Evidence for a single, ancient origin of a genus-wide alternative life history strategy.

Understanding the evolutionary origins and factors maintaining alternative life history strategies (ALHS) within species is a major goal of evolutionary research. While alternative alleles causing discrete ALHS are expected to purge or fix over time, one-third of the ~90 species of Colias butterflies are polymorphic for a female-limited ALHS called Alba. Whether Alba arose once, evolved in parallel, or has been exchanged among taxa is currently unknown. Using comparative genome-wide association study (GWAS) and population genomic analyses, we placed the genetic basis of Alba in time-calibrated phylogenomic framework, revealing that Alba evolved once near the base of the genus and has been subsequently maintained via introgression and balancing selection. CRISPR-Cas9 mutagenesis was then used to verify a putative cis-regulatory region of Alba, which we identified using phylogenetic foot printing. We hypothesize that this cis-regulatory region acts as a modular enhancer for the induction of the Alba ALHS, which has likely facilitated its long evolutionary persistence.

Hybrid incompatibility arises in a sequence-based bioenergetic model of transcription factor binding.

Postzygotic isolation between incipient species results from the accumulation of incompatibilities that arise as a consequence of genetic divergence. When phenotypes are determined by regulatory interactions, hybrid incompatibility can evolve even as a consequence of parallel adaptation in parental populations because interacting genes can produce the same phenotype through incompatible allelic combinations. We explore the evolutionary conditions that promote and constrain hybrid incompatibility in regulatory networks using a bioenergetic model (combining thermodynamics and kinetics) of transcriptional regulation, considering the bioenergetic basis of molecular interactions between transcription factors (TFs) and their binding sites. The bioenergetic parameters consider the free energy of formation of the bond between the TF and its binding site and the availability of TFs in the intracellular environment. Together these determine fractional occupancy of the TF on the promoter site, the degree of subsequent gene expression and in diploids, and the degree of dominance among allelic interactions. This results in a sigmoid genotype-phenotype map and fitness landscape, with the details of the shape determining the degree of bioenergetic evolutionary constraint on hybrid incompatibility. Using individual-based simulations, we subjected two allopatric populations to parallel directional or stabilizing selection. Misregulation of hybrid gene expression occurred under either type of selection, although it evolved faster under directional selection. Under directional selection, the extent of hybrid incompatibility increased with the slope of the genotype-phenotype map near the derived parental expression level. Under stabilizing selection, hybrid incompatibility arose from compensatory mutations and was greater when the bioenergetic properties of the interaction caused the space of nearly neutral genotypes around the stable expression level to be wide. F2's showed higher hybrid incompatibility than F1's to the extent that the bioenergetic properties favored dominant regulatory interactions. The present model is a mechanistically explicit case of the Bateson-Dobzhansky-Muller model, connecting environmental selective pressure to hybrid incompatibility through the molecular mechanism of regulatory divergence. The bioenergetic parameters that determine expression represent measurable properties of transcriptional regulation, providing a predictive framework for empirical studies of how phenotypic evolution results in epistatic incompatibility at the molecular level in hybrids.

Specific-gene studies of evolutionary mechanisms in an age of genome-wide surveying.

The molecular tools of genomics have great power to reveal patterns of genetic difference within or among species, but must be complemented by the mechanistic study of the genetic variants found if these variants' evolutionary meaning is to be well understood. Central to this purpose is knowledge of the organisms' genotype-phenotype-environment interactions, which embody biological adaptation and constraint and thus drive natural selection. The history of this approach is briefly reviewed. Strategies embracing the complementarity of genomics and specific-gene studies in evolution are considered. Implementation of these strategies, and examples showing their feasibility and power, are discussed. Initial generalizations emphasize: (1) reproducibility of adaptive mechanisms; (2) evolutionary co-importance of variation in protein sequences and expression; (3) refinement of rudimentary molecular functions as an origin of evolutionary innovations; (4) identification of specific-gene mechanisms as underpinnings of genomic or quantitative genetic variation; and (5) multiple forms of adaptive or constraining epistasis among genes. Progress along these lines will advance understanding of evolution and support its use in addressing urgent medical and environmental applications.

A genetic polymorphism evolving in parallel in two cell compartments and in two clades.

BACKGROUND: The enzyme phosphoenolpyruvate carboxykinase, PEPCK, occurs in its guanosine-nucleotide-using form in animals and a few prokaryotes. We study its natural genetic variation in Colias (Lepidoptera, Pieridae). PEPCK offers a route, alternative to pyruvate kinase, for carbon skeletons to move between cytosolic glycolysis and mitochondrial Krebs cycle reactions. RESULTS: PEPCK is expressed in both cytosol and mitochondrion, but differently in diverse animal clades. In vertebrates and independently in Drosophila, compartment-specific paralogous genes occur. In a contrasting expression strategy, compartment-specific PEPCKs of Colias and of the silkmoth, Bombyx, differ only in their first, 5', exons; these are alternatively spliced onto a common series of following exons. In two Colias species from distinct clades, PEPCK sequence is highly variable at nonsynonymous and synonymous sites, mainly in its common exons. Three major amino acid polymorphisms, Gly 335 ↔ Ser, Asp 503 ↔ Glu, and Ile 629 ↔ Val occur in both species, and in the first two cases are similar in frequency between species. Homology-based structural modelling shows that the variants can alter hydrogen bonding, salt bridging, or van der Waals interactions of amino acid side chains, locally or at one another's sites which are distant in PEPCK's structure, and thus may affect its enzyme function. We ask, using coalescent simulations, if these polymorphisms' cross-species similarities are compatible with neutral evolution by genetic drift, but find the probability of this null hypothesis is 0.001 ≤ P ≤ 0.006 under differing scenarios. CONCLUSION: Our results make the null hypothesis of neutrality of these PEPCK polymorphisms quite unlikely, but support an alternative hypothesis that they are maintained by natural selection in parallel in the two species. This alternative can now be justifiably tested further via studies of PEPCK genotypes' effects on function, organismal performance, and fitness. This case emphasizes the importance, for evolutionary insight, of studying gene-specific mechanisms affected by natural genetic variation as an essential complement to surveys of such variation.

Evolutionary genomics of Colias Phosphoglucose Isomerase (PGI) introns.

Little is known of intron sequences' variation in cases where eukaryotic gene coding regions undergo strong balancing selection. Phosphoglucose isomerase, PGI, of Colias butterflies offers such a case. Its 11 introns include many point mutations, insertions, and deletions. This variation changes with intron position and length, and may leave little evidence of homology within introns except for their first and last few basepairs. Intron position is conserved between PGIs of Colias and the silkmoth, but no intron sequence homology remains. % GC content and length are functional properties of introns which can affect whole-gene transcription; we find a relationship between these properties which may indicate selection on transcription speed. Intragenic recombination is active in these introns, as in coding sequences. The small extent of linkage disequilibrium (LD) in the introns decays over a few hundred basepairs. Subsequences of Colias introns match subsequences of other introns, untranslated regions of cDNAs, and insect-related transposons and pathogens, showing that a diverse pool of sequence fragments is the source of intron contents via turnover due to deletion, recombination, and transposition. Like Colias PGI's coding sequences, the introns evolve reticulately with little phylogenetic signal. Exceptions are coding-region allele clades defined by multiple amino acid variants in strong LD, whose introns are closely related but less so than their exons. Similarity of GC content between introns and flanking exons, lack of small introns despite mutational bias toward deletion, and findings already mentioned suggest constraining selection on introns, possibly balancing transcription performance against advantages of higher recombination rate conferred by intron length.

Emergence of complex haplotypes from microevolutionary variation in sequence and structure of Colias phosphoglucose isomerase.

A molecular evolutionary explanation of natural genetic variation requires analysis of specific variants' evolutionary dynamics. To pursue this for phosphoglucose isomerase (PGI) of Colias butterflies, whose polymorphism is maintained by strong natural selection, we assembled a large data set of wild haplotypes, highly variable at the amino acid and DNA levels. The most common electrophoretic, i.e., charge macrostate, allele class, 3, is conserved in its pattern of charged amino acid residues. The next most common macrostate, 4, has multiple patterns of charge, i.e., microstates, while less common (1, 2, 5, 6) macrostates are very diverse. Macrostate 4 shows significant linkage disequilibrium (LD) among its variants, especially for two groups of five haplotypes each. We find extensive intragenic recombination among all haplotypes except the two high-LD groups of macrostate 4, which display none. Phyletic relations among haplotypes are largely reticulate, again except for the high-LD groups of macrostate 4, which form clades with strong bootstrap support. Charge-changing and linked charge-neutral amino acid variants occur in diverse parts of PGI's sequence. Homology-based modeling of PGI's structure shows that these regions are related spatially in ways suggesting functional interaction. The high-LD groups of macrostate 4 display parallel amino acid variation in these regions. This pattern of haplotype clades with high LD among multiple varying sites, emerging from chaotically recombining variation, may be a "signature" of refinement of complex adaptive sequences by recombination and selection. It should be tested further in this study system and others as a possibly general feature of the evolution of living complexity.

A mitochondrial-DNA-based phylogeny for some evolutionary-genetic model species of Colias butterflies (Lepidoptera, Pieridae).

We study the phylogenetic relationships among some North American Colias ("sulfur") butterflies, using mitochondrial gene sequences (ribosomal RNA, cytochrome oxidase I+II) totaling about 20% of the mitochondrial genome. We find that (1) the lowland species complex shows a branching order different from earlier views; (2) several montane and northern taxa may be more distinct than in earlier views; (3) one morphologically conservative Holarctic assemblage, C. hecla, is differentiated at the molecular-genetic level into at least three taxa which occupy distinct positions in the phylogeny and are sisters to diverse other taxa. These conclusions, constituting phylogenetic hypotheses, are supported by parsimony, maximum-likelihood, and Bayesian reconstruction algorithms. They are tested formally, by interior branch tests and paired-site tests, against alternative hypotheses derived from conventional species and subspecies naming combinations. In all cases our hypotheses are supported by these tests and the conventional alternatives are rejected. The "barcoding" subset of cytochrome oxidase I sequence identifies only some of the taxa supported by our full data set. Comparison of genetic divergence values among Colias taxa with those among related Pierid butterflies suggests that species radiations within Colias are comparatively younger. This emerging Colias phylogeny facilitates comparisons of genetic polymorphism and other adaptive mechanisms among taxa, thereby connecting micro- and macro-evolutionary processes.

From DNA to fitness differences: sequences and structures of adaptive variants of Colias phosphoglucose isomerase (PGI).

Colias eurytheme butterflies display extensive allozyme polymorphism in the enzyme phosphoglucose isomerase (PGI). Earlier studies on biochemical and fitness effects of these genotypes found evidence of strong natural selection maintaining this polymorphism in the wild. Here we analyze the molecular features of this polymorphism by sequencing multiple alleles and modeling their structures. PGI is a dimer with rotational symmetry. Each monomer provides a critical residue to the other monomer's catalytic center. Sequenced alleles differ at multiple amino acid positions, including cryptic charge-neutral variation, but most consistent differences among the electromorph alleles are at the charge-changing amino acid sites. Principal candidate sites of selection, identified by structural and functional analyses and by their variants' population frequencies, occur in interpenetrating loops across the interface between monomers, where they may alter subunit interactions and catalytic center geometry. Comparison to a second (and basal) species, Colias meadii, also polymorphic for PGI under natural selection, reveals one fixed amino acid difference between their PGIs, which is located in the interpenetrating loop and accompanies functional differences among their variants. We also study nucleotide variability among the PGI alleles, comparing these data to similar data from another glycolytic enzyme gene, glyceraldehyde-3-phosphate dehydrogenase. Despite extensive nonsynonymous and synonymous polymorphism at PGI in each species, the only base changes fixed between species are the two causing the amino acid replacement; this absence of synonymous fixation yields a significant McDonald-Kreitman test. Analyses of these data suggest historical population expansion. Positive peaks of Tajima's D statistic, representing regions of neutral "hitchhiking," are found around the principal candidate sites of selection. This study provides novel views of molecular-structural mechanisms, and beginnings of historical evidence, for a long-persistent balanced enzyme polymorphism at PGI in these and perhaps other species.

Thermal physiological ecology of Colias butterflies in flight.

As a comparison to the many studies of larger flying insects, we carried out an initial study of heat balance and thermal dependence of flight of a small butterfly (Colias) in a wind tunnel and in the wild.Unlike many larger, or facultatively endothermic insects, Colias do not regulate heat loss by altering hemolymph circulation between thorax and abdomen as a function of body temperature. During flight, thermal excess of the abdomen above ambient temperature is weakly but consistently coupled to that of the thorax. Total heat loss is best expressed as the sum of heat loss from the head and thorex combined plus heat loss from the abdomen because the whole body is not isothermal. Convective cooling is a simple linear function of the square root of air speed from 0.2 to 2.0 m/s in the wind tunnel. Solar heat flux is the main source of heat gain in flight, just as it is the exclusive source for warmup at rest. The balance of heat gain from sunlight versus heat loss from convection and radiation does not appear to change by more than a few percent between the wings-closed basking posture and the variable opening of wings in flight, although several aspects require further study. Heat generation by action of the flight muscles is small (on the order of 100 m W/g tissue) compared to values reported for other strongly flying insects. Colias appears to have only very limited capacity to modulate flight performance. Wing beat frequency varies from 12-19 Hz depending on body mass, air speed, and thoracic temperature. At suboptimal flight temperatures, wing beat frequency increases significantly with thoracic temperature and body mass but is independent of air speed. Within the reported thermal optimum of 35-39°C, wing beat frequency is negatively dependent on air speed at values above 1.5 m/s, but independent of mass and body temperature. Flight preference of butterflies in the wind tunnel is for air speeds of 0.5-1.5 m/s, and no flight occurs at or above 2.5 m/s. Voluntary flight initiation in the wild occurs only at air speeds ≦1.4 m/s.In the field, Colias fly just above the vegetation at body temperatures of 1-2°C greater than when basking at the top of the vegetation. These measurements are consistent with our findings on low heat gain from muscular activity during flight. Basking temperatures of butterflies sheltered from the wind within the vegetation were 1-2°C greater than flight temperatures at vegetation height.

Population structure of pierid butterflies IV. Genetic and physiological investment in offspring by male Colias.

Population structure encompasses all the rules by which a population's gametes come together, including genetic and physiological investment in offspring. We document female use of nutrients donated by males at mating, and complete sperm precedence, in Colias eurytheme Boisduval. The effect of these phenomena on the population structure of this species is discussed.

Individual variation in oviposition preference in the butterfly, Colias eurytheme.

We examined intra-population variation in oviposition preference in the pierid butterfly, Colias eurytheme. Females' preferences were tested in the laboratory, using two-way choice tests between the potential hosts, alfalfa and vetch. There were consistent differences in oviposition preference among females within a population. Larval and adult experience had little or no effect on females' preference. These results suggest that the intra-population variation in oviposition preference is genetically based, but the results of experiments designed to estimate the heritability of oviposition preference were not conclusive. We suggest that intra-population variation in host selection characters may play a key role in shifts to new host plants.

Population structure of pierid butterflies : II. A "Native" population of Colias philodice eriphyle in Colorado.

The structure of a bivoltine, discrete-generation population of Colias philodice eriphyle, occurring in relatively undisturbed habitat, has been examined by mark-release-recapture techniques. The population's general ecology is briefly discussed. Males eclose before females as in other Colias, and a measure of physical wear on adults is related to age of individuals and to the overall position of a sample in the flight period, again as in other Colias. Densities of adults fluctuate drastically, with the first (overwintering) brood always being less dense than the second brood. Dispersal radius of those dispersing does not vary with brood, sex, or year, although the proportion of dispersants does: more males than females disperse in the first brood, while the reverse is true in the second. A tentative behavioral explanation for this is proposed. Adult mortality is unusually high compared to other Colias. The population displays area continuity with adjacent population areas. The Wrightian neighborhood size varies in geographic extent, due to change in dispersant proportions, from 70 to 260 hectares. In adult numbers, it varies from 4-500 (or possibly fewer in very severe first-brood conditions) to upwards of 20,000 in some second broods (though not all adults present always reproduce successfully). Two possible models for the dispersal behavior of Colias are presented. One, the "excited state" model, is so far supported over the other, "continuous activity" model, by the present data and by earlier work on C. alexandra. The adult mortality level is consistent with the conclusion that this population is ecologically marginal for the species. Possible selective pressures preventing further extension of the species' distribution, including possible competition with other Colias, are discussed.

Population structure of pierid butterflies : I. Numbers and movements of some montane Colias species.

The numbers, dispersal behavior, aging and residence, and Wrightian neighborhood configurations of three species of Colias butterflies have been studied in central Colorado, using mark-release-recapture techniques as major tools. All populations studied have nonoverlapping generations and mature one brood each year. A brief general review of these species' autecology is given. A system for measuring degree of physical damage to the adults is introduced. This "wear rating" varies with temporal position of any given sample in the course of a brood's flight season, the insects becoming progressively more damaged with time. The sex ratio also changes with brood aging: males eclose before females, and are in the majority early in the flight season, while females may predominate at the end of flight. Local population numbers for the montane grassland species C. alexandra may reach peak levels of 700-900 insects in favorable years, but be much lower in other years as a result of, e.g., drought. Peak densities are no more than 2/ha. The montane bog species, C. scudderi, maintains comparable low density but has much smaller local populations. The subalpine/alpine grassland species C. meadii displays peak local numbers as high as 3000, with peak density as high as 120/hectare. Dispersal varies both among and within species. Those C. alexandra who disperse show an average dispersal radius of about 1.3 km, with a radius for the whole population of about 0.6 km; maximum distance moved was 8 km. Dispersal proportions among recaptures are sharply curtailed by adverse weather, but the dispersal radius of those moving is unaffected by weather. C. scudderi's dispersal is strongly influenced by the geometry of its bog and streamside habitats. Some C. meadii populations approach isolated "island" status, but others show much dispersal. Dispersal radius of those dispersing ranges from 0.3 to 0.7 km in different populations, but the proportion of dispersals varies greatly. The longest observed movement by this species is 1.3 km, although up to 2.6 km could have been detected. Colias normally display constant loss (death plus emigration) rates with average residence expectations of 4-6 days; few insects reach their maximum physiological lifespan of approximately 1 month. Bad weather can increase the loss rate drastically. Females show shorter residence than males, appearently as a result of greater mortality. Total-numbers-per-brood estimates are given for our better studied populations. The reproductive strategy of Colias is such that Wright's models for neighborhood size apply. Neighborhood size for C. alexandra varied sixfold in numbers, and from 3 to 1.3 km in physical extent, between a favorable year and a drought year. One localized C. scudderi habitat is only 200 m in diameter, but a streamside population has a neighborhood length of 4.8 km. In C. meadii, one population of 2000-2500 insects is an 8-ha "island", while another of similar numbers extends a single neighborhood across 1.9 km distance, 450 m altitude, and a major ecological boundary (timberline). Factors such as weather, individuals' visual cueing, and thermoregulatory behavior can influence population structure. For some Colias populations, selection may be very uniform within neighborhoods, while for others, single neighborhoods cross sharp discontinuities in selective forces. These patterns may differ for different selective forces, and may also vary with stages of the insects' life cycle. these populations will now prove a valuable resource for studying evolutionary population genetics.

Nectar resource use by Colias butterflies : Chemical and visual aspects.

Nectar foraging preferences of Colias butterflies in two different mountain ecosystems are examined with respect to plant distribution, nectar quantity, carbohydrate (and amino acid) content of nectar, and visual pattern of the plants utilized and avoided. Colias, and apparently numerous other small, ectothermic, low-energy-demand pollinators, "patronize" plants producing relatively dilute nectars containing a high proportion of monosaccharide sugars and significant amounts of polar, nitrogen-rich amino acids. These plants also converge on a common "target" flower pattern in ultraviolet and human-visible light. High-energy demand, endothermic pollinators, by contrast, appear to require higher concentration nectars and/or higher proportions of di- and oligosaccharide sugars. These results are discussed in the light of water balance and energy budget demands of different pollinator classes. Questions are also raised concerning behavioral aspects of pollinator search for resources and the pertinence of these data to the concept of floral mimicry.