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Tales from the crypt(ic)



Neutral mutations can breathe life into evolutionary adaptation
By Jessica A. Lee1and Christopher J. Marx2
Adaptation through natural selection
requires inherited changes in an or-
ganism’s phenotype. However, neu-
tral or “cryptic” mutations—changes
in genotype that do not affect phe-
notype—can influence adaptation
outcomes, because genotype-to-phenotype
mapping is inherently dependent on con-
text. The phenotypic consequence of a
mutation might change as a result of in-
teractions either with other mutations in
the genome (epistasis) or with the physical
environment [a genotype-by-environment
(G×E) interaction]. On page 347 of this is-
sue, Zheng et al. (1) demonstrate that the
accumulation of mutations that yield neu-
tral changes in a protein promotes faster
adaptation in an environment selecting
for a new function, and that this effect
requires the combined impact of epistasis
and G×E interactions.
The impact of neutral mutations on ad-
aptation is often framed from the pheno-
typic, rather than the genotypic, point of
view; an ancestral phenotype that remains
unchanged in the face of genomic muta-
tions is considered mutationally robust.
However, scientists have debated whether
mutational robustness spurs or suppresses
adaptation over the long term. Although a
broader array of genotypes can be tolerated
in a mutationally robust phenotype, the
mutations form a “neutral network” that
might serve as a genetic resource should
the population be confronted with a new
environment. Shielding cryptic mutations
from G×E interactions in the organism’s
original environment can allow them to
accumulate (see the figure). Theoretical
analysis has revealed that mutational ro-
bustness can either speed or slow adapta-
tion, depending on whether high-fitness
mutations are rare or common, respec-
tively, across the neutral network (2).
Until recently, there have been few ex-
perimental tests of whether or how cryptic
mutations function in the adaptation of
populations. Zheng et al. provide an empiri-
cal test to ask whether generating a broad
pool of cryptic genetic variations accelerates
and diversifies adaptive outcomes when
that population requires a specific protein
to acquire a new activity. The authors ex-
amined yellow fluorescent protein (YFP)
function in living Escherichia coli cells by
using fluorescence-activated cell sorting
(FACS) to select cells that display yellow
fluorescence in vivo. To generate cryptic
genetic variation, they mutagenized the yfp
gene, introduced the resulting pool into E.
coli cells, and then selected
for the 20% of cells with yel-
low fluorescence levels that
mirrored most closely that of
the ancestral phenotype. Af-
ter four rounds of selection,
they created a new selective
environment by switching
the FACS to select for a green
fluorescence—ancestral YFP
is weakly fluorescent at the
green wavelength—and car-
ried the cells through four
rounds of selection for the top 0.1% of YFP
variants with the highest green fluores-
cence activity.
The generation of cryptic variation in YFP
before selection for green fluorescence in-
creased the rate of adaptation compared to
control populations initiated without prior
generation of diversity. The benefit of cryp-
tic variation was most prominent in the first
round after the transition to selection for
green fluorescence. This stands in contrast
to results from the in vitro evolution of a ri-
bozyme selected for its ability to use a new
substrate, in which boosts in adaptation con-
tinued through five rounds of selection (3).
Thus, the quantitative effect of cryptic ge-
netic variation is likely to be different across
systems and selective pressures.
Zheng et al. also found that
the cryptic genetic diversity
generated in the first environ-
ment permitted evolutionary
trajectories that would not
otherwise have been acces-
sible. This crucial finding was
uncovered by reconstructing
all possible mutational inter-
mediates that preceded the
end combination, a process
that culminated in yfp genes
that expressed high green
fluorescence in the final E. coli populations.
When green fluorescence was directly se-
lected from the ancestral yfp without cryptic
variation, the network of mutational inter-
mediates almost exclusively featured steps
that, in any order, would have created cells
that survive the selection process (“benefi-
cial” mutations). This observation, along with
Tales from the crypt(ic)
Neutral mutations can breathe life into evolutionary adaptation
1Global Viral, San Francisco, CA 94104, USA. 2Department of
Biological Sciences, University of Idaho, Moscow, ID 83844,
USA. Email:;
Under selection for yellow uorescence
(yellow glow), mutations in the ancestral
gene (ANC) that do not change the selection
phenotype (neutral; black arrows) can
accumulate as cryptic genetic variation.
When the selection phenotype is changed
(gray arrow) to green uorescence (green
glow), every path from ANC to the (brightest)
ABC variant (such as ANC A) is blocked by
deleterious mutations (red arrows).
Neutral mutations accumulated
in the AB variant enable rapid
evolution (blue arrows) to ABC
(high green uorescence).
E. col i
“Zheng et al.
greatly advance
of how cryptic
can aid in
318 26 JULY 2019 • VOL 365 ISSUE 6451
Cryptic mutations facilitate adaptation
Accumulation of mutations that yield neutral changes in a protein
promotes adaptation when selecting for a new function.
Published by AAAS
on August 1, 2019 from
the fact that these populations all ended up
with very similar genotypes, demonstrated a
constraint on selection. By contrast, nearly
all trajectories observed from the pool with
cryptic variation featured steps that would
have been deleterious in the environment se-
lecting for green fluorescence and, therefore,
would not have survived without the initial
generation of diversity (see the figure).
Fluorescent proteins and ribozymes ma-
nipulated under laboratory conditions rep-
resent excellent model systems; but does
evidence exist to show that cryptic genetic
variation has contributed to the evolution
of new traits throughout Earth’s history?
The answer appears to be yes. An analysis
of reconstructed evolutionary intermedi-
ates for a family of hormone receptors re-
vealed mutations in the genes that encode
these proteins that did not change activity
on their own, but were essential for the
evolution of new hormone-binding proper-
ties more than 400 million years ago (4). A
clever genetic screen even allowed research-
ers to attempt to “replay the tape” to deter-
mine the number of possible mutations
that would have set the stage for novelty to
arise without disrupting the current protein
function. They found only one amino acid
change that fit these criteria, and it was the
one known to have occurred historically (5).
By demonstrating the role of epista-
sis and the avoidance of G×E interactions
through the change of selective conditions,
Zheng et al. greatly advance understanding
of how cryptic variation—phenotypes that
are mutationally robust—can aid in adap-
tation. The authors suggest that future ef-
forts to use directed evolution for practical
purposes incorporate these principles, as is
already being done when considering the
folding stability and directed evolution of
proteins (6). From a fundamental perspec-
tive, perhaps the most important question
is whether the observations from evolving
single RNA or protein molecules also apply
at the level of the whole cell; if so, we can
expect to move toward a predictive under-
standing of these phenomena. j
1. J. Zheng et al., Science 365, 347 (2019).
2. J. A. Drag hi, T. L. Parson s, G. P. Wagner, J. B. Pl otki n, Nature
463, 353 (2010).
3. D. P. Bendixse n, J. Coll et, B. Øst man, E . J. Hayden , PLOS
Biol. 17, e3000300 (2019).
4. E . A. Or tlun d, J. T. Bri dgha m, M. R. Re dinb o, J. W. Thor nton ,
Science 317, 1544 (2007).
5. M. J. Ha rms, J. W. Tho rnto n, Nature 512, 203 (2014).
6. P. A. Romero, F. H. Arnold, Nat. Rev. Mol. Cell Biol. 10, 866
The authors are supported by NSF MCB-1714949 (C.J.M.) and
John Templeton Foundation grant 60973 (J.A.L.).
Lowering ceramides
to overcome diabetes
Lowering toxic lipid concentrations in mice has a promising
impact on obesity-associated metabolic disorder
By Christine M. Kusminski and
Philipp E. Scherer
Excess nutrient intake leads to a dis-
ruption in metabolic homeostasis.
In particular, prolonged periods of
excess glucose intake can directly
contribute to deterioration of insulin
sensitivity. Insulin is a key player in
the disposal of carbohydrates from food.
Frequently associated with this insulin re-
sistance is a dysregulation of lipid metabo-
lism that can lead to lipotoxicity, whereby
excess lipids wreak havoc on important
intracellular signaling pathways. How-
ever, it is largely unknown what types of
lipids trigger these cytotoxic effects, which
result in further deterioration of glucose
and lipid homeostasis. Concentrations of
ceramide lipids in blood plasma and tis-
sues are strongly associated with the risk
of developing type 2 diabetes (T2D), he-
patic steatosis, and cardiovascular disease,
which are caused by lipotoxicity and insu-
lin resistance (1, 2). On page 386 of this is-
sue, Chaurasia et al. (3) provide evidence
that therapeutically intervening in the ce-
ramide biosynthesis pathway in mice can
improve metabolic homeostasis.
The authors focused on a critical, rate-
limiting step in the ceramide biosynthesis
pathway. Dihydroceramide desaturase 1
(DEGS1) is an enzyme that inserts a dou-
ble bond into dihydroceramide to produce
ceramide. The authors showed that the re-
moval of DEGS1 in mice causes an increase
in ceramides lacking a double bond in
tissues and plasma. Deletion of the Degs1
gene in adult mice did not elicit any del-
eterious effects (an important consider-
ation for possible therapeutic targets, the
inhibition of which may lead to unwanted
side effects). Furthermore, complete or
partial loss of DEGS1 activity substantially
improved glucose and lipid metabolism in
mice exposed to a high-fat diet.
Chaurasia et al. showed that inhibiting
ceramide synthesis in adipocytes or hepa-
tocytes leads to a system-wide improve-
ment in metabolic parameters. This was
also observed for components of the ce-
ramide catabolism pathway, such as cell
type–specific overexpression of acid ce-
ramidase, in addition to overexpression
of adiponectin receptors (which also have
ceramidase activity) (4, 5). Together, this
reveals a strong exchange of ceramides
throughout the body, such that depleting
these lipid species in either hepatocytes or
adipocytes lowers ceramides across many
tissues involved in maintaining metabolic
homeostasis. Adiponectin is a circulating
adipokine that signals through its cognate
receptors to enhance insulin sensitivity by
reducing intracellular ceramides (6). Many
clinical studies demonstrate inverse corre-
lations between the amounts of ceramides
in plasma and adiponectin in healthy in-
dividuals or those with T2D (79) (see the
Chaurasia et al. further implicate cera -
mides in a phenomenon called selective
Acid ceramidase
Adiponectin receptors
Adiponectin Cellular insulin
Pancreatic b-cell
b-cell apoptosis
26 JULY 2019 • VOL 365 ISSUE 6451 319
Intervening in
In mice, removing
desaturase 1 (DEGS1) or
the ceramide synthases
reduces the amount of
available ceramide, which
improves metabolic
Published by AAAS
on August 1, 2019 from
Tales from the crypt(ic)
Jessica A. Lee and Christopher J. Marx
DOI: 10.1126/science.aay2727
(6451), 318-319.365Science
This article cites 6 articles, 2 of which you can access for free
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... Neutral effects with respect to phenotypic expression may also occur for most other genetic levels and effects (Paaby and Rockman, 2014), a general phenomenon called cryptic genetic variation (CGV), which may serve as a genetic reservoir for future evolutionary change (Zheng et al., 2019). In populations of bacteria, CGV facilitated adaptation when the population faced rapid environmental change as enacted by a change in selection (Lee and Marx, 2019;Zheng et al., 2019). ...
... For the connections that remain, their mappings can be altered by epigenetic processes. When noise is introduced into the G-P map for the neurocontrollers of simulated quadrupedal robots, the resulting stochastic ontogenesis (SO), a developmental process, can have surprising and positive evolutionary consequences (Lee and Marx, 2019). As is true with populations possessing CGV, populations with SO respond better, as measured by evolutionary fitness, to changes in the environment, apparently by providing a reservoir of solutions. ...
Full-text available
Given that selection removes genetic variance from evolving populations, thereby reducing exploration opportunities, it is important to find mechanisms that create genetic variation without the disruption of adapted genes and genomes caused by random mutation. Just such an alternative is offered by random epigenetic error, a developmental process that acts on materials and parts expressed by the genome. In this system of embodied computational evolution, simulated within a physics engine, epigenetic error was instantiated in an explicit genotype-to-phenotype map as transcription error at the initiation of gene expression. The hypothesis was that transcription error would create genetic variance by shielding genes from the direct impact of selection, creating, in the process, masquerading genomes. To test this hypothesis, populations of simulated embodied biorobots and their developmental systems were evolved under steady directional selection as equivalent rates of random mutation and random transcriptional error were covaried systematically in an 11 × 11 fully factorial experimental design. In each of the 121 different experimental conditions (unique combinations of mutation and transcription error), the same set of 10 randomly created replicate populations of 60 individuals were evolved. Selection for the improved locomotor behavior of individuals led to increased mean fitness of populations over 100 generations at nearly all levels and combinations of mutation and transcription error. When the effects of both types of error were partitioned statistically, increasing transcription error was shown to increase the final genetic variance of populations, incurring a fitness cost but acting on variance independently and differently from genetic mutation. Thus, random epigenetic errors in development feed back through selection of individuals with masquerading genomes to the population’s genetic variance over generational time. Random developmental processes offer an additional mechanism for exploration by increasing genetic variation in the face of steady, directional selection.
... If cladistic structure (meaning a branching clade) equivalent to paraphyly (Funk, Omland, 2003) can be shown to be exhibited between molecular strains of the same species, at times including different species among the strains and even different genera, then the mechanism of molecular strains of the same species giving rise to different species or even different genera as suggested by Darwin (Haskell, Adhikari, 2009) is supported as an evolutionary process. Increasing numbers of tracking traits should parallel increasing numbers of neutral mutations with potential for future adaptive evolution (Lee, Marx, 2019). This also supports the idea that molecular paraphyly implies ancestor-descendant serial generation of species, and advances the concept of the dissilient (radiative) genus (Zander, 2013(Zander, : 92, 2018 as an empirically supported scientific reality. ...
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The analytic orientation of this paper is intended as a replacement for the antiquated but still prevalent phylogenetic inferential models and techniques of the late 20th century that are focused entirely on shared descent. Serial descent, that is, progenitor to descendant, may occur at the species or infraspecies level. In molecular systematics, species level paraphyly occurs when two examples of the same species are separated on a cladogram by a second species. This implies linear macroevolution of the second species from the first. Molecular cladograms often show cladistic structure (branching) among examples of the same species. If well-supported, such indicates a potential for evolution. A range of infraspecific and intraspecific cladistic structure in species of Pottiaceae (Bryophyta) was demonstrated in previously published molecular cladograms and data sets of other authors. This includes well-supported cladistic structure of molecular strains, and well-supported paraphyly involving other species. Large numbers of base pair changes among strains are considered here evidence of evolvability and increasing age of a species. Infraspecific strains are apparently lost in older species through speciation and extinction. Cluster analysis using DNA metadata of Oxystegus species matched published molecular cladograms to a large extent. The fact that apparent molecular strains are present in both nonparaphyletic and paraphyletic species, about half the species studied, shore up the theory that internal racial differentiation at the molecular level leads to or signals serial descent of multiple extant morphotaxa. It is because much infraspecific molecular cladistic structure exists that newly speciated taxa are already strongly cladistically dichotomized. Thus, the ultimate source of molecular paraphyly is internal to each species, and does not imply polyphyly by convergent species or cryptic taxa. Molecular systematics cannot effectively model progenitor-descendant radiation. Species with many strains are potential sources of future biological diversity. Recognition of differential evolvability may allow facilitation of complex, interactive, diverse ecosystems successfully tracking climate change.
... If cladistic structure (meaning a branching clade) equivalent to paraphyly (Funk, Omland, 2003) can be shown to be exhibited between molecular strains of the same species, at times including different species among the strains and even different genera, then the mechanism of molecular strains of the same species giving rise to different species or even different genera as suggested by Darwin (Haskell, Adhikari, 2009) is supported as an evolutionary process. Increasing numbers of tracking traits should parallel increasing numbers of neutral mutations with potential for future adaptive evolution (Lee, Marx, 2019). This also supports the idea that molecular paraphyly implies ancestor-descendant serial generation of species, and advances the concept of the dissilient (radiative) genus (Zander, 2013(Zander, : 92, 2018 as an empirically supported scientific reality. ...
Full-text available
General recommendations regarding proper type designation of names of taxa described by Turczaninow in his Animadversiones series of articles (as well as in some other publications) are provided. It is concluded that, as clearly indicated in the protologues, all (or almost all) taxa described in these publications are based on specimens from the private herbarium of Turczaninow which was donated in the 1840s to the Kharkiv University (CWU) and in the 1940s was transferred to the Institute of Botany in Kyiv (KW). Consequently, holotypes and syntypes of these taxa are now almost exclusively in KW. Several cases of correct and incorrect type designations are discussed, specifically of some South American Brassicaceae, Geraniaceae and Hypericaceae, Central American Malvaceae, and southern African Polygalaceae. Information on the re-discovered holotype (KW) of Abelmoschus achanioides Turcz. (now accepted as Malvaviscus achanioides (Turcz.) Fryxell, Malvaceae) is provided, and an earlier lectotypification of that name with a specimen from G is considered ineffective. The holotype of Stenocalyx involutus Turcz. (now considered a synonym of Mezia includens (Benth.) Cuatrec., Malpighiaceae) was originally in the Turczaninow herbarium, but the whole folder with that specimen is now missing in KW (considered lost or destroyed), and it was already missing in the mid-1920s, when the collection was still in CWU. Because of that the lectotype of S. involutus is designated here, the specimen from MPU, to replace the lost or destroyed holotype. The need for thorough analysis of protologues, available original material, and associated information for correct type designation/indication is emphasized.
Beyond the major biochemical connections that make up the framework of the metabolic network is crosstalk. Metabolic crosstalk consists of the subtle, hard-to predict interactions between discrete metabolic processes/pathways. These interactions, which are mediated by metabolites and proteins, coordinate metabolic processes to produce a robust metabolic network. Crosstalk interactions can be identified genetically in vivo and are further characterized with a cross-disciplinary approach. Probing metabolic crosstalk aids in characterizing genes of unknown function and revealing the next tier of metabolic paradigms.
Full-text available
Evolutionary innovations are qualitatively novel traits that emerge through evolution and increase biodiversity. The genetic mechanisms of innovation remain poorly understood. A systems view of innovation requires the analysis of genotype networks-the vast networks of genetic variants that produce the same phenotype. Innovations can occur at the intersection of two different genotype networks. However, the experimental characterization of genotype networks has been hindered by the vast number of genetic variants that need to be functionally analyzed. Here, we use high-throughput sequencing to study the fitness landscape at the intersection of the genotype networks of two catalytic RNA molecules (ribozymes). We determined the ability of numerous neighboring RNA sequences to catalyze two different chemical reactions, and we use these data as a proxy for a genotype to fitness map where two functions come in close proximity. We find extensive functional overlap, and numerous genotypes can catalyze both functions. We demonstrate through evolutionary simulations that these numerous points of intersection facilitate the discovery of a new function. However, the rate of adaptation of the new function depends upon the local ruggedness around the starting location in the genotype network. As a consequence, one direction of adaptation is more rapid than the other. We find that periods of neutral evolution increase rates of adaptation to the new function by allowing populations to spread out in their genotype network. Our study reveals the properties of a fitness landscape where genotype networks intersect and the consequences for evolutionary innovations. Our results suggest that historic innovations in natural systems may have been facilitated by overlapping genotype networks.
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Understanding how chance historical events shape evolutionary processes is a central goal of evolutionary biology. Direct insights into the extent and causes of evolutionary contingency have been limited to experimental systems, because it is difficult to know what happened in the deep past and to characterize other paths that evolution could have followed. Here we combine ancestral protein reconstruction, directed evolution and biophysical analysis to explore alternative 'might-have-been' trajectories during the ancient evolution of a novel protein function. We previously found that the evolution of cortisol specificity in the ancestral glucocorticoid receptor (GR) was contingent on permissive substitutions, which had no apparent effect on receptor function but were necessary for GR to tolerate the large-effect mutations that caused the shift in specificity. Here we show that alternative mutations that could have permitted the historical function-switching substitutions are extremely rare in the ensemble of genotypes accessible to the ancestral GR. In a library of thousands of variants of the ancestral protein, we recovered historical permissive substitutions but no alternative permissive genotypes. Using biophysical analysis, we found that permissive mutations must satisfy at least three physical requirements-they must stabilize specific local elements of the protein structure, maintain the correct energetic balance between functional conformations, and be compatible with the ancestral and derived structures-thus revealing why permissive mutations are rare. These findings demonstrate that GR evolution depended strongly on improbable, non-deterministic events, and this contingency arose from intrinsic biophysical properties of the protein.
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Robustness seems to be the opposite of evolvability. If phenotypes are robust against mutation, we might expect that a population will have difficulty adapting to an environmental change, as several studies have suggested. However, other studies contend that robust organisms are more adaptable. A quantitative understanding of the relationship between robustness and evolvability will help resolve these conflicting reports and will clarify outstanding problems in molecular and experimental evolution, evolutionary developmental biology and protein engineering. Here we demonstrate, using a general population genetics model, that mutational robustness can either impede or facilitate adaptation, depending on the population size, the mutation rate and the structure of the fitness landscape. In particular, neutral diversity in a robust population can accelerate adaptation as long as the number of phenotypes accessible to an individual by mutation is smaller than the total number of phenotypes in the fitness landscape. These results provide a quantitative resolution to a significant ambiguity in evolutionary theory.
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Directed evolution circumvents our profound ignorance of how a protein's sequence encodes its function by using iterative rounds of random mutation and artificial selection to discover new and useful proteins. Proteins can be tuned to adapt to new functions or environments by simple adaptive walks involving small numbers of mutations. Directed evolution studies have shown how rapidly some proteins can evolve under strong selection pressures and, because the entire 'fossil record' of evolutionary intermediates is available for detailed study, they have provided new insight into the relationship between sequence and function. Directed evolution has also shown how mutations that are functionally neutral can set the stage for further adaptation.
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The structural mechanisms by which proteins have evolved new functions are known only indirectly. We report x-ray crystal structures of a resurrected ancestral protein—the ∼450 million-year-old precursor of vertebrate glucocorticoid (GR) and mineralocorticoid (MR) receptors. Using structural, phylogenetic, and functional analysis, we identify the specific set of historical mutations that recapitulate the evolution of GR's hormone specificity from an MR-like ancestor. These substitutions repositioned crucial residues to create new receptor-ligand and intraprotein contacts. Strong epistatic interactions occur because one substitution changes the conformational position of another site. “Permissive” mutations—substitutions of no immediate consequence, which stabilize specific elements of the protein and allow it to tolerate subsequent function-switching changes—played a major role in determining GR's evolutionary trajectory.
Cryptic alleles make a bridge for adaptation Protein function is generally constrained by selective parameters that can inhibit evolutionary potential. It has thus been difficult to determine how novelties arise. Zheng et al. allowed bacterial populations to accumulate mutations and then used directed evolution to evolve green fluorescent protein function from a gene that expressed yellow fluorescent protein (see the Perspective by Lee and Marx). Protein alternatives could evolve in cases where cryptic alleles—selectively neutral or mildly deleterious genetic variants with no apparent phenotypic differences—were present in the population. Thus, cryptic alleles provide an evolutionary bridge between diversity and selection and facilitate the generation of novel adaptations. Science , this issue p. 347 ; see also p. 318
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