCTURE seemed to be less sensitive to the proportion of hybrids included in the sample, while NEWHYBRIDS seemed to perform slightly better when individuals from both backcross and F1 hybrid classes were present in the sample. However, separating backcrosses from purebred parental individuals requires a considerable genotyping effort (at least 48 loci), even when divergence between parental populations is high.     

The strength of reproductive isolation in two hybridizing food-deceptive orchid species

Reproductive isolation is of fundamental importance for maintaining species boundaries in sympatry. In orchids, the wide variety of pollination systems and highly diverse floral traits have traditionally suggested a prominent role for pollinator isolation, and thus for prezygotic isolation, as an effective barrier to gene flow among species. Here, we examined the nature of reproductive isolation between Anacamptis morio and Anacamptis papilionacea, two sister species of Mediterranean food-deceptive orchids, in two natural hybrid zones. Comparative analyses of the two hybrid zones that are located on soils with volcanic origin and have different and well-dated ages consistently revealed that all hybrid individuals were morphologically and genetically intermediate between the parental species, but had strongly reduced fitness. Molecular analyses based on nuclear ITS1 and (amplified fragment length polymorphism) AFLP markers clearly showed that all examined hybrids were F1 hybrids, and that no introgression occurred between parental species. The maternally inherited plastid DNA markers indicated that hybridization between A. morio and A. papilionacea was bidirectional, as confirmed by the molecular analysis of seed families. The genetic architecture of the two hybrid zones suggests that the two parental species easily and frequently hybridize in sympatry as a consequence of partial pollinator overlap but that strong postzygotic barriers reduce hybrid fitness and prevent gene introgression. These results corroborate that chromosomal divergence is instrumental for reproductive isolation between these food-deceptive orchids and suggest that hybridization is of limited importance for their diversification.     

Molecular criteria for determining new hybrid species--an application to the Sonneratia hybrids

The possible hybrid origin of new species can usually be corroborated by molecular means. Here, we suggest that the segregation patterns of the molecular markers be further analyzed. A true hybrid species should show the patterns under continuous breeding among its members, at least beyond the F2 generation. We applied the guidelines to the putative hybrid species of Sonneratia, a widespread mangrove genus, and concluded that all the observed hybrids in this genus are simple F1's. Thus, S. x gulngai and S. x hainanensis are not true hybrid species. The segregation patterns of molecular markers should be heeded in interpreting the existence of hybrid species.     

Estimating population structure from AFLP amplification intensity

In the last decade, amplified fragment length polymorphisms (AFLPs) have become one of the most widely used molecular markers to study the genetic structure of natural populations. Most of the statistical methods available to study the genetic structure of populations using AFLPs consider these markers as dominant and are thus unable to distinguish between individuals being heterozygous or homozygous for the dominant allele. Some attempts have been made to treat AFLPs as codominant markers by using AFLP band intensities to infer the most likely genotype of each individual. These two approaches have some drawbacks, the former discarding potentially valuable information and the latter being sometimes unable to correctly assign genotypes to individuals. In this study, we propose an alternative likelihood‐based approach, which does not attempt at inferring the genotype of each individual, but rather incorporate the uncertainty about genotypes into a Bayesian framework leading to the estimation of population‐specific F_IS and F_ST coefficients. We show with simulations that the accuracy of our method is much higher than one using AFLP as dominant markers and is generally close to what would be obtained by using the same number of Single‐Nucleotide Polymorphism (SNP) markers. The method is applied to a data set of four populations of the common vole (Microtus arvalis) from Grisons in Switzerland, for which we obtained 562 polymorphic AFLP markers. Our approach is very general and has the potential to make AFLP markers as useful as SNP data for nonmodel species.     

Population genetics of the yellow fever mosquito in Trinidad: comparisons of amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers

Recent development of DNA markers provides powerful tools for population genetic analyses. Amplified fragment length polymorphism (AFLP) markers result from a polymerase chain reaction (PCR)-based DNA fingerprinting technique that can detect multiple restriction fragments in a single polyacrylamide gel, and thus are potentially useful for population genetic studies. Because AFLP markers have to be analysed as dominant loci in order to estimate population genetic diversity and genetic structure parameters, one must assume that dominant (amplified) alleles are identical in state, recessive (unamplified) alleles are identical in state, AFLP fragments segregate according to Mendelian expectations and that the genotypes of an AFLP locus are in Hardy-Weinberg equilibrium (HWE). The HWE assumption is untestable for natural populations using dominant markers. Restriction fragment length polymorphism (RFLP) markers segregate as codominant alleles, and can therefore be used to test the HWE assumption that is critical for analysing AFLP data. This study examined whether the dominant AFLP markers could provide accurate estimates of genetic variability for the Aedes aegypti mosquito populations of Trinidad, West Indies, by comparing genetic structure parameters using AFLP and RFLP markers. For AFLP markers, we tested a total of five primer combinations and scored 137 putative loci. For RFLP, we examined a total of eight mapped markers that provide a broad coverage of mosquito genome. The estimated average heterozygosity with AFLP markers was similar among the populations (0.39), and the observed average heterozygosity with RFLP markers varied from 0.44 to 0.58. The average FST (standardized among-population genetic variance) estimates were 0.033 for AFLP and 0.063 for RFLP markers. The genotypes at several RFLP loci were not in HWE, suggesting that the assumption critical for analysing AFLP data was invalid for some loci of the mosquito populations in Trinidad. Therefore, the results suggest that, compared with dominant molecular markers, codominant DNA markers provide better estimates of population genetic variability, and offer more statistical power for detecting population genetic structure.     

Hybrid origin of Audubon's warbler

Several animal species have recently been shown to have hybrid origins, but no avian examples have been documented with molecular evidence. We investigate whether the Audubon’s warbler (Dendroica auduboni), one of four visually distinct species in the yellow‐rumped warbler complex, has originated through hybridization between two other species in this group, the myrtle warbler (D. coronata) and black‐fronted warbler (D. nigrifrons). Analysis of nuclear amplified fragment length polymorphism (AFLP) and sequence markers shows that Audubon’s warblers are genetically intermediate and carry a mixture of alleles otherwise found only in one or the other of their putative parental species. Audubon’s warblers also carry two deeply divergent mitochondrial DNA lineages, each shared with only one putative parental form. Broad clines between Audubon’s and black‐fronted warblers in AFLP markers call into question the validity of these two forms as full species; nevertheless, our results suggest that the Audubon’s warbler probably originated through hybridization between two long‐diverged species. It is likely that more cases of avian species of hybrid origin will be revealed by surveys of variation in nuclear DNA and other traits.     

Genetic variability of Siberian fir (Abies sibirica Ledeb.) inferred from AFLP markers

Genetic variability of AFLP markers was studied in 20 populations of Siberian fir (Abies sibirica (Pinaceae) and in two populations of Far-Eastern Manchurian fir A. nephrolepis and Sakhalin fir A. sachalinensis each. Four pairs of selective primers were used. In total, 168 samples from three fir species were genotyped for 117 polymorphic loci. According to the AMOVA results, the variability proportion characterizing the differences between three Abies species was several times higher (F(CT) = 0.53) than that accounting for among-population differences within the species (F(SC) = 0.125). Differentiation of the A. sibirica populations based on AFLP markers exceeded 14% (F(ST) = 0.141). Significant correlation between the genetic distances calculated from the AFLP data and the geographic distances between populations was found. The results of AFLP variability analysis supported and supplemented the conclusions inferred previously from allozyme and cpSSR data: several genetically similar geographic groups of Siberian fir were identified. These groups differ both in allele frequencies and in the levels of genetic variation.     

Segregation distortion and linkage analysis in eggplant (Solanum melongena L.)

An anther-derived doubled haploid (DH) population and an F2 mapping population were developed from an intraspecific hybrid between the eggplant breeding lines 305E40 and 67/3. The former incorporates an introgressed segment from Solanum aethiopicum Gilo Group carrying the gene Rfo-sa1, which confers resistance to Fusarium oxysporum; the latter is a selection from an intraspecific cross involving two conventional eggplant varieties and lacks Rfo-sa1. Initially, 28 AFLP primer combinations (PCs) were applied to a sample of 93 F2 individuals and 93 DH individuals, from which 170 polymorphic AFLP fragments were identified. In the DH population, the segregation of 117 of these AFLPs as well as markers closely linked to Rfo-sa1 was substantially distorted, while in the F2 population, segregation distortion was restricted to just 10 markers, and thus the latter was chosen for map development. A set of 141 F2 individuals was genotyped with 73 AFLP PCs (generating 406 informative markers), 32 SSRs, 4 tomato RFLPs, and 3 CAPS markers linked to Rfo-sa1. This resulted in the assignment of 348 markers to 12 major linkage groups. The framework map covered 718.7?cM, comprising 238 markers (212 AFLPs, 22 SSRs, 1 RFLP, and the Rfo-sa1 CAPS). Marker order and inter-marker distances in this eggplant map were largely consistent with those reported in a recently published SSR-based map. From an eggplant breeding perspective, DH populations produced by anther culture appear to be subject to massive segregation distortion and thus may not be very efficient in capturing the full range of genetic variation present in the parental lines.     

Using AFLP markers for species differentiation and assessment of genetic variability of in vitro-cultured Papaver bracteatum (section Oxytona)

Summary Amplified fragment length polymorphism (AFLP) markers were employed to deteet genetic variation among species of Papever (section Oxytona) and assess genetic fidelity between in vitro cell lines of Papaver bracteatum and mature plants derived from the propagation of their callus cultures. Regenerated plants exhibited morphological and phytochemical characteristics dissimilar to those of their source material. Thebaine, the dominant alkaloid produced by Papaver bracteatum, was not detected in capsules from mature regenerated accessions, indicating that there may have been a loss of genetic uniformity. Instead, the dominant alkaloid produced by the regenerated plant was shown to be isothebaine (by TLC and GC/MS), a metabolic characteristic of P. pseudo-orientale. A Neighbor-Joining tree constructed from AFLP fingerprints distinetly separates the three species of Oxytona while firmly grouping the in vitro-cultured plants with P. pseudo-orientale. Additionally phytochemical data and chromosome counts indicate that the seed used to initiate cultures was of hybrid origin and ihat the loss in genetic uniformity was not due to somaclonal variation occurring during the in vitro culture process. AFLP fingerprinting was therefore able to differentiate Oxytona species and invesgigate allopolyploidy in closely related papaver species.     

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.