Generational stability of epigenetic transgenerational inheritance facilitates adaptation and evolution

ABSTRACT

The epigenome and epigenetic inheritance were not included in the original modern synthesis theory or more recent extended evolutionary synthesis of evolution. In a broad range of species, the environment has been shown to play a significant role in natural selection, which more recently has been shown to occur through epigenetic alterations and epigenetic inheritance. However, even with this evidence, the field of epigenetics and epigenetic inheritance has been left out of modern evolutionary synthesis, as well as other current evolutionary models. Epigenetic mechanisms can direct the regulation of genetic processes (e.g. gene expression) and also can be directly changed by the environment. In contrast, DNA sequence cannot be directly altered by the environment. The goal of this review is to present the evidence of how epigenetics and epigenetic inheritance can alter phenotypic variation in numerous species. This can occur at a significantly higher frequency than genetic change, so correlates with the frequency of evolutionary change. In addition, the concept and importance of generational stability of transgenerational inheritance is incorporated into evolutionary theory. For there to be a better understanding of evolutionary biology, we must incorporate all aspects of molecular (e.g. genetics and epigenetics) and biological sciences (e.g. environment and adaptation).

KEYWORDS:

• Epigenetic
• inheritance
• transgenerational
• generational stability
• adaptation
• evolutionary biology
• review

Introduction

Modern synthesis, extended evolutionary synthesis, and the unified theory of evolution

Evolutionary biologists in the 16th and 17th centuries described the world and its natural laws [1]. First came Copernicus’ and his two main points of natural laws were ‘origin and design organisms’ [1]. Charles Darwin stated that organisms came from natural selection and thus published four postulates of natural selection and adaptive evolution [2]. In 1866, Gregor Mendel published his work on plant hybridization [3,4]. From his work, the explanation of how genetic materials such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) were discovered [3]. This also began to explain how traits could be inherited through molecular mechanisms [3,4]. Julian Huxley coined the term modern synthesis in the mid-1900s in a book called Evolution: the modern synthesis [5]. The publication of this book was before there were discoveries in molecular biology [6]. Theories and conceptualization of non-genetic inheritance were not included in the theory of modern synthesis. The incorporation of other areas of studies into modern synthesis such as epigenomic theory would help us have a broader understanding of evolutionary theory as a whole.

In 1896, the organism, daphnia, and the presence of predators in their environment were examined by James Mark Baldwin [7]. Baldwin saw that when predators are in the daphnia environment, the daphnia will adapt to the changing environment [7]. Thus, developing adaptive traits that were passed onto their offspring [7]. This term was then named the Baldwin effect and was mainly used for psychological work and it wasn’t until later that this term was used in evolutionary biology [8]. Another observational study in 1910 was Paul Kammerer who demonstrated an aqueous environment in the Midwife Toad altered reproduction phenotypic variation that was inherited to the next generation [9]. Conrad Waddington coined the term ‘developmental epigenetics’ in the 1940s [10]. Waddington had exposed drosophila flies to extreme heat shock for several generations [10]. The heat exposure was then stopped for several generations, the flies began to develop a new phenotype [10]. The wings of the flies in the later generations of flies exposed to the extreme heat had changed in shape and structure [10]. These changes in wing structure and shape persisted over sixteen generations, and still exist today [10]. Waddington concluded that the flies had adapted to extreme heat exposure and the genotype had not been imposed through a non-genetic process he termed epigenetics [10]. There have been other accounts of non-genetic inheritance observed that appear to impact evolutionary biology. This includes gestating female effects, where the fitness of the offspring can be determined by the maternal environment [11]. The offspring’s fitness derived from the maternal environment is considered an example of non-genetic inheritance [11]. Research with maize done in the 1950s, resulted in additional observations of non-genetic inheritance [12]. These researchers discovered that maize has a paramutation of the b1 gene [12]. When the b1 gene would be expressed, this would lead to the maize adding anthocyanin [12]. Researchers examined that when the b1 gene was not highly expressed, the maize would give off a low purple color [12]. However, when the b1 gene was highly expressed, the maize showed to have a very deep purple color [12]. Researchers then took a homozygous maize which had high expressions of the b1 gene and a homozygous plant which had a low expression of the b1 gene and crossed them [12,13]. The F1 generation offspring were also crossed and given the F2 generation offspring [12,13]. The F2 generation offspring showed to have low color in the maize (low purple pigmentation) [12,13]. Researchers concluded that when looking at a comparison of the b1 gene that was highly expressed in the maize and the b1 gene that had low levels of expression in the maize, there were no differences in the DNA sequences examined [14]. However, researchers did find differences in the chromatin structure when looking into other epigenetic mechanisms [14]. Even with the research showing that changes happen in organisms through apparently non-genetic mechanisms, the conclusions of this research were targeted toward genetic inheritance concepts.

The formation of a new proposal of the evolutionary model was coined the extended evolutionary synthesis (EES) [15]. Modern synthesis did not include the discoveries of epigenetics or non-genetic inheritance. EES theory incorporated the view of modern synthesis, but did not effectively add the discoveries of all epigenetic mechanisms [15]. Although we do not dismiss the ideas of modern synthesis, as they are not wrong, they are simply incomplete. For there to be progress within the theories of the evolutionary model, there must be an acknowledgment of the influence epigenetics and non-genetic inheritance have on phenotypes in organisms. Another theory that has emerged is the Unified Theory [16]. The unified theory incorporates epigenetic inheritance theory [16], as well as the newer concepts of EES which includes the epigenetics [15]. However, unified theory also includes how the environment can directly change or alter the phenotype, independent of genetics [16], Figure 1. Later in this discussion, examples of direct environmental influences on the phenotype and evolution will be discussed. In all, the incorporation of the modern synthesis, theories of the extended evolutionary synthesis, and the unified theory which incorporates epigenetics will help better understand evolutionary biology and the molecular mechanisms involved.

Figure 1. Unified theory of evolution with incorporation of epigenetics and epigenetic transgenerational inheritance. Inclusion of both darwinian concepts and neo-lamarckian concepts to expand evolutionary theory. Schematic of the unified theory of evolution. No dominance is suggested by the appearance of specific circles (e.g., epimutations versus genetics) such that all are equally important components. Modified from Skinner [16].

Epigenetic mechanisms

Epigenetics are heritable changes in gene expression that occur without modifications at the DNA level [17]. However, this definition is incomplete and does not clarify the molecular level understanding of epigenetics. The definition of epigenetics is ‘molecular factors and processes around DNA that regulate genome activity, independent of DNA sequence, and are mitotically stable’ [17]. This incorporates the molecular factors involved and the integration with genetics to regulate genome activity, (e.g., what genes are on or off). This also incorporates the concept when a cell undergoes mitosis not only is the DNA sequence replicated, but the epigenome is also replicated to maintain cell specificity and identity [17]. The epigenetic impacts on somatic cells can alter the functions of these cells and influence the phenotypes of the individual throughout life, but somatic cell effects cannot influence subsequent generations unless an epigenetic alteration develops in the germline (sperm or egg) [17]. In contrast, an epigenetic effect in the germline can transmit epigenetic information to the zygote at fertilization and subsequent embryonic stem cells and developing embryo to impact the next generation through non-genetic mechanisms of epigenetic inheritance [17].

There are several different epigenetics mechanisms that are mitotically stable including DNA methylation, chromatin structure, histone modification, and non-coding RNA [18–20], Figure 2. One of the first epigenetic mechanisms discovered and frequently studied is DNA methylation [21,22]. DNA methylation occurs where nucleotides cytosine and guanine are held together by a phosphate group [21,23]. The site of a cytosine and guanine is also known as a CpG site [24]. When a methyl group attaches to the C5 position of the cytosine by the enzyme methyltransferase, this is called DNA methylation [22,24]. A methylation of the C5 position of the cytosine will produce a 5-methylcytosine (5mC) [24,25]. The methylation of the 5mC can directly regulate gene expression [24]. This can involve suppression of gene expression in heterochromatin or increase in expression in euchromatin. During early development, the process of transcription and the suppression of transcription, an enzyme family named ten-eleven translocation will drive the erasure of DNA methylation [24]. The partial erasure of DNA methylation has been proposed to mainly occur during the time of early embryonic development [24,25]. Once this process occurs, the 5mC is enzymatically modified and the product is called a hydroxymethyl cytosine (5hmC) [24]. Although the purpose during adaptation has not yet been found for 5hmC, it is also to be considered an epigenetic factor [26]. Although the concept DNA methylation erasure was developed genome-wide [24,25], recently lower density CpG regions that have been shown to not be erased, but increase in DNA methylation [27]. Therefore, high-density CpG islands are erased during embryo development [24,25] but the majority of the epigenome DNA methylation either increases or does not get erased during early embryonic development in the morula embryo [27]. DNA methylation is important for transcription and replication since it takes place during the time somatic cells are being developed [25]. Each somatic cell type has a unique DNA methylation profile and epigenetics to drive the somatic cell septicity [22]. In addition to methylation at CpG sites, there is also a process of methylation of another nucleotide, adenine [28]. This process is less frequent and is also found in the RNA [28]. The methylation of an adenine site is called a m6A [28]. In a drosophila model, researchers found that when an m6A modification takes place the process of biogenesis and splicing within the RNA occurs [29]. Methylation of an adenine was found to involve a gene called sex lethal (Sxl) within drosophila [29]. Adenine methylation associated with the Sxl gene was found to determine changes to the organisms’ functions [29]. M6A is an epigenetic mechanism and has been shown to play a critical role in the process of sex determination in drosophila flies [29].

Figure 2. Schematic of epigenetic processes. Representation of the primary epigenetic factors and processes schematic of non-coding RNA, DNA methylation, chromatin structure, histone modifications, and DNA structure presented. Modified from Nilsson, et al. [21].

Another well-known epigenetic mechanism is the modification of histones [30,31], Figure 2. Histones as a whole, are important to the structure of DNA [30,31]. DNA is wrapped tightly around histones that form a nucleosome [31]. These same histones can then be chemically modified with methylation or acetylation or phosphorylation to change gene expression [31]. Histone modifications have been associated with changing the structure of chromatin [25,31]. In addition to changing the structure of chromatin, histone modifications can also lead to the recruitment of transcriptional cofactors to facilitate gene expression or repression of gene expression [18,32]. Therefore, the histone modifications can turn off gene activity through hypermethylation and turn on gene expression through acetylation histone modifications [31,32]. In addition to the ability of histone modifications to act as epigenetic factors and regulate gene expression, these histone modifications can alter chromatin structure that is also another epigenetic factor [33]. A combination of increased DNA methylation and histone methylation can promote the condensation of DNA to generate heterochromatin and shut down gene expression [33]. Alternately, the removal of DNA methylation and histone acetylation can provide euchromatin de-condensed DNA structure to allow gene activation and transcription [33]. In addition to DNA condensation, coils and loop structures in the DNA can also influence gene expression to act as an epigenetic factor as well [34]. Therefore, chromatin structure is a critical epigenetic factor as well [33,34], Figure 2.

Another epigenetic factor involves non-coding RNA (ncRNA), which are also an important epigenetic mechanisms [20], Figure 2. ncRNA has a role to enable or facilitate proteins to bind to DNA which will change the expression of genes [35]. The epigenetic aspect of ncRNA is that there is no DNA sequence required for action to regulate gene expression [35]. The most common type of methylation on an RNA is through ncRNA methylation on adenine sites [36]. The process of methylation on the ncRNA will alter the secondary structure of the ncRNA and subsequently alter its function [37,38]. Research has shown that ncRNA methylation can have an association with some cancers and non-cancerous diseases [36]. As will be discussed, epigenetic mechanisms and the inheritance of these epigenetic mechanisms can drive phenotypic variation and genetic variation. The methylation of cytosine is known to increase the frequency and promote the point mutation of a cytosine to tyrosine transition [39]. Environmentally induced epigenetic transgenerational inheritance of DNA methylation changes in sperm were found to associate with cytosine to tyrosine mutations termed tertiary epimutations [40]. The environmentally induced epigenetic transgenerational inheritance of sperm genetic mutation involving copy number variation increases has also been observed [41]. Therefore, epigenetic change can promote low levels of genetic mutation and variation. The integration of epigenetic mechanisms needs to be involved in the revised and unified theory of evolution, Figure 1.

Many models for biological organisms have been inbred and developed in the mid 1900s including mice, drosophila flies and C-elegans worms [42]. Although these inbred models have been ideal for genetic studies, inbred lines develop altered molecular and physiological parameters associated with the inbreeding. Epigenetics is dramatically impacted by inbreeding depression [43,44], and the cause for phenotypic variation and loss of biological viability. This was first identified in plants [45,46], but also shown in other species like fish [47]. The role of epigenetics in inbreeding depression has led evolutionary biologists to propose a role in evolution and ecology [48]. This inbreeding depression alters the epigenetics to allow survival of the organism after thousands of inbreedings such as observed in the drosophila and C-elegans, or hundreds in mice and plants. Therefore, the lack of cytosine methylation in drosophila and C-elegans, due to loss of CpG methylation and DNA methyltransferase, comes from the inbreeding depression and required to find alternate epigenetic processes for survival [49]. Therefore, the alternate epigenetics in inbred species such as drosophila and C-elegans should not be seen as a normal biology process, but due to the inbreeding depression phenomenon of epigenetic change required. Although these models have been compared to normal biological processes, the nearest phylogenetic relations of the inbred lines still have the normal epigenetics of methylation of cytosine and other epigenetic processes. This needs to be considered in the use of these model organisms.

Epigenetic transgenerational inheritance and generational stability

Epigenetic transgenerational inheritance is the process of germline transmission of epigenetics between generations [25]. Examples of this include when a female is gestating, and the fetus is exposed to environmental exposures of stressors or toxicants [50]. Environmental exposures to toxicants or stressors can range from alcohol, fungicides, low or high-caloric diet, and stress (Table 1). During the time a female is gestating, exposure to a toxicant becomes critical in the development of the fetus during sex determination [25]. In rats, sex determination occurs 10 to 11 days of embryonic development [51]. The directly exposed generations will be the F0 generation (gestating female), the F1 generation (fetus), and the F2 generation (germline cells of the fetus) [52]. The first generation to be transgenerational will be the offspring of the F2 generation, the F3 generation [52]. The F3 generation is considered to be ancestrally exposed to that toxicant or stressor [52], see Figure 3. The subsequent offspring will inherit epigenetic alterations from their parents through epigenetic changes in the germline which alters the epigenetics of all somatic cells to promote the phenotypic variation [52].

Figure 3. Clarification of intergenerational and transgenerational epigenetic inheritance. Environmentally induced transgenerational epigenetic inheritance: schematic of environmental exposure and affected generations for both gestating female and adult male or female. The multigenerational direct exposures are indicated in contrast to the transgenerational generation without direct exposure. Modified from Nilsson, et al. [21].

Table 1. Environmental toxicant exposure induced epigenetic transgenerational inheritance and generational toxicology. (A) The toxicant exposure, pathologies, and literature references are provided. Rodent species were used in these studies. (B) Other non-model species with demonstrated environmentally induced epigenetic transgenerational inheritance.

Exposure	Pathology	Reference
A. Toxicant exposure induced epigenetic transgenerational inheritance and generational toxicology
Vinclozolin (agricultural fungicide)	Testis, Ovary, Kidney, Prostate	[25,94,95]
Methoxychlor (agricultural pesticide)	Obesity, Testis, Ovary, Kidney, Prostate	[94,96]
TCDD/dioxin (industrial contaminant)	Testis, Ovary, Kidney, Prostate	[97–100]
Plastics (BPA, phthalate-DEHP and DBP)	Obesity, Testis, Ovary, Kidney, Prostate	[101–103]
Jet fuel [JP8] (hydrocarbon mixture)	Testis, Ovary, Kidney, Prostate	[104,105]
Permethrin and DEET (pesticide and insect repellent)	Testis, Ovary, Kidney, Prostate	[106,107]
DDT (pesticide)	Obesity, Testis, Ovary, Kidney, Prostate	[108–110]
BPA (plastic toxicant)	Obesity	[111,112]
Phthalates (plastic toxicant)	Testis, Ovary, Kidney, Prostate	[113]
Tributyltin (industrial toxicant)	Obesity	[114]
Glyphosate	Obesity, Testis, Ovary, Kidney, Prostate	[59,115]
Smoking	Obesity, Lean, Asthma	[116–119]
Alcohol	Endocrine, Anxiety	[120,121]
B. Non-model species showing epigenetic inheritance and variation
Plants	Snap dragon: (Antirrhium)	[70]
Marine Animals	Oyster: (Vrassostrea gigas)	[71]
	Mollusks: New Zealand mud snail	[72,73]
	Fish: Poecilia, Steelhead trout (Oncorhynchus mykiss)	[74,75]
	Fish: Atlantic salmon (Salmo Salar)	[47,76]
	Tubeworm: (Hydroidea Diramphus)	[77]
Birds	House Sparrow: (Passer domesticus)	[78]
	Darwin Finches	[79]

Examples of transgenerational inheritance have been shown through many studies in many different species and models for multiple generations [17,25,29,35,52–56], Table 1 and, Figure 4. Therefore, transgenerational inheritance is widespread in biology and causes generational stability of phenotypic variation. Observations have shown epigenetic transgenerational inheritance is stable through generations affected by toxicant or environmental stressor exposure during development. An example of this was found in the C. elegans worms [57]. C. elegans were found to have stable inheritance of piRNAs which triggered long-term silencing that persisted for twenty generations [57]. Another example of generational stability was in drosophila flies [58]. Waddington exposed drosophila flies to ether vapor for several generations which led to the flies to begin to develop a phenotypic change of a bithorax [58]. Before this experiment, drosophila was not known to produce a bithorax [58]. After Waddington stopped the exposure [58], the flies themselves produced offspring that had a bithorax [58]. Due to environmental exposure of the ether vapors, the fly embryos began to form a new phenotype that persisted for over twenty generations [58]. Without continuous exposure, the flies began to present an altered phenotype [58]. In addition to ether vapor exposure, Waddington had performed another experiment with drosophila flies using heat shock exposure [10]. Waddington began to expose flies to extreme heat shock and carried on for several generations [10]. When the heat exposure stopped for several generations the flies’ wings began to change their physical appearance [10]. The later generations of flies began to show changes in their wing’s phenotype. Compared to the earlier generations directly exposed to heat shock, the later generations began to produce different structures and shapes on their wings [10]. There have also been studies in mammalian models that showed environmental induced generational stability in epigenetic and phenotypic alterations [59–61]. Dichlorodiphenyldichloroethane (DDT) has been used globally to prevent malaria throughout north America [62]. Direct exposure to DDT was not found to cause effects on the F1 generation offspring [62]. Health effects began to show up in the offspring after the first transgenerational F3 generation [62]. A recent study exposed a gestating female rat (F0 generation) to DDT [61]. The lineages were carried out to the first transgenerational generation (F3 generation) without a continuous exposure to DDT. Results showed that DNA methylation can cause alterations in adipocyte size in the F3 generation which promotes an increase of obesity [61], Table 1. Fungicides have also caused transgenerational effects on the offspring. The agricultural fungicide vinclozolin has been used globally and been shown to cause promotion of transgenerational diseases [63]. Vinclozolin blocks androgen receptors and is a testosterone antagonist [63]. Using a rat model, researchers exposed a gestating female rat (F0 generation) to vinclozolin [63]. Impacts of the exposure were observed on male fertility through reduced sperm number within the subsequent generations, that involved altered DNA methylation patterns [63], Table 1. Spermatogenic cell death analysis was observed through quantification of apoptosis at the single sperm cell level [63]. This process showed that within the testes, many spermatogenic cells had undergone apoptosis to reduce sperm number [63]. In conclusion, exposure to vinclozolin can induce germline epigenetic changes [63], and demonstrate these epigenetic changes can be inherited transgenerationally and contribute to transgenerational phenotypes [63]. A recent study done with multiple-generational exposure to different toxicants for three generations promoted high levels of transgenerational phenotypes [60]. The F0 generation was exposed to vinclozolin, F1 generation was exposed to jet fuel, and F2 generation was exposed to DDT [60]. Exposure to toxicants then stopped for the F3 generation, F4 generation, and the F5 generation [60]. The F5 generation would be the first transgenerational generation, not directly exposed to a toxicant [60]. Observations demonstrated that the F3–F5 generations sperm had a similarity of differential DNA methylation regions (DMRs) [60]. However, within the F1-F3 generations, there was minimal overlap of DMRs [60]. This suggests that there was a reprogramming of epigenetics at the start of each generation being directly exposed [60]. Researchers concluded that the multiple generational exposure promoted higher levels of disease and pathology, even more so in the subsequent transgenerational generations [60]. In addition to toxicants shown in Table 1, a wide range of environmental exposures from temperature, trauma and stress, to nutrition can also promote the epigenetic transgenerational inheritance of phenotypic variation and pathology [52], Figure 4

Figure 4. Environmentally induced epigenetic transgenerational inheritance and generational toxicology. A variety of toxicants and exposures are listed that promote epigenetic transgenerational inheritance. A number of different organisms the environmental exposure promotes epigenetic transgenerational inheritance are presented, from plants to humans. Modified from Nilsson, et al. [21].

Transgenerational inheritance of epigenetics and generational stability of phenotypes explains how an environmental exposure can promote a generationally stable phenotypic change in all species examined transgenerationally, Figure 4. Therefore, a wide variety of environmental factors from toxicants to nutrition to temperature can promote transgenerational phenotypic variation and pathology in a wide variety of species from plants, flies, worms, fish, birds, rodents, pigs, and humans [52], Table 1B and Figure 4. Adding epigenetic inheritance into the discussion of evolution is essential for a better understanding of the evolutionary models as a whole. Generational stability of transgenerational epigenetics in species has been shown to promote specific phenotype development in every generation of subsequent offspring from the same lineage. We would not call this generational stability if the same phenotype or pathology was not exhibited in all subsequent generations after exposure. Observations indicate environmentally induced phenotypic and epigenetic alterations have been found to be generationally stable [60,61,63]. Epigenetic transgenerational inheritance facilitates this generational stability and thus can impact phenotypic variation and natural selection [16,64], Figure 1.

Evolution and epigenetic transgenerational inheritance

Epigenetic transgenerational inheritance is induced by the environment through all organisms and species investigated, Table 1B and Figure 4. An example in plants was done when researchers exposed pathogens, heavy metals, and severe temperature changes to their plant subjects [65]. When the plants were exposed to these different stressors, the plants were shown to alter photosynthesis and overall growth processes [65]. Other recent studies have shown that epigenetic mechanisms are important to the plant’s phenotypic variation [66–68]. Another study done in plants dealt with floral symmetry and epigenetic mutations [69]. A gene called Lcyc is found in a flowering plant named Antirrhinum [70]. The Lcyc gene is the driving force of the growth rate of the flower and the symmetry of the flower [70]. Researchers found that when the Lcyc gene is methylated the flowering plant phenotype changes through several generations [69]. In a comparison of the wild-type flowers to the mutant peloric, the wild-type flowers had a stunted stamen, however in the mutant peloric the stamen was normal [69]. These examples in plants have been shown to exhibit phenotypic changes when there is an environmental and epigenetic change.

Marine animals have also been shown to inherit epigenetic alterations. In wild Pacific oysters (Crassostrea gigas) heat shock has been shown to cause physiological phenotypes [71]. Wild Pacific oyster collected from intertidal pools and bred for two generations in a subtidal pool were exposed to heat shock [71]. Researchers found that transgenerational plasticity could influence physiological phenotypes that are linked to oxidation status and energy within the non-stress subtidal offspring [71]. Differentially methylated genes that were found in the F0 generation were passed down to the F2 generation [71]. Researchers suggested that due to this factor, this may help control the organism’s growth and development [71]. Intertidal experience also influenced the thermal plasticity of physiological phenotypes within and across generations [71]. In mollusk species, an asexual clonal snail invasive to North America was investigated [72,73]. Researchers found that when changing the flow of water in the snail’s environment, these changes induced alterations to the epigenetics and phenotype of the snail’s shell shape [72]. In another study using a snail model, researchers collected asexual snails from three separate lakes found in the Northwest USA and compared DNA methylation variation using differentially methylated regions (DMRs) [73]. Snails found in lake 1 (rural lake) and lake 2 (urban lake) had higher number of epigenetic variation than lake 1 (rural lake) and lake 3 (urban lake) [73]. Researchers suggested that dispersal distance among the lakes may be a cause for epigenetic alterations and may be associated with phenotypic changes in shell shape and size of the species [73]. Fish species have also been found to show adaptive phenotypic variation and epigenetic variations [74]. Poecilia is a species of fish that primarily lives in freshwater environments with high levels of sulfur and has been shown to have generational epigenetic changes found in the assessment of DMRs [74,75]. In Atlantic salmon, early hatchery induced maturation was hypothesized to be occurring due to high levels of DNA methylation and epigenetic processes [76]. Hatchery spawned steelhead trout (Oncorhynchus mykiss) were compared to wild caught fish on a molecular level [75]. Results showed that there were epigenetic changes between the hatchery fish and the wild fish, however, negligible genetic differences were observed [75]. This suggested that the environment at the hatcheries can alter development due to epigenetic changes in the fish [75]. Another species dealing with high levels of compounds in their environments was the marine tubeworm with high salinity [77]. The marine tubeworm (Hydroides diramphus) was faced with salinity stress before reproduction and then was assessed for phenotypic changes in their gametes and their offspring phenotypes [77]. Results showed that the mother and father would adapt and alter the phenotype of their gametes in order for the best survival for their offspring [77]. Due to these numerous observations, epigenetic transgenerational inheritance and epigenetic mechanisms must be discussed and considered within the subject of evolutionary biology.

A similar trend of epigenetic inheritance can be found in other animals, Table 1B. Examples in birds have shown alterations in epigenetic variation and phenotype of the house sparrow (Passer domesticus) where there was higher epigenetic variation compared to genetic variation [78,79]. A bird study examined the urban and rural populations of Darwin’s finches in the Galapagos and their epigenetic and phenotypic variation [56]. The researchers found the finches in the rural environment and the finches in the urban environment had no genetic variation but had epigenetic variation, and different phenotypes [56]. A similar study looked at five different species of Darwin’s finches and the same conclusion was found [55]. There was less genetic variation compared to epigenetic variation [55]. There has been ample research done on epigenetic variation and epigenetic inheritance in mammalian models as well. In rats, an F0 generation pregnant rat was exposed to an agricultural pesticide (N-(phosphonomethyl) glycine) glyphosate [59]. The directly exposed generations (F0 and F1 generations) did not have many pathologies or disease [59]. The F2 and F3 subsequent generations had a significant increase in pathology of disease including kidney disease, ovarian disease, prostate disease, and obesity, Table 1. This correlated with the F1, F2 and F3 generations sperm alterations in differential DNA methylation regions (DMRs) [59]. The DMRs found have previously been linked to the pathologies observed [59]. Observations suggest that glyphosate can cause generational toxicology [59]. A similar epigenetic transgenerational inheritance study was done using a rat model and exposure of the gestating female to vinclozolin [80]. When F0 generation rats were exposed to vinclozolin and outcrossed for multiple generations, the later generations had significant alterations in epigenetics and phenotypes [80], Table 1A. The later transgenerational generations were not directly exposed to vinclozolin [80]. In the transgenerational generations, there was an increase in spermatogenic cell apoptosis in the testes [80]. Results suggested that exposure to vinclozolin during the time of male sex determination can promote decreased sperm production being epigenetically inherited transgenerationally [80]. Another study done in rats showed how diet can influence the inheritance of epigenetic factors [81]. The paternal male rats were under a specific high-fat diet. Results showed that the daughters of the father under a high-fat diet promoted glucose intolerance and insulin secretion problems in offspring [81]. Another study done in mice investigated four genes that regulate the color of fur in mice [82]. Researchers found that the Agouti mice having darker coat color will have more methylation on the allele Avy than that of the mice that had yellow colored fur [82]. These results suggested that since there is not a complete erasure when silenced Avy allele passes through the female reproductive cells, there will be an inheritance of epigenetic changes [82].

There have also been studies done on humans and epigenetic inheritance. During the time of World War two (WWII), the Dutch population in the Netherlands were forced into a famine due to German forced closure of all railways [83]. The daily calorie intake for the people of Netherlands fell below 1000 calories [83]. The worst of the Dutch famine spanned from the end of 1944 to beginning of 1945 [83]. During the Dutch famine, many people were restricted to daily rations of at most 800 calories a day [83]. Researchers found that the offspring of the women that experienced the Dutch famine had issues with glucose intolerance, increased obesity, and coronary heart disease [83]. Specifically, when a woman was in early gestation and exposed to famine, the offspring had an increased risk of breast cancer [83]. Likewise, when a woman was exposed to famine during mid gestation her offspring had increased cases of kidney damage and airway diseases [83]. These results suggested that depending on when a woman was gestating and exposed to famine would impact the outcome of the offspring overall health [83]. Subsequent generation analyses have demonstrated inherited epigenetics and metabolic disease phenotypes [83], Table 1 and Figure 4. From the years 1961–1971, hundreds of thousands of soldiers in Vietnam were exposed to the pesticide, agent orange (2,3,7,8-tetrachlorodibenzo[p]dioxin, TCDD) [84]. Due to this exposure to the harmful industrial compound around 400,000 people died or the offspring of those exposed were born with birth defects [84]. Humans exposed to high levels of DDT have also had offspring with issues of reproductive disease, cancers, neurological disease, and abnormalities in development [85–88]. Other human studies involving reproductive disorders have been done highlighting a prominent disorder called polycystic ovary syndrome (PCOS) [89]. PCOS is heritable through offspring; however, the exact pathway of how was undetermined [89]. Researchers have found DNA hypomethylation is one of the factors that regulate genes that are aligned with PCOS [89]. Endocrine disruptor chemicals have also been found to be associated with human health and more specifically, birth outcomes [90]. PCOS affects birth outcomes such as pre-term birth and decreased birth weight [90]. Therefore, the human population also has demonstrated environmentally induced epigenetic transgenerational inheritance phenomenon of phenotypic variation.

Conclusion

It is important to incorporate all aspects of science to better understand adaptation and evolution. Since the first publication of the modern synthesis, there have been advancements made to incorporate other theories. The extended evolutionary model had the original view of modern synthesis, however, included the concepts and mechanisms of the epigenome, but not as drivers of the evolution process. From the examples above, epigenetics and the epigenome can drive phenotypic variation independent of genetic mutations. However, it is noted that the extended evolutionary theory did not include the concept that the environment can directly impact phenotypic variation. In a previous review, the topic of a Unified theory of evolution was discussed [16]. Like the extended evolutionary theory, the unified theory of evolution adds the epigenome, but also epigenetic transgenerational inheritance [16]. The unified theory integrated that the environment drives molecular systems to change phenotypes, independent of genetic change [16]. Having a unified theory of evolution that integrates the epigenome, epigenetics, and how the environment can influence biology broadens our outlook on evolution. Epigenetics has been shown to pass traits down from one generation to the next without changing the DNA sequence [91]. Epigenetic transgenerational inheritance across generations offers a way for traits and phenotypic variation to be passed down and thus influences how the evolution process occurs [91], Figure 1. Understanding how epigenetic changes vary and how they can affect evolution is important for predicting how well organisms can adapt to their changing environments [92].

From the examples above, one may conclude that the epigenome, epigenetic inheritance, and the impact of the environment has on altering a species’ phenotype and pathology need to be added to the discussion of the evolutionary theory. It is to be noted that we do not dismiss the ideas of the modern synthesis or Darwinian theory as a whole. However, the addition of newer concepts and theories need to be added to make a unified theory of evolution. Previous studies discussed have shown that when there is environmental exposure to a toxicant, stressor, or environment, the species will exhibit epigenetic variation that promotes phenotypic variation [55,56,59,64–84,89,90,93], Table 1 and Figure 4. Darwin’s evolutionary theory and postulates are supported by phenotypic variation, epigenetic variation, and epigenetic transgenerational inheritance. As mentioned previously, with the novel research that has been done in a wide range of species, denying that there is no evidence of an environmentally responsive non-genetic inheritance (epigenetic) that drives phenotypic variation will not advance our understanding of evolution as a whole. Incorporating epigenetics and epigenetic transgenerational inheritance into the discussion and study of evolution will provide a deeper comprehensive understanding of how adaptation, inheritance, and evolutionary change progresses, Figure 1. This gives detailed insights into how organisms change in response to their environment while providing insight into the driving forces of the evolutionary process.

Author contributions

SK data analysis, wrote and edited manuscript.

MKS funding acquisition and edited manuscript.

Acknowledgments

We acknowledge Ms. Heather Johnson for assistance in preparation of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this article as no new data were created or analysed in this study.

Additional information

Funding

This study was supported by the John Templeton Foundation (50183 and 61174) (https://templeton.org/) grants to MKS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.