E. coli long-term evolution experiment

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https://en.wikipedia.org/wiki/E._coli_long-term_evolution_experiment

 

The 12 E. coli LTEE populations on June 25, 2008.

 

The E. coli long-term evolution experiment (LTEE) is an ongoing study in experimental evolution led by Richard Lenski that has been tracking genetic changes in 12 initially identical populations of asexual Escherichia coli bacteria since 24 February 1988. The populations reached the milestone of 50,000 generations in February 2010 and 66,000 in November 2016. Lenski performed the 10,000th transfer of the experiment on March 13, 2017.

Over the course of the experiment, Lenski and his colleagues have reported a wide array of phenotypic and genotypic changes in the evolving populations. These have included changes that have occurred in all 12 populations and others that have only appeared in one or a few populations. For example, all 12 populations showed a similar pattern of rapid improvement in fitness that decelerated over time, faster growth rates, and increased cell size. Half of the populations have evolved defects in DNA repair that have caused mutator phenotypes marked by elevated mutation rates. The most striking adaptation reported so far is the evolution of aerobic growth on citrate, which is unusual in E. coli, in one population at some point between generations 31,000 and 31,500.

Experimental approach

The long-term evolution experiment was designed as an open-ended means of empirical examination of central features of evolution. The experiment was begun with three principal goals:

1. To examine the dynamics of evolution, including the rate of evolutionary change.
2. To examine the repeatability of evolution.
3. To better understand the relationship between change on the phenotypic and genotypic levels.

As the experiment has continued, its scope has grown as new questions in evolutionary biology have arisen that it can be used to address, as the populations' evolution has presented new phenomena to study, and as technology and methodological techniques have advanced.

The use of E. coli as the experimental organism has allowed many generations and large populations to be studied in a relatively short period of time. Moreover, due to the long use of E. coli as a principle model organism in molecular biology, a wide array of tools, protocols, and procedures were available for studying changes at the genetic, phenotypic, and physiological levels. The bacteria can also be frozen and preserved while remaining viable. This has permitted the creation of what Lenski describes as a "frozen fossil record" of samples of evolving populations that can be revived at any time. This frozen fossil record allows populations to be restarted in cases of contamination or other disruption in the experiment, and permits the isolation and comparison of living exemplars of ancestral and evolved clones. Lenski chose an E. coli strain that reproduces only asexually, lacks any plasmids that could permit bacterial conjugation, and has no viable prophage. As a consequence, evolution in the experiment occurs only by the core evolutionary processes of mutation, genetic drift, and natural selection. This strict asexuality also means that genetic markers persist in lineages and clades by common descent, but cannot otherwise spread in the populations.

Lenski chose to carry out the experiment with the bacteria grown in a glucose-limited minimal medium called DM25, which was initially developed by Bernard Davis for use in isolating auxotrophic mutants of E. coli using penicillin as a selective agent. DM25 is supplemented with a low concentration of glucose. Lenski chose this concentration to simplify analysis of the populations' evolution by reducing clonal interference, in which multiple versions of alleles are competing in an evolving population, while also reducing the possibility of the evolution of ecological interactions. This concentration of glucose used supports a maximum population of 500 million cells of the ancestor in a 10 mL culture, though the maximum now varies among the evolved populations. DM25 also contains a large amount of citrate (about 11 times the concentration of glucose), which was originally included by Davis because it improved the killing efficiency of penicillin during his experiments, though it is now known to aid in E. coli's acquisition of iron from the medium.

Methods

The 12 populations are maintained in a 37 °C (99 °F) incubator in Lenski's laboratory at Michigan State University. Each day, 1% of each population is transferred to a flask of fresh DM25 growth medium. The dilution means that each population experiences 6.64 generations, or doublings, each day. Large, representative samples of each population are frozen with glycerol as a cryoprotectant at 500-generation (75-day) intervals. The bacteria in these samples remain viable, and can be revived at any time. This collection of samples is referred to as the "frozen fossil record", and provides a history of the evolution of each population through the entire experiment. The populations are also regularly screened for changes in mean fitness, and supplemental experiments are regularly performed to study interesting developments in the populations. As of April 2016, the E. coli populations have been under study for over 64,500 generations, and are thought to have undergone enough spontaneous mutations that every possible single point mutation in the E. coli genome has occurred multiple times.

Founding strain

The strain of E. coli Lenski chose to use in the long-term evolution experiment was derived from "strain Bc251", as described in a 1966 paper by Seymour Lederberg, via Bruce Levin, who had used it in a bacterial ecology experiment in 1972. The defining genetics traits of this strain were: T6^r, Str^r, r⁻m⁻, Ara⁻ (unable to grow on arabinose). Lenski designated the original founding strain as REL606. Before the beginning of the experiment, Lenski isolated an Ara⁺ variant of the strain in which a point mutation in the ara operon had restored growth on arabinose, which he designated as strain REL607. When beginning the long-term evolution experiment, Lenski founded six populations with six individual Ara⁻ colonies of REL606. These populations are referred to as Ara-1 through Ara-6. Lenski also founded six more populations from six individual Ara⁺ colonies of REL607. These are referred to as populations Ara+1 through Ara+6. The marker differences permit strains to be differentiated on Tetrazolium Arabinose plates, on which Ara⁻ colonies appear red, while Ara⁺ colonies appear white to pink. Over the course of the experiment, each population has accumulated a large number of distinct mutations, which permit further means of identifying strains by their population of origin.

Results

 

Changes in fitness

Timeline of the E. coli long-term evolution experiment, showing relationship between years and generations of evolution, as well as significant events and findings.

 

Much analysis of the experiment has dealt with how the fitness of the populations relative to their ancestral strain has changed. All populations showed a pattern of rapid increase in relative fitness during early generations, with this increase decelerating over time. By 20,000 generations the populations grew approximately 70% faster than the ancestral strain. This increase and deceleration in increase has continued in subsequent generations. A 2013 study by Wiser et al. reported ongoing improvement at 50,000 generations relative to samples isolated at 40,000 generations. They found that the fitness increase fit to a power law model much better than the hyperbolic models that had been used earlier. As a power law model describes an ever-slowing increase that has no upper limit, while a hyperbolic model implies a hard limit, the work suggested that the increase would continue without bound as progressively lower benefit mutations were fixed in the populations. Further work published in 2015 reported the results of over 1100 new fitness assays that examined fitness changes through 60,000 generations. The data once again fit the proposed power law model, and, indeed, fit within predictions of the model from earlier data. These results suggest that, contrary to previous thinking, adaptation and adaptive divergence can potentially increase indefinitely, even in a constant environment.

Genome evolution

Of the 12 populations, six have so far been reported to have developed defects in their ability to repair DNA, greatly increasing the rate of mutation in those strains. Although the bacteria in each population are thought to have generated hundreds of millions of mutations over the first 20,000 generations, Lenski has estimated that within this time frame, only 10 to 20 beneficial mutations achieved fixation in each population, with fewer than 100 total point mutations (including neutral mutations) reaching fixation in each population. In 2009, Barrick et al. reported the results of genome sequences from multiple time points in population Ara-1. They found that, unlike the declining rate of fitness improvement, mutation accumulation was linear and clock like, even though several lines of evidence suggested that much of the accumulation was beneficial, rather than neutral.

Evolution of increased cell size in all twelve populations

Growth in cell size of bacteria in the Lenski experiment

All twelve of the experimental populations show an increase in cell size concurrent with a decline in maximum population density, and in many of the populations, a more rounded cell shape.^[24] This change was partly the result of a mutation that changed the expression of a gene for a penicillin-binding protein, which allowed the mutant bacteria to outcompete ancestral bacteria under the conditions in the long-term evolution experiment. However, although this mutation increased fitness under these conditions, it also increased the bacteria's sensitivity to osmotic stress and decreased their ability to survive long periods in stationary phase cultures.

Ecological specialization

Over the course of the experiment, the populations have evolved to specialize on the glucose resource on which they grow. This was first described in 2000, when Cooper and Lenski demonstrated that all populations had experienced decay of unused metabolic functions after 20,000 generations, restricting the range of substances on which the bacteria could grow. Their analysis suggested that this decay was due to antagonistic pleiotropy, in which mutations that improved ability to grow on glucose had reduced or eliminated the ability to grow on other substances.^[25] A later study by Leiby and Marx that used more advanced techniques showed that much of the decay Cooper and Lenski had identified were experimental artifacts, that loss of unused functions was not as extensive as first thought, and that some unused functions had improved. Moreover, they concluded that the metabolic losses were not due to antagonistic pleiotropy, but the neutral accumulation of mutations in unused portions of the genome, suggesting that adaptation to a simple environment might not necessarily lead to specialization.

Evolution of balanced polymorphism and simple ecosystems

Two distinct variants, S and L, were identified in the population designated Ara-2 at 18,000 generations based on their formation of small and large colonies, respectively. Clones of the S and L types could co-exist stably in co-culture with each other, indicating they occupied distinct niches in the population. This was verified by the finding that the L type had an advantage during growth on glucose, but that S had an advantage during stationary phase, after glucose had run out. The two types were found to have initially evolved prior to 6,000 generations, and then co-existed thereafter. Phylogenetic analysis of clones of the two types isolated from different generations demonstrated that the S and L types belonged to distinct, co-existing lineages in the population, and might be undergoing incipient speciation.

Evolution of aerobic citrate usage in one population

Background

The population designated Ara-3 (center) is more turbid because that population evolved to use the citrate present in the growth medium.

 

E. coli is normally unable to grow aerobically on citrate due to the inability to express a citrate transporter when oxygen is present. However, E. coli has a complete citric acid cycle, and therefore metabolizes citrate as an intermediate during aerobic growth on other substances, including glucose. Most E. coli can grow anaerobically on citrate via fermentation, if a co-substrate such as glucose is available to provide reducing power. The anaerobic growth is possible due to the expression of a transmembrane citrate-succinate antiporter gene, citT, which was first identified in 1998. This gene is co-regulated with other genes involved in citrate fermentation found on the cit operon, which is turned on only when oxygen is absent.

The inability to grow aerobically on citrate, referred to as a Cit⁻ phenotype, is considered a defining characteristic of E. coli as a species, and one that has been a valuable means of differentiating E. coli from pathogenic Salmonella. Although Cit⁺ strains of E. coli have been isolated from environmental and agricultural samples, in every such case, the trait was found to be due to the presence of a plasmid that carries a foreign citrate transporter. A single, spontaneous Cit⁺ mutant of E. coli was reported by Hall in 1982. This mutant had been isolated during prolonged selection for growth on another novel substance in a growth broth that also contained citrate. Hall's genetic analysis indicated the underlying mutation was complex, but he was ultimately unable to identify the precise changes or genes involved, leading him to hypothesize activation of a cryptic transporter gene. The genome regions to which Hall was able to narrow down the locations of the changes do not correspond to the known location of the citT gene identified 16 years later, nor did the physiological characteristics in transport assays of Hall's Cit⁺ mutants match those to be expected for aerobic expression of the CitT transporter.

Cit⁺ evolves in the LTEE

In 2008, Lenski's team, led by Zachary D. Blount, reported that the ability to grow aerobically on citrate had evolved in one population. Around generation 33,127, a dramatic increase in turbidity was observed in the population designated Ara-3. They found that the population contained clones that were able to grow aerobically on citrate (Cit⁺). This metabolic capacity permitted the population to grow several-fold larger than it had previously, due to the large amount of citrate present in the medium. Examination of frozen fossil samples of the populations showed that Cit⁺ clones could be isolated as early as 31,500 generations. The Cit⁺ variants in the population were found to possess a number of genetic markers unique to the Ara-3 population; this observation excluded the possibility that the clones were contaminants, rather than spontaneous mutants. In a series of experiments that "replayed" the tape of Ara-3 evolution from Cit⁻ clones isolated from samples frozen at various time points in the population's history, they demonstrated that the ability to grow aerobically on citrate was more likely to re-evolve in a subset of genetically pure, evolved clones. In these experiments, they observed 19 new, independent instances of Cit⁺ re-evolution, but only when starting from clones isolated from after generation 20,000. Fluctuation tests showed that clones from this generation and later displayed a rate of mutation to the Cit⁺ trait which was significantly higher than the ancestral rate. Even in these later clones, the rate of mutation to Cit⁺ was on the order of one occurrence per trillion cell divisions.

Lenski and his colleagues concluded that the evolution of the Cit⁺ function in this one population arose due to one or more earlier, possibly nonadaptive, "potentiating" mutations that increased the rate of mutation to an accessible level. The data suggested that citrate usage involved at least two mutations subsequent to these "potentiating" mutations. More generally, the authors suggest these results indicate, following the argument of Stephen Jay Gould, "that historical contingency can have a profound and lasting impact" on the course of evolution. These findings have come to be considered a significant instance of the impact of historical contingency on evolution.

Genomic analysis of the Cit⁺ trait and implications for evolutionary innovation

The Cit⁺ trait was actualized by a duplication mutation that created a new regulatory module by placing a copy of the citT gene that encodes a citrate-succinate antiporter under the control of a promoter that supports expression under aerobic conditions. This mutation results in the CitT transporter being expressed when oxygen is present, permitting growth on citrate.

 

In 2012, Lenski and his team reported the results of a genomic analysis of the Cit⁺ trait that shed light on the genetic basis and evolutionary history of the trait. The researchers had sequenced the entire genomes of twenty-nine clones isolated from various time points in the Ara-3 population's history. They used these sequences to reconstruct the phylogenetic history of the population; this reconstruction showed that the population had diversified into three clades by 20,000 generations. The Cit⁺ variants had evolved in one of these, which they called Clade 3. Clones that had been found to be potentiated in earlier research were distributed among all three clades, but were over-represented in Clade 3. This led the researchers to conclude that there had been at least two potentiating mutations involved in Cit⁺ evolution.

The researchers also found that all Cit⁺ clones had mutations in which a 2933-base-pair segment of DNA was duplicated or amplified. The duplicated segment contained the gene citT for the citrate transporter protein used in anaerobic growth on citrate. The duplication is tandem, and resulted in copies that were head-to-tail with respect to each other. This new configuration placed a copy of the previously silent, unexpressed citT under the control of the adjacent rnk gene's promoter, which directs expression when oxygen is present. This new rnk-citT module produced a novel regulatory pattern for citT, activating expression of the citrate transporter when oxygen was present, and thereby enabled aerobic growth on citrate.

Movement of this rnk-citT module into the genome of a potentiated Cit⁻ clone was shown to be sufficient to produce a Cit⁺ phenotype. However, the initial Cit⁺ phenotype conferred by the duplication was very weak, and only granted a ~1% fitness benefit. The researchers found that the number of copies of the rnk-citT module had to be increased to strengthen the Cit⁺ trait sufficiently to permit the bacteria to grow well on the citrate. Further mutations after the Cit⁺ bacteria became dominant in the population continued to accumulate improved growth on citrate. 

The researchers concluded that the evolution of the Cit⁺ trait occurred in three distinct phases: (1) mutations accumulated that increased the rate of mutation to Cit⁺, (2) the trait itself appeared in a weak form, and (3) the trait was improved by later mutations. Blount et al. suggested that this pattern might be typical of how novel traits in general evolve, and proposed a three-step model of evolutionary innovation:

1. Potentiation: a genetic background evolves in which a trait is mutationally accessible, making the trait's evolution possible.
2. Actualization: a mutation occurs that produces the trait, making it manifest, albeit likely in a weak form.
3. Refinement: Once the trait exists, if it provides selective benefit, mutations will accumulate that improve the trait, making it effective. This phase is open-ended, and will continue so long as refining mutations arise and the trait remains beneficial.

This model has seen acceptance in evolutionary biology. In 2015 paleontologist Douglas Erwin suggested a modification to a four-step model to better reflect a possible distinction between evolutionary novelty and evolutionary innovation, and to highlight the importance of environmental conditions: potentiation, generation of novel phenotypes (actualization), adaptive refinement, and exploitation (conversion of a novelty to an innovation as it becomes important for the ecological establishment of possessing organisms).

Investigation of potentiation

In 2014, a research team led by Eric Quandt in the lab of Jeffrey Barrick at the University of Texas at Austin described the application of a new technique called Recursive Genomewide Recombination and Sequencing (REGRES) to identify potentiating mutations among the 70 present in the Ara-3 lineage that evolved Cit⁺. This method used multiple rounds of a process in which F plasmid based conjugation between a 33,000 generation Cit⁺ clone, CZB154, and the Cit⁻ founding clone of the LTEE to purge mutations not required for either manifestation of a weak or strong form of the Cit⁺ trait, which they refer to as Cit⁺⁺. They found that the rnk-citT module responsible for the phenotypic switch to Cit⁺ was sufficient to produce a weak Cit⁺ phenotype in the ancestor. They also identified a mutation that had occurred in the lineage leading to CZB154 after the initial evolution of Cit⁺ that conferred a strong, Cit⁺⁺ phenotype in the ancestor absent any mutation but the rnk-citT module. This mutation, found in the regulatory region of a gene called dctA, caused a massive increase in the expression of the DctA transporter, which functions to import C₄-dicarboxylates into the cell. This increased DctA expression, they found, permitted Cit⁺ cells to re-uptake succinate, malate, and fumarate released into the medium by the CitT transporter during import of citrate. They identified a similar mutation in Cit⁺⁺ clones in the Ara-3 population that increased DctA expression by restoring function to a gene that regulates it, dcuS, that had been deactivated in the ancestral clone. Quandt et al. concluded that the dctA mutation was not involved in potentiation, but refinement. This led them to suggest that evolution of Cit⁺ in the Ara-3 population might have been contingent upon a genetic background and population-specific ecology that permitted the early, weak Cit⁺ variants to persist in the population long enough for refining mutations to arise and render growth on citrate strong enough to provide a significant fitness benefit. 

Quandt and colleagues later published findings definitively identifying a mutation that did potentiate Cit⁺ evolution. This mutation was in the gltA gene, which encodes citrate synthase, an enzyme involved in the flow of carbon into the citric acid cycle. It had the effect of increasing citrate synthase activity, and they showed that it permitted improved growth on acetate. Moreover, with the gltA mutation, the rnk-citT module that causes the Cit⁺ trait has a neutral-to-slightly beneficial fitness effect, while, without it, the module was strongly detrimental. The gltA mutation therefore seems to have permitted early, weak Cit⁺ variants to persist in the population until later refining mutations could occur, consistent with their earlier conclusions. After a strong Cit⁺⁺ phenotype evolved, the increased citrate synthase activity became detrimental. The researchers found that later mutations in gltA countered the first mutation, reducing citrate synthase activity, and further improving growth on citrate. They concluded that the series of mutation in gltA first potentiated, and then refined growth on citrate. They also suggested that the lineage in which Cit⁺ arose might have occupied a niche in Ara-3 based on growth on acetate, and that the potentiating mutations that led to evolution of Cit⁺ in Ara-3 were originally adaptive for acetate use. 

Investigation of post-Cit⁺ ecology and persistent diversity

A small subpopulation of Cit⁻ cells unable to grow on citrate, and belonging to a separate clade persisted in the population after the Cit⁺ cells became dominant. Early findings showed that this diversity was partly due to the Cit⁻ cells being better at growing on the glucose in the medium. Turner et al. later found that another factor behind the coexistence was that the Cit⁻ cells evolved the ability to cross feed on the Cit⁺ majority. They showed that the Cit⁺ cells release succinate, malate, and fumarate during growth on citrate, as the CitT transporter pumps these substances out of the cell while pumping citrate into the cell. The Cit⁻ cells had rapidly evolved the ability to grow on these substances due to a mutation that restored expression of an appropriate transporter protein that was silent in the ancestor.

The Cit⁻ subpopulation eventually went extinct in the population between 43,500 and 44,000 generations. This extinction was shown to not be due to the Cit⁺ majority evolving to be able to invade the niche occupied by the Cit⁻ minority. Indeed, Cit⁻ clones could invade Cit⁺ populations from after the extinction event. Moreover, in an experiment in which they restarted twenty replicates of the Ara-3 population from the sample frozen 500 generations before the extinction, Turner et al. found that the Cit⁻ subpopulation had not gone extinct in any of the replicates after 500 generations of evolution. One of these replicates was continued for 2,500 generations, over which Cit⁻ continued to coexist. The researchers concluded that the extinction of Cit⁻ had been due to some unknown "rare environmental perturbation", similar to that which can impact natural populations. The final replicate was integrated into the main LTEE experiment, becoming the thirteenth population, Ara-7.

Criticism of citrate-usage findings

Other researchers have experimented on evolving aerobic citrate-utilizing E. coli. Dustin Van Hofwegen et al., working in the lab of intelligent design proponent Scott Minnich, were able to isolate 46 independent citrate-utilizing mutants of E. coli in just 12 to 100 generations using highly prolonged selection under starvation, during which the bacteria would sample more mutations more rapidly. In their research, the genomic DNA sequencing revealed an amplification of the citT and dctA loci, and rearrangement of DNA were the same class of mutations identified in the experiment by Richard Lenski and his team. They concluded that the rarity of the citrate-utilizing mutant in Lenski's research was likely a result of the selective experimental conditions used by his team rather than being a unique evolutionary speciation event.

John Roth and Sophie Maisnier-Patin reviewed the approaches in both the Lenski team's delayed mutations and the Van Hofweges team's rapid mutations on E. coli. They argue that both teams experienced the same sequence of potentiation, actualization, and refinement leading up to similar Cit⁺ variants. According to them, the period of less than a day during which citrate usage would be under selection, followed by 100-fold dilution, and a period of growth on glucose that would not select for citrate use, ultimately lowered the probability of E. coli being able to accumulate early adaptive mutations from one period of selection to the next. On the other hand, Van Hofwegen's team allowed for a continuous selection period of 7 days, which yielded a more rapid development of citrate-using E. coli. Roth and Maisnier-Patin suggest that the serial dilution of E. coli and short period of selection for citrate-use under the conditions of the LTEE perpetually impeded each generation of E. coli from reaching the next stages of aerobic citrate utilization.

In response, Blount and Lenski acknowledge that the problem is not with the experiments or the data, but with the interpretations made by Van Hofwegen et al. and Maisnier-Patin and Roth. Lenski notes that the rapid evolution of Cit⁺ was not necessarily unexpected since his team was also able to produce multiple Cit⁺ mutants in a few weeks during the replay experiments they reported in the 2008 paper in which his team first described the evolution of aerobic citrate use in the LTEE. Furthermore, Lenski criticizes Van Hofwegen et al.'s description of the initial evolution of Cit⁺ as a "speciation event" by pointing out that the LTEE was not designed to isolate citrate-using mutants or to deal with speciation since in their 2008 paper they said "that becoming Cit⁺ was only a first step on the road to possible speciation", and thus did not propose that the Cit⁺ mutants were a different species, but that speciation might be an eventual consequence of the trait's evolution. Lenski acknowledges that scientists, including him and his team, often use short hand and jargon when discussing speciation, instead of writing more carefully and precisely on the matter, and this can cause issues. However, he notes that speciation is generally considered by evolutionary biologists to be a process, and not an event. He also criticizes Van Hofwegen et al. and Roth and Maisnier-Patin for positing "false dichotomies" regarding the complex concept of historical contingency. He argues that historical contingency means that history matters, and that their 2008 paper presented data that showed that the evolution of Cit⁺ in the LTEE was contingent upon mutations that had accumulated earlier. He concludes that "...historical contingency was invoked and demonstrated in a specific context, namely that of the emergence of Cit⁺ in the LTEE—it does not mean that the emergence of Cit⁺ is historically contingent in other experimental contexts, nor for that matter that other changes in the LTEE are historically contingent—in fact, some other evolved changes in the LTEE have been highly predictable and not (or at least not obviously) contingent on prior mutations in the populations."

Hypoxia (environmental)

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https://en.wikipedia.org/wiki/Hypoxia_(environmental)

Hypoxia refers to low oxygen conditions. Normally, 20.9% of the gas in the atmosphere is oxygen. The partial pressure of oxygen in the atmosphere is 20.9% of the total barometric pressure. In water, oxygen levels are much lower, approximately 1%, and fluctuate locally depending on the presence of photosynthetic organisms and relative distance to the surface (if there is more oxygen in the air, it will diffuse across the partial pressure gradient).

Atmospheric hypoxia

Atmospheric hypoxia occurs naturally at high altitudes. Total atmospheric pressure decreases as altitude increases, causing a lower partial pressure of oxygen which is defined as hypobaric hypoxia. Oxygen remains at 20.9% of the total gas mixture, differing from hypoxic hypoxia, where the percentage of oxygen in the air (or blood) is decreased. This is common in the sealed burrows of some subterranean animals, such as blesmols. Atmospheric hypoxia is also the basis of altitude training which is a standard part of training for elite athletes. Several companies mimic hypoxia using normobaric artificial atmosphere.

Aquatic hypoxia

Oxygen depletion is a phenomenon that occurs in aquatic environments as dissolved oxygen (DO; molecular oxygen dissolved in the water) becomes reduced in concentration to a point where it becomes detrimental to aquatic organisms living in the system. Dissolved oxygen is typically expressed as a percentage of the oxygen that would dissolve in the water at the prevailing temperature and salinity (both of which affect the solubility of oxygen in water; see oxygen saturation and underwater). An aquatic system lacking dissolved oxygen (0% saturation) is termed anaerobic, reducing, or anoxic; a system with low concentration—in the range between 1 and 30% saturation—is called hypoxic or dysoxic. Most fish cannot live below 30% saturation. Hypoxia leads to impaired reproduction of remaining fish via endocrine disruption. A "healthy" aquatic environment should seldom experience less than 80%. The exaerobic zone is found at the boundary of anoxic and hypoxic zones. 

Hypoxia can occur throughout the water column and also at high altitudes as well as near sediments on the bottom. It usually extends throughout 20-50% of the water column, but depending on the water depth and location of pycnoclines (rapid changes in water density with depth). It can occur in 10-80% of the water column. For example, in a 10-meter water column, it can reach up to 2 meters below the surface. In a 20-meter water column, it can extend up to 8 meters below the surface.

Causes of hypoxia

Decline of oxygen saturation to anoxia, measured during the night in Kiel Fjord, Germany. Depth = 5 m

Oxygen depletion can result from a number of natural factors, but is most often a concern as a consequence of pollution and eutrophication in which plant nutrients enter a river, lake, or ocean, and phytoplankton blooms are encouraged. While phytoplankton, through photosynthesis, will raise DO saturation during daylight hours, the dense population of a bloom reduces DO saturation during the night by respiration. When phytoplankton cells die, they sink towards the bottom and are decomposed by bacteria, a process that further reduces DO in the water column. If oxygen depletion progresses to hypoxia, fish kills can occur and invertebrates like worms and clams on the bottom may be killed as well.

Still frame from an underwater video of the sea floor. The floor is covered with crabs, fish, and clams apparently dead or dying from oxygen depletion.

 

Hypoxia may also occur in the absence of pollutants. In estuaries, for example, because freshwater flowing from a river into the sea is less dense than salt water, stratification in the water column can result. Vertical mixing between the water bodies is therefore reduced, restricting the supply of oxygen from the surface waters to the more saline bottom waters. The oxygen concentration in the bottom layer may then become low enough for hypoxia to occur. Areas particularly prone to this include shallow waters of semi-enclosed water bodies such as the Waddenzee or the Gulf of Mexico, where land run-off is substantial. In these areas a so-called "dead zone" can be created. Low dissolved oxygen conditions are often seasonal, as is the case in Hood Canal and areas of Puget Sound, in Washington State. The World Resources Institute has identified 375 hypoxic coastal zones around the world, concentrated in coastal areas in Western Europe, the Eastern and Southern coasts of the US, and East Asia, particularly in Japan.

Jubilee photo from Mobile Bay

Hypoxia may also be the explanation for periodic phenomena such as the Mobile Bay jubilee, where aquatic life suddenly rushes to the shallows, perhaps trying to escape oxygen-depleted water. Recent widespread shellfish kills near the coasts of Oregon and Washington are also blamed on cyclic dead zone ecology.

Phytoplankton breakdown

Scientists have determined that high concentrations of minerals dumped into bodies of water causes significant growth of phytoplankton blooms. As these blooms are broken down by bacteria, such as Phanerochaete chrysosprium, oxygen is depleted by the enzymes of these organisms.

Breakdown of lignin

Tetrapyrrol ring, the active site of Ligninperoxidase enzyme

 

Phytoplankton are mostly made up of lignin and cellulose, which are broken down by enzymes present in organisms such as P. chrysosprium, known as white-rot. The breakdown of cellulose does not deplete oxygen concentration in water, but the breakdown of lignin does. This breakdown of lignin includes an oxidative mechanism, and requires the presence of dissolved oxygen to take place by enzymes like ligninperoxidase. Other fungi such as brown-rot, soft-rot, and blue stain fungi also are necessary in lignin transformation. As this oxidation takes place, CO₂ is formed in its place.

Active site of tetrapyrrol ring binding oxygen

 

Oxyferroheme is converted to Ferri-LiP with the addition of veratric alcohol, and gives off diatomic oxygen radical.

 

This is the breakdown of a confieryl alcohol by a hydrogen ion to make propanol and ortho-methoxyphenol.

 

Ligninperoxidase (LiP) serves as the most import enzyme because it is best at breaking down lignin in these organisms. LiP disrupts C-C bonds and C-O bonds within Lignin's three-dimensional structure, causing it to break down. LiP consists of ten alpha helices, two Ca²⁺ structural ions, as well as a heme group called a tetrapyrrol ring. Oxygen serves an important role in the catalytic cycle of LiP to form a double bond on the Fe²⁺ ion in the tetrapyrrol ring. Without the presence of diatomic oxygen in the water, this breakdown cannot take place because Ferrin-LiP will not be reduced into Oxyferroheme. Oxygen gas is used to reduce Ferrin-LiP into Oxyferroheme-LiP. Oxyferroheme and veratric alcohol combine to create oxygen radical and Ferri-LiP, which can now be used to degrade lignin. Oxygen radicals cannot be used in the environment, and are harmful in high presence in the environment.

Once Ferri-LiP is present in the ligninperoxidase, it can be used to break down lignin molecules by removing one phenylpropane group at a time through either the LRET mechanism or the mediator mechanism. The LRET mechanism (long range electron transfer mechanism) transfers an electron from the tetrapyrrol ring onto a molecule of phenylpropane in a lignin. This electron moves onto a C-C or C-O bond to break one phenylpropane molecule from the lignin, breaking it down by removing one phenylpropane at a time.

In the mediator mechanism, LiP enzyme is activated by the addition of hydrogen peroxide to make LiP radical, and a mediator such as veratric alcohol is added and activated creating veratric alcohol radical. Veratric alcohol radical transfers one electron to activate the phenylpropane on lignin, and the electron dismantles a C-C or C-O bond to release one phenylpropane from the lignin. As the size of a lignin molecule increases, the more difficult it is to break these C-C or C-O bonds. Three types of phenyl propane rings include coniferyl alcohol, sinapyl alcohol, and-coumaryl alcohol.

LiP has a very low MolDock score, meaning there is little energy required to form this enzyme and stabilize it to carry out reactions. LiP has a MolDock score of -156.03 kcal/mol. This is energetically favorable due to its negative free energy requirements, and therefore this reaction catalyzed by LiP is likely to take place spontaneously. Breakdown of propanol and phenols occur naturally in the environment because they are both water-soluble.

Environmental factors

The breakdown of phytoplankton in the environment depends on the presence of oxygen, and once oxygen is no longer in the bodies of water, ligninperoxidases cannot continue to break down the lignin. When oxygen is not present in the water, the breakdown of phytoplankton changes from 10.7 days to a total of 160 days for this to take place.

The rate of phytoplankton breakdown can be represented using this equation: 

${\displaystyle G(t)=G(0)e^{-kt}}$

In this equation, G(t) is the amount of particulate organic carbon (POC) overall at a given time, t. G(0) is the concentration of POC before breakdown takes place. k is a rate constant in year-1, and t is time in years. For most POC of phytoplankton, the k is around 12.8 years-1, or about 28 days for nearly 96% of carbon to be broken down in these systems. Whereas for anoxic systems, POC breakdown takes 125 days, over four times longer. It takes approximately 1 mg of Oxygen to break down 1 mg of POC in the environment, and therefore, hypoxia takes place quickly as oxygen is used up quickly to digest POC. About 9% of POC in phytoplankton can be broken down in a single day at 18 °C, therefore it takes about eleven days to completely break down a full phytoplankton.

After POC is broken down, this particulate matter can be turned into other dissolved organic carbon, such as carbon dioxide, bicarbonate ions, and carbonate. As much as 30% of phytoplankton can be broken down into dissolved organic carbon. When this particulate organic carbon interacts with 350 nm ultraviolet light, dissolved organic carbon is formed, removing even more oxygen from the environment in the forms of carbon dioxide, bicarbonate ions, and carbonate. Dissolved inorganic carbon is made at a rate of 2.3-6.5 mg/(m^3)day.

As phytoplankton breakdown, free phosphorus and nitrogen become available in the environment, which also fosters hypoxic conditions. As the breakdown of these phytoplankton takes place, the more phosphorus turns into phosphates, and nitrogens turn into nitrates. This depletes the oxygen even more so in the environment, further creating hypoxic zones in higher quantities. As more minerals such as phosphorus and nitrogen are displaced into these aquatic systems, the growth of phytoplankton greatly increases, and after their death, hypoxic zones are formed.

Solutions

To combat hypoxia, it is essential to reduce the amount of land-derived nutrients reaching rivers in runoff. This can be done by improving sewage treatment and by reducing the amount of fertilizers leaching into the rivers. Alternately, this can be done by restoring natural environments along a river; marshes are particularly effective in reducing the amount of phosphorus and nitrogen (nutrients) in water. Other natural habitat-based solutions include restoration of shellfish populations, such as oysters. Oyster reefs remove nitrogen from the water column and filter out suspended solids, subsequently reducing the likelihood or extent of harmful algal blooms or anoxic conditions. Foundational work toward the idea of improving marine water quality through shellfish cultivation was conducted by Odd Lindahl et al., using mussels in Sweden. More involved than single-species shellfish cultivation, integrated multi-trophic aquaculture mimics natural marine ecosystems, relying on polyculture to improve marine water quality. 

Graphs of oxygen and salinity levels at Kiel Fjord in September 1998.

Technological solutions are also possible, such as that used in the redeveloped Salford Docks area of the Manchester Ship Canal in England, where years of runoff from sewers and roads had accumulated in the slow running waters. In 2001 a compressed air injection system was introduced, which raised the oxygen levels in the water by up to 300%. The resulting improvement in water quality led to an increase in the number of invertebrate species, such as freshwater shrimp, to more than 30. Spawning and growth rates of fish species such as roach and perch also increased to such an extent that they are now amongst the highest in England.

In a very short time the oxygen saturation can drop to zero when offshore blowing winds drive surface water out and anoxic depth water rises up. At the same time a decline in temperature and a rise in salinity is observed (from the longterm ecological observatory in the seas at Kiel Fjord, Germany). New approaches of long-term monitoring of oxygen regime in the ocean observe online the behavior of fish and zooplankton, which changes drastically under reduced oxygen saturations (ecoSCOPE) and already at very low levels of water pollution.

Exaptation (updated)

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Exaptation

Exaptation and the related term co-option describe a shift in the function of a trait during evolution. For example, a trait can evolve because it served one particular function, but subsequently it may come to serve another. Exaptations are common in both anatomy and behaviour. Bird feathers are a classic example: initially they may have evolved for temperature regulation, but later were adapted for flight. Interest in exaptation relates to both the process and products of evolution: the process that creates complex traits and the products (functions, anatomical structures, biochemicals, etc.) that may be imperfectly developed. Exaptation was proposed by Stephen Jay Gould and Elisabeth Vrba as a replacement for what they considered to be a teleologically loaded term 'pre-adaptation'.

History and definitions

Charles Darwin

The idea that the function of a trait might shift during its evolutionary history originated with Charles Darwin (Darwin 1859). For many years the phenomenon was labeled "preadaptation", but since this term suggests teleology in biology, appearing to conflict with natural selection, it has been replaced by the term exaptation.

The idea had been explored by several scholars when in 1982 Stephen Jay Gould and Elisabeth Vrba introduced the term "exaptation". However, this definition had two categories with different implications for the role of adaptation.

(1) A character, previously shaped by natural selection for a particular function (an adaptation), is coopted for a new use—cooptation. (2) A character whose origin cannot be ascribed to the direct action of natural selection (a nonaptation), is coopted for a current use—cooptation. (Gould and Vrba 1982, Table 1)

The definitions are silent as to whether exaptations had been shaped by natural selection after cooption, although Gould and Vrba cite examples (e.g., feathers) of traits shaped after cooption. Note that the selection pressure upon a trait is likely to change if it is (especially, primarily or solely) used for a new purpose, potentially initiating a different evolutionary trajectory.

To avoid these ambiguities, Buss et al. (1998) suggested the term "co-opted adaptation", which is limited to traits that evolved after cooption. However, the commonly used terms of "exaptation" and "cooption" are ambiguous in this regard. 

Preadaptation

In some circumstances, the "pre-" in preadaptation can be interpreted as applying, for non-teleological reasons, prior to the adaptation itself, creating a meaning for the term that is distinct from exaptation. For example, future environments (say, hotter or drier ones), may resemble those already encountered by a population at one of its current spatial or temporal margins. This is not actual foresight, but rather the luck of having adapted to a climate which later becomes more prominent. Cryptic genetic variation may have the most strongly deleterious mutations purged from it, leaving an increased chance of useful adaptations, but this represents selection acting on current genomes with consequences for the future, rather than foresight.

Function may not always come before form: developed structures could change or alter the primary functions they were intended for due to some structural or historical cause.

Examples

Bird feathers of various colors

Exaptations include the co-option of feathers, which initially evolved for heat regulation, for display, and later for use in bird flight. Another example is the lungs of many basal fish, which evolved into the lungs of terrestrial vertebrates but also underwent exaptation to become the gas bladder, a buoyancy control organ, in derived fish. A third is the repurposing of two of the three bones in the reptilian jaw to become the malleus and incus of the mammalian ear, leaving the mammalian jaw with just one hinge.

A behavioural example pertains to subdominant wolves licking the mouths of lead wolves as a sign of submissiveness. (Similarly, dogs, which are wolves who through a long process were domesticated, lick the faces of their human owners.) This trait can be explained as an exaptation of wolf pups licking the faces of adults to encourage them to regurgitate food.

Arthropods provide the earliest identifiable fossils of land animals, from about 419 million years ago in the Late Silurian, and terrestrial tracks from about 450 million years ago appear to have been made by arthropods. Arthropods were well pre-adapted to colonize land, because their existing jointed exoskeletons provided support against gravity and mechanical components that could interact to provide levers, columns and other means of locomotion that did not depend on submergence in water.

Metabolism can be considered an important part of exaptation. As one of the oldest biological systems and being central to life on the Earth, studies have shown that metabolism may be able to use exaptation in order to be fit, given some new set of conditions or environment. Studies have shown that up to 44 carbon sources are viable for metabolism to successfully take place and that any one adaptation in these specific metabolic systems is due to multiple exaptations. Taking this perspective, exaptations are important in the origination of adaptations in general. A recent example comes from Richard Lenski's E. coli long-term evolution experiment, in which aerobic growth on citrate arose in one of twelve populations after 31,000 generations of evolution. Genomic analysis by Blount and colleagues showed that this novel trait was due to a gene duplication that caused oxic expression of a citrate transporter gene that is normally only expressed under anoxic conditions, thus exapting it for aerobic use. Metabolic systems have the potential to innovate without adaptive origins. 

Gould and Brosius took the concept of exaptation to the genetic level. It is possible to look at a retroposon, originally thought to be simply junk DNA, and deduce that it may have gotten a new function to be termed as an exaptation. Given an emergency situation in the past, a species may have used junk DNA for a useful purpose in order to evolve and be able to survive. This may have occurred with mammalian ancestors when confronted with a large mass extinction about 250 million years ago and substantial increase in the level of oxygen in Earth's atmosphere. More than 100 loci have been found to be conserved only among mammalian genomes and are thought to have essential roles in the generation of features such as the placenta, diaphragm, mammary glands, neocortex, and auditory ossicles. It is believed that as a result of exaptation, or making previously "useless" DNA into DNA that could be used in order to increase survival chance, mammals were able to generate new brain structures as well as behavior to better survive the mass extinction and adapt to new environments. Similarly, viruses and their components have been repeatedly exapted for host functions. The functions of exapted viruses typically involve either defense from other viruses or cellular competitors or transfer of nucleic acids between cells, or storage functions. Koonin and Krupovic suggested that virus exaptation can reach different depths, from recruitment of a fully functional virus to exploitation of defective, partially degraded viruses, to utilization of individual virus proteins.

Adaptation and exaptation cycle

It was speculated by Gould and Vrba in one of the first papers written about exaptation, that when an exaptation arises, it may not be perfectly suited for its new role and may therefore develop new adaptations to promote its use in a better manner. In other words, the beginning of developing a particular trait starts out with a primary adaptation toward a fit or specific role, followed by a primary exaptation (a new role is derived using the existing feature but may not be perfect for it), which in turn leads to the development of a secondary adaptation (the feature is improved by natural selection for better performance), promoting further development of an exaptation, and so forth.

Once again, feathers are an important example, in that they may have first been adapted for thermoregulation and with time became useful for catching insects, and therefore served as a new feature for another benefit. For instance, large contour feathers with specific arrangements arose as an adaptation for catching insects more successfully, which eventually led to flight, since the larger feathers served better for that purpose.

Implications

Evolution of complex traits

One of the challenges to Darwin's theory of evolution was explaining how complex structures could evolve gradually, given that their incipient forms may have been inadequate to serve any function. As George Jackson Mivart (a critic of Darwin) pointed out, 5 percent of a bird wing would not be functional. The incipient form of complex traits would not have survived long enough to evolve to a useful form.

As Darwin elaborated in the last edition of The Origin of Species, many complex traits evolved from earlier traits that had served different functions. By trapping air, primitive wings would have enabled birds to efficiently regulate their temperature, in part, by lifting up their feathers when too warm. Individual animals with more of this functionality would more successfully survive and reproduce, resulting in the proliferation and intensification of the trait.

Eventually, feathers became sufficiently large to enable some individuals to glide. These individuals would in turn more successfully survive and reproduce, resulting in the spread of this trait because it served a second and still more beneficial function: that of locomotion. Hence, the evolution of bird wings can be explained by a shifting in function from the regulation of temperature to flight.

Jury-rigged design

Darwin explained how the traits of living organisms are well-designed for their environment, but he also recognized that many traits are imperfectly designed. They appear to have been made from available material, that is, jury-rigged. Understanding exaptations may suggest hypotheses regarding subtleties in the adaptation. For instance, that feathers evolved initially for thermal regulation may help to explain some of their features unrelated to flight (Buss et al., 1998). However, this is readily explained by the fact that they serve a dual purpose.

Some of the chemical pathways for physical pain and pain from social exclusion overlap.^[26] The physical pain system may have been co-opted to motivate social animals to respond to threats to their inclusion in the group.

Evolution of technology

Exaptation has received increasing attention in innovation and management studies inspired by evolutionary dynamics, where it has been proposed as a mechanism that drives the serendipitous expansion of technologies and products in new domains.

Heteroplasmy

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Heteroplasmy

Heteroplasmy is the presence of more than one type of organellar genome (mitochondrial DNA or plastid DNA) within a cell or individual. It is an important factor in considering the severity of mitochondrial diseases. Because most eukaryotic cells contain many hundreds of mitochondria with hundreds of copies of mitochondrial DNA, it is common for mutations to affect only some mitochondria, leaving most unaffected.

Although detrimental scenarios are well-studied, heteroplasmy can also be beneficial. For example, centenarians show a higher than average degree of heteroplasmy.

Microheteroplasmy is present in most individuals. This refers to hundreds of independent mutations in one organism, with each mutation found in about 1–2% of all mitochondrial genomes.

 

 

Types of heteroplasmy

In order for heteroplasmy to occur, organelles must contain a genome and, in turn, a genotype. In animals, mitochondria are the only organelles that contain their own genomes, so these organisms will only have mitochondrial heteroplasmy. In contrast, photosynthetic plants contain mitochondria and chloroplasts, each of which contains plastid genomes. Therefore, plant heteroplasmy occurs in two dimensions.

Organelle inheritance patterns

In 1909, while studying chloroplast genomes, Erwin Baur made the first observations about organelle inheritance patterns. Organelle genome inheritance differs from nuclear genome, and this is illustrated by four violations of Mendel's laws.

1. During asexual reproduction, nuclear genes never segregate during cellular divisions. This is to ensure that each daughter cell gets a copy of every gene. However, organelle genes in heteroplasmic cells can segregate because they each have several copies of their genome. This may result in daughter cells with differential proportions of organelle genotypes.
2. Mendel states that nuclear alleles always segregate during meiosis. However, organelle alleles may or may not do this.
3. Nuclear genes are inherited from a combination of alleles from both parents, making inheritance biparental. Conversely, organelle inheritance is uniparental, meaning the genes are all inherited from one parent.
4. It is also unlikely for organelle alleles to segregate independently, like nuclear alleles do, because plastid genes are usually on a single chromosome and recombination is limited by uniparental inheritance.

There is a wide variety of mitochondrial DNA genotypes in the maternal pool, which is represented by the bottle. The two genotypes in this maternal pool are represented by blue and yellow. When generated, each oocyte receives a small subsampling of mitochondrial DNA molecules in differing proportions. This is represented by the conveyor belt with oocytes, each one unique, as they are produced.

 

Vegetative segregation

Vegetative segregation, the random partitioning of cytoplasm, is a distinguishable characteristic of organelle heredity. During cell division, the organelles are divided equally, providing each daughter cell with a random selection of plasmid genotypes.

Uniparental inheritance

Uniparental inheritance refers to the fact that, in most organisms, many offspring inherit organelle genes from only one parent. However, this is not a general law. Many organisms that have the ability to differentiate maternal and paternal sexes will produce offspring with a mixture of maternal, paternal, and biparental mitochondrial DNA.

Mitochondrial bottleneck

Entities undergoing uniparental inheritance and with little to no recombination may be expected to be subject to Muller's ratchet, the inexorable accumulation of deleterious mutations until functionality is lost. Animal populations of mitochondria avoid this buildup through a developmental process known as the mtDNA bottleneck. The bottleneck exploits stochastic processes in the cell to increase in the cell-to-cell variability in mutant load as an organism develops: a single egg cell with some proportion of mutant mtDNA thus produces an embryo where different cells have different mutant loads. Cell-level selection may then act to remove those cells with more mutant mtDNA, leading to a stabilisation or reduction in mutant load between generations. The mechanism underlying the bottleneck is debated, with a recent mathematical and experimental metastudy providing evidence for a combination of random partitioning of mtDNAs at cell divisions and random turnover of mtDNA molecules within the cell.

The mitochondrial bottleneck concept refers to the classic evolutionary term, which is used to explain an event that reduces and specifies a population. It was developed to describe why mitochondrial DNA in an embryo might be drastically different from that of its mother. When a large population of DNA is subsampled, each sample population will receive a slightly different proportion of mitochondrial genotypes. Consequently, when paired with a high degree of replication, a rare or mutated allele can begin to proportionally dominate. In theory, this makes possible a single-generation shift of overall mitochondrial genotype.

Selection

Although it is not well characterized, selection can occur for organelle genomes in heteroplasmic cells. Intracellular ("within cells") selection occurs within individual cells. It refers to the selective segregation of certain genotypes in mitochondrial DNA that allows the favoured genotype to thrive. Intercellular ("between cells") selection occurs on a larger scale, and refers to the preferential growth of cells that have greater numbers of a certain mitochondrial genotype. Selective differences can occur between naturally occurring, non-pathological mtDNA types when mixed in cells, and may depend on tissue type, age, and genetic distance. Selective differences between naturally occurring mtDNA types may pose challenges for gene therapies.

In mitochondrial DNA, there is evidence for potent germline purifying selection, as well as purifying selection during embryogenesis. Additionally, there is a dose-dependent decrease in reproduction ability for females that have mutations in mitochondrial DNA. This demonstrates another selection mechanism to prevent the evolutionary preservation of harmful mutations.

Reduced recombination

It is very rare for organelle genes from different lineages to recombine. These genomes are usually inherited uniparentally, which does not provide a recombination opportunity. If they are inherited biparentally, it is unlikely that the organelles from the parents will fuse, meaning they will not share genomes. 

However, it is possible for organelle genes from the same lineage to recombine. Intramolecular and intermolecular recombination can cause inversions and repeats in chloroplast DNA, and can produce subgenomic circles in mitochondrial DNA.

Mitochondrial mutations in disease

Mutations in mitochondrial DNA are usually single nucleotide substitutions, single base insertions, or deletions.

Because each cell contains thousands of mitochondria, nearly all organisms house low levels of mitochondrial variants, conferring some degree of heteroplasmy. Although a single mutational event might be rare in its generation, repeated mitotic segregation and clonal expansion can enable it to dominate the mitochondrial DNA pool over time. When this occurs, it is known as reaching threshold, and it usually results in physiological consequences.

Severity and time to presentation

Symptoms of severe heteroplasmic mitochondrial disorders do not usually appear until adulthood. Many cell divisions and a great deal of time are required for a cell to accumulate enough mutant mitochondria to cause symptoms. An example of this phenomenon is Leber optic atrophy. Generally, individuals with this condition do not experience vision difficulties until they have reached adulthood. Another example is MERRF syndrome (or Myoclonic Epilepsy with Ragged Red Fibers). In MELAS, heteroplasmy explains the variation in severity of the disease among siblings. 

Screening

Preimplantation genetic screening (PGS) can be used to quantitate the risk of a child of being affected by a mitochondrial disease. In most cases, a muscle mutation level of approximately 18% or less confers a 95% risk reduction.

Sequence illustrating heteroplasmy genotype of 16169 C/T in Nicholas II of Russia.

 

Notable cases

One notable example of an otherwise healthy individual whose heteroplasmy was discovered incidentally is Nicholas II of Russia, whose heteroplasmy (and that of his brother) served to convince Russian authorities of the authenticity of his remains.