Genomic basis of parallel adaptation varies with divergence in Arabidopsis and its relatives

Parallel adaptation provides valuable insight into the predictability of evolutionary change through replicated natural experiments. A steadily increasing number of studies have demonstrated genomic parallelism, yet the magnitude of this parallelism varies depending on whether populations, species, or genera are compared. This led us to hypothesize that the magnitude of genomic parallelism scales with genetic divergence between lineages, but whether this is the case and the underlying evolutionary processes remain unknown. Here, we resequenced seven parallel lineages of two Arabidopsis species, which repeatedly adapted to challenging alpine environments. By combining genome-wide divergence scans with model-based approaches, we detected a suite of 151 genes that show parallel signatures of positive selection associated with alpine colonization, involved in response to cold, high radiation, short season, herbivores, and pathogens. We complemented these parallel candidates with published gene lists from five additional alpine Brassicaceae and tested our hypothesis on a broad scale spanning ∼0.02 to 18 My of divergence. Indeed, we found quantitatively variable genomic parallelism whose extent significantly decreased with increasing divergence between the compared lineages. We further modeled parallel evolution over the Arabidopsis candidate genes and showed that a decreasing probability of repeated selection on the same standing or introgressed alleles drives the observed pattern of divergence-dependent parallelism. We therefore conclude that genetic divergence between populations, species, and genera, affecting the pool of shared variants, is an important factor in the predictability of genome evolution.

Evolution is driven by a complex interplay of deterministic and stochastic forces whose relative importance is a matter of debate (1). Being largely a historical process, we have limited ability to experimentally test for the predictability of evolution in its full complexity (i.e., in natural environments) (2). Distinct lineages that independently adapted to similar conditions by similar phenotype (termed parallel,” considered synonymous to “convergent” here) can provide invaluable insights into the issue (3, 4). An improved understanding of the probability of parallel evolution in nature may inform on constraints on evolutionary change and provide insights relevant for predicting the evolution of pathogens (5–7), pests (8, 9), or species in human-polluted environments (10, 11). Although the past few decades have seen an increasing body of work supporting the parallel emergence of traits by the same genes and even alleles, we know surprisingly little about what makes parallel evolution more likely and, by extension, what factors underlie evolutionary predictability (1, 12).A wealth of literature describes the probability of “genetic” parallelism, showing why certain genes are involved in parallel adaptation more often than others (13). There is theoretical and empirical evidence for the effect of pleiotropic constraints, availability of beneficial mutations or position in the regulatory network all having an impact on the degree of parallelism at the level of a single locus (3, 13–18). In contrast, we know little about causes underlying “genomic” parallelism (i.e., what fraction of the genome is reused in adaptation and why). Individual case studies demonstrate large variation in genomic parallelism, ranging from absence of any parallelism (19), similarity in functional pathways but not genes (20, 21), and reuse of a limited number of genes (22–24) to abundant parallelism at both gene and functional levels (25, 26). Yet, there is little consensus about what determines variation in the degree of gene reuse (fraction of genes that repeatedly emerge as selection candidates) across investigated systems (1).Divergence (the term used here to consistently describe both intra- and interspecific genetic differentiation) between the compared instances of parallelism appears as a potential driver of the variation in gene reuse (14, 27, 28). Phenotype-oriented meta-analyses suggest that both phenotypic convergence (28) and genetic parallelism underlying phenotypic traits (14) decrease with increasing time to the common ancestor. Although a similar targeted multiscale comparison is lacking at the genomic level, our brief review of published studies (29 cases, Dataset S1) suggests that also gene reuse tends to scale with divergence (Fig. 1A and SI Appendix, Fig. S1). Moreover, allele reuse (repeated sweep of the same haplotype that is shared among populations either via gene flow or from standing genetic variation) frequently underlies parallel adaptation between closely related lineages (29–32), while parallelism from independent de novo mutations at the same locus dominates between distantly related taxa (13). Similarly, previous studies reported a decreasing probability of hemiplasy (apparent convergence resulting from gene tree discordance) with divergence in phylogeny-based studies (33, 34). This suggests that the degree of allele reuse may be the primary factor underlying the hypothesized divergence-dependency of parallel genome evolution, possibly reflecting either weak hybridization barriers, widespread ancestral polymorphism between closely related lineages (35), or ecological reasons (lower niche differentiation and geographical proximity) (36, 37). However, the generally restricted focus of individual studies of genomic parallelism on a single level of divergence does not lend itself to a unified comparison across divergence scales. Although different ages of compared lineages affect a variety of evolutionary–ecological processes such as diversification rates, community structure, or niche conservatism (37), the hypothesis that genomic parallelism scales with divergence has not yet been systematically tested, and the underlying evolutionary processes remain poorly understood.Open in a separate windowFig. 1.Hypotheses regarding relationships between genomic parallelism and divergence and the Arabidopsis system used to address these hypotheses. (A) Based on our literature review, we propose that genetically closer lineages adapt to a similar challenge more frequently by gene reuse, sampling suitable variants from the shared pool (allele reuse), which makes their adaptive evolution more predictable. Color ramp symbolizes rising divergence between the lineages (∼0.02 to 18 Mya in this study); the symbols denote different divergence levels tested here using resequenced genomes of 22 Arabidopsis populations (circles) and meta-analysis of candidates in Brassicaceae (asterisks). (B) Spatial arrangement of lineages of varying divergence (neutral F_ST; bins only aid visualization; all tests were performed on a continuous scale) encompassing parallel alpine colonization within the two Arabidopsis outcrossers from central Europe: A. arenosa (diploid: aVT; autotetraploid: aNT, aZT, aRD, and aFG) and A. halleri (diploid: hNT and hFG). Note that only two of the ten between-species pairs (dark green) are shown to aid visibility. The color scale corresponds to the left part of the color ramp used in A. (C) Photos of representative alpine and foothill habitat. (D) Representative phenotypes of originally foothill and alpine populations grown in common garden demonstrating phenotypic convergence. Scale bar corresponds to 4 cm. (E) Morphological differentiation among 223 A. arenosa individuals originating from foothill (black) and alpine (gray) populations from four regions after two generations in a common garden. Principal component analysis was run using 16 morphological traits taken from ref. 45.Here, we aimed to test this hypothesis and investigate whether allele reuse is a major factor underlying the relationship. We analyzed replicated instances of adaptation to a challenging alpine environment, spanning a range of divergence from populations to tribes within the plant family Brassicaceae (38–43) (Fig. 1A). First, we took advantage of a unique naturally multireplicated setup in the plant model genus Arabidopsis that was so far neglected from a genomic perspective (Fig. 1B). Two predominantly foothill-dwelling Arabidopsis outcrossers (A. arenosa, A. halleri) exhibit scattered, morphologically distinct alpine occurrences at rocky outcrops above the timberline (Fig. 1C). These alpine forms are separated from the widespread foothill population by a distribution gap spanning at least 500 m of elevation. Previous genetic and phenotypic investigations and follow-up analyses presented here showed that the scattered alpine forms of both species represent independent alpine colonization in each mountain range, followed by parallel phenotypic differentiation (Fig. 1 D and E) (44–46). Thus, we sequenced genomes from seven alpine and adjacent foothill population pairs, covering all European lineages encompassing the alpine ecotype. We discovered a suite of 151 genes from multiple functional pathways relevant to alpine stress that were repeatedly differentiated between foothill and alpine populations. This points toward a polygenic, multifactorial basis of parallel alpine adaptation.We took advantage of this set of well-defined parallel selection candidates and tested whether the degree of gene reuse decreases with increasing divergence between the compared lineages (Fig. 1A). By extending our analysis to five additional alpine Brassicaceae species, we further tested whether there are limits to gene reuse above the species level. Finally, we inquired about possible underlying evolutionary processes by estimating the extent of allele reuse using a designated modeling approach. Overall, our empirical analysis provides a perspective to the ongoing discussion about the variability in the reported magnitude of parallel genome evolution and identifies allele reuse as an important evolutionary process shaping the extent of genomic parallelism between populations, species, and genera.     

From the Cover: The genomes of ancient date palms germinated from 2,000 y old seeds

Seven date palm seeds (Phoenix dactylifera L.), radiocarbon dated from the fourth century BCE to the second century CE, were recovered from archaeological sites in the Southern Levant and germinated to yield viable plants. We conducted whole-genome sequencing of these germinated ancient samples and used single-nucleotide polymorphism data to examine the genetics of these previously extinct Judean date palms. We find that the oldest seeds from the fourth to first century BCE are related to modern West Asian date varieties, but later material from the second century BCE to second century CE showed increasing genetic affinities to present-day North African date palms. Population genomic analysis reveals that by ∼2,400 to 2,000 y ago, the P. dactylifera gene pool in the Eastern Mediterranean already contained introgressed segments from the Cretan palm Phoenix theophrasti, a crucial genetic feature of the modern North African date palm populations. The P. theophrasti introgression fraction content is generally higher in the later samples, while introgression tracts are longer in these ancient germinated date palms compared to modern North African varieties. These results provide insights into crop evolution arising from an analysis of plants originating from ancient germinated seeds and demonstrate what can be accomplished with the application of a resurrection genomics approach.

Genome sequencing of ancient samples has provided an unprecedented window into the evolutionary history and biology of past populations and even extinct species. Data from ancient genomes has given us glimpses into human evolution, including extinct hominin species such as Neanderthals (1), and also helped us understand the evolutionary history of species as divergent as mammoths (2), horses (3), grapes (4), and maize (5). Sequencing ancient DNA, however, comes with numerous challenges (6, 7). DNA from fossilized samples is degraded—the molecules are short and chemically modified—and the amount of recovered endogenous DNA is small, even under hospitable preservation conditions (8). Plants present particular challenges (9); in general, plant materials do not have the protective bone tissue found in vertebrates that helps in preserving ancient DNA, and the limited size of archaeobotanical remains often yields only small quantities of endogenous DNA (10). Nevertheless, studies of ancient plant DNA have been conducted successfully, particularly in maize (5), emmer wheat (11), barley (12), and rice (13) as well as other crop species (14, 15). Most of these studies center on archaeobotanical samples found in arid (12), heavily waterlogged (16), or even mineralized environments (17) that have helped to preserve DNA material.An alternative approach to ancient DNA studies in plants is to germinate ancient seeds from archaeological sites (18), permafrost (19), or historical collections (11) and study the genomics of these revived individuals—an approach we refer to as resurrection genomics. Seeds ranging in age from ∼1,300 y old in the sacred lotus Nelumnbo nucifera (20) to ∼30,000 y old for Silene stenophylla (19) have been germinated successfully, providing the ability to obtain living, intact biological material from otherwise ancient samples. Access to such material may lead to information on earlier phenotypes and (for genomic analysis) would circumvent sequencing errors that arise from degraded ancient DNA, overcome the low amount of endogenous DNA, and allow sequencing at deep coverage.We applied this resurrection genomics approach to date palms (Phoenix dactylifera L.), which represent one of the best examples of successful germination of ancient seeds (21, 22). Date palms are a domesticated fruit crop species that are the iconic perennial plant of the arid lands of West Asia and North Africa (Fig. 1A). This species is believed to have been domesticated ∼7,000 y ago in the region around the Arabian Gulf (23, 24). From there, dates presumably spread westward and were widely cultivated in Egypt from at least the mid-second millennium BCE and further west to the Maghreb at least by the first millennium BCE (23, 25–27).Open in a separate windowFig. 1.Map of date palms. (A) Geographical distribution of P. dactylifera and P. theophrasti. The Southern Levant is indicated, and the box in this area indicates where the archaeological samples were collected (see next panel). (B) Location of archaeological sites where the germinated Judean date palm seeds were collected.The Southern Levant (modern-day Israel, Palestine, and Jordan) (Fig. 1B) is situated at the crossroads of Asia, Africa, and Europe, and during the 11th century BCE, this area saw the rise of the ancient geopolitical entity of Judea. Date palms grown in antiquity around Jericho and along the Dead Sea in Judea were referred to as Judean date palms, although it is unclear whether this referred to a distinct genetic population. These Judean date palms were discussed by classical writers such as Josephus and Pliny, who described them as producing superior fruit (28). Indeed, analysis from archaeological excavations in the region do show that the sizes of ancient seeds from these palms were significantly larger than those from modern date varieties (22).Judean date cultivation is thought to have continued sporadically through the Byzantine and Arab periods (fourth to 11th century CE). By the 19th century, however, no trace of the Judean date palms remained, as date palm agriculture was extinguished in the area (28). Although Judean date palms are believed to be extinct (21, 22), it is unclear if some of their descendants still exist among modern date palm varieties.Given their presence in a pivotal geographic location at the edge of West Asia and North Africa, the study of Judean date palm genomes could provide insights into the nature and timing of the spread of domesticated dates across the region. Studying the genomes of Judean date palms, as well as reviving this hitherto extinct population, became possible in 2008 when we reported the germination of a ∼2,000 y old date seed recovered from the historic archaeological site of Masada overlooking the Dead Sea (21). Six additional ancient date palm seeds from archaeological sites in Masada, Qumran, and Wadi Makukh in the Judean Desert and dated from the same time period were subsequently germinated (22) (Fig. 1B).The genetic analyses of these germinated Judean date palms using microsatellite markers established that they represent a mix of North African and West Asian ancestry (22). Modern date palm varieties are genetically differentiated into West Asian [previously referred to by us as Middle Eastern (29–31)] and North African populations (29, 32–34) (Fig. 1A), the latter resulting in part from introgressive hybridization of the Cretan palm Phoenix theophrasti, or a theophrasti-like population, into P. dactylifera (30). The location and timing of this interspecies hybridization, which may be associated with the rise and spread of North African dates, remains unclear (35). In the Nile Valley, date palms were present as early as the predynastic period ∼5,000 y ago but were not extensively cultivated in Egypt until at least the New Kingdom ∼3,500 y ago (26, 27). Further west, date palm remains are dated to about ∼2,800 to 2,400 y ago in Libya (36) but are not found in the Saharan Maghreb and the sub-Saharan Sahel until much later (30, 37).We do not know if these early examples of date palm cultivation in Africa were already impacted by genetic contributions from P. theophrasti, and determining when this interspecific hybridization occurred could tell us whether this took place during antiquity or much later in the evolution of this domesticated fruit species. An analysis of the full genome sequences of the Judean date palms germinated from ∼2,000 y old seeds could also advance our understanding of the role that interspecies hybridization has played in the evolution of this fruit crop species. Here, we report whole-genome sequencing of the seven germinated ancient Judean date palm samples, which provides an opportunity to study the change in genomic composition of date palms in the Southern Levant two millennia ago. We demonstrate that hybridization between P. dactylifera and P. theophrasti took place at least by the second century BCE, and we show increasing levels of Cretan palm introgression in the Southern Levant date palm populations across a period that spanned the fourth century BCE to mid-second century CE. We also use genome data to examine genes associated with fruit color and sugar composition, providing information on genetic characteristics of previously extinct (but now resurrected) Judean dates from ∼2,000 y ago.     

Genomic stability through time despite decades of exploitation in cod on both sides of the Atlantic

The mode and extent of rapid evolution and genomic change in response to human harvesting are key conservation issues. Although experiments and models have shown a high potential for both genetic and phenotypic change in response to fishing, empirical examples of genetic responses in wild populations are rare. Here, we compare whole-genome sequence data of Atlantic cod (Gadus morhua) that were collected before (early 20th century) and after (early 21st century) periods of intensive exploitation and rapid decline in the age of maturation from two geographically distinct populations in Newfoundland, Canada, and the northeast Arctic, Norway. Our temporal, genome-wide analyses of 346,290 loci show no substantial loss of genetic diversity and high effective population sizes. Moreover, we do not find distinct signals of strong selective sweeps anywhere in the genome, although we cannot rule out the possibility of highly polygenic evolution. Our observations suggest that phenotypic change in these populations is not constrained by irreversible loss of genomic variation and thus imply that former traits could be reestablished with demographic recovery.

As anthropogenic activities rapidly transform the environment, a fundamental question is whether wild populations have the capacity to adapt and evolve fast enough in response (1–3). Phenotypic change can result from phenotypic plasticity, but emerging examples of genomic change over only a few generations have made clear that rapid evolution is also possible (4–6). In the literature, one of the most dramatic and widely cited cases involves the declining age and size at maturation of Atlantic cod (Gadus morhua) following several generations of high fishing pressure (3, 7–10). Fisheries produce some of the fastest rates of phenotypic change ever observed in wild populations (2, 11), but the extent to which fisheries-induced evolution has occurred in the wild and the degree to which it is reversible remain strongly debated (12).The hypothesis that evolution underlies these phenotypic changes is supported by a range of observations. For example, theory on the selective nature of many fisheries reveals that higher rates of harvesting will—with only a few exceptions—favor earlier sexual maturation, greater investment in reproduction, and slower growth (13). In addition, experiments in the laboratory that selectively remove large or small individuals from a population reveal rapid evolution of body size and maturation time in only a few generations, as well as substantial impacts on fishery yields (14–16). Fisheries-induced evolution experiments in the laboratory also reveal selective sweeps through dramatic shifts in allele frequencies, loss of genetic diversity, and increases in linkage disequilibrium at specific locations in the genome (15, 17, 18).However, translating these findings to wild populations has been substantially more difficult. One concern is that phenotypic plasticity, gene flow, or spatial shifts in populations can also explain the substantial phenotypic and limited genotypic changes reported from the wild to date (10, 13, 19–23). The magnitude and rate of fisheries-induced evolution may also be quite small in the wild (19). While theory provides strong evidence that fishing can be a potent driver of evolutionary changes, a clear empirical demonstration of fisheries-induced evolution would require evidence that the observed change is genetic (13). Whether and to what extent the widespread genomic reorganization observed in experiments also occurs in wild-harvested populations therefore remain unknown.Genomic analyses of temporal samples before and after selective events have provided key opportunities to test for rapid adaptive evolution from standing genetic variation in wild populations by identifying unusually strong shifts in allele frequencies over time (4, 5). In addition, the history of genomic research with Atlantic cod (24, 25) provides a unique opportunity to test for genomic signatures of fisheries-induced evolution in particular. Archival samples collected by fisheries scientists decades or even centuries ago represent a valuable source of historical genomic material that can provide rare insight into the genetic patterns of the past (26). Here, we obtained whole-genome sequence data from well-preserved archives of Atlantic cod scales and otoliths (ear bones) that were originally collected from two populations on either side of the Atlantic Ocean: the northeast Arctic population sampled near Lofoten, Norway in 1907 and the Canadian northern cod population sampled near Twillingate, Newfoundland in 1940 (Fig. 1A and SI Appendix, Table S1). The Canadian northern population collapsed from overfishing in the early 1990s, while the northeast Arctic population experienced high fishing rates but smaller declines in biomass (10, 27, 28). Both populations have shown marked reductions in age at maturation, though with slight increases in maturation age in northeast Arctic cod after 2005 (Fig. 1B). We compared these historical genomes with modern data from the same locations (Fig. 1A and SI Appendix, Table S2). In total, we analyzed 113 individual genomes (Methods) from these two unique populations that had independently experienced intensive fishing during the last century (7, 10). We found a marked lack of large genomic changes or selective sweeps through time, suggesting instead that phenotypic plasticity or, potentially, highly polygenic evolution can explain the observed changes in phenotype.Open in a separate windowFig. 1.Spatiotemporal population structure based on genome-wide data in Atlantic cod. (A) In total, 113 modern and historical specimens were analyzed from northern cod collected in Newfoundland, Canada (1940, yellow; 2013, dark yellow) and from northeast Arctic cod collected in the Lofoten archipelago, Norway (1907, orange; modern: 2011, red; 2014, dark red). (B) Age at 50% maturity over time in each population. (C) PCA as implemented in PCAngsd. Velicier’s minimum average partial (MAP) test identified a single significant PC and only one PC is shown. Individuals are colored according to A. (D) Model-based ADMIXTURE ancestry components for historical (1907, 1940) and modern (2013, 2014) populations (k = 2; NGSadmix). Each individual is represented by a column colored to show the proportion of each ancestry component for Canada (dark yellow) and Norway (orange). Population differentiation based on pairwise weighted F_ST is also shown. (E) The correlation between the allele frequencies in historical and modern populations. Colors reflect the relative density of points, from darker (more density) to lighter (less density). R², coefficient of correlation.     

A switch to feeding on cycads generates parallel accelerated evolution of toxin tolerance in two clades of Eumaeus caterpillars (Lepidoptera: Lycaenidae)

We assembled a complete reference genome of Eumaeus atala, an aposematic cycad-eating hairstreak butterfly that suffered near extinction in the United States in the last century. Based on an analysis of genomic sequences of Eumaeus and 19 representative genera, the closest relatives of Eumaeus are Theorema and Mithras. We report natural history information for Eumaeus, Theorema, and Mithras. Using genomic sequences for each species of Eumaeus, Theorema, and Mithras (and three outgroups), we trace the evolution of cycad feeding, coloration, gregarious behavior, and other traits. The switch to feeding on cycads and to conspicuous coloration was accompanied by little genomic change. Soon after its origin, Eumaeus split into two fast evolving lineages, instead of forming a clump of close relatives in the phylogenetic tree. Significant overlap of the fast evolving proteins in both clades indicates parallel evolution. The functions of the fast evolving proteins suggest that the caterpillars developed tolerance to cycad toxins with a range of mechanisms including autophagy of damaged cells, removal of cell debris by macrophages, and more active cell proliferation.

The genus Eumaeus Hübner (Lycaenidae, Theclinae) arguably contains the most aposematically colored caterpillars and butterflies among the ∼4,000 Lycaenidae in the world (1–6). The brilliant red and gold gregarious caterpillars (Fig. 1) sequester cycasin from the leaves of their cycad food plants (Zamiaceae), which deters predators (3–9). Other secondary metabolites in cycads (e.g., 10‒11) may also deter predators. Eumaeus adults have a bright orange-red abdomen and an orange-red hindwing spot (except for one species) (Fig. 2). Blue and green iridescent markings are especially conspicuous on a black ground color. Eumaeus adults are among the largest lycaenids and have more rounded wings and a slower, more gliding flight than most Theclinae (1). Cycads are among the most primitive extant seed-plants (9), and the “plethora of aposematic attributes suggests a very ancient association between Eumaeus and the cycad host plants” (3).Open in a separate windowFig. 1.Caterpillars and pupae of Theorema eumenia (Top) and Eumaeus godartii (Bottom) in Costa Rica. Clockwise from Upper Left, second or third instar (length, ∼13 mm), fourth (final) instar (∼20 mm), pupa (∼18 mm), pupa (∼24 mm), fourth (final) instar (∼27 mm), second or third instar (∼20 mm). (Images from authors W.H. and D.H.J.).Open in a separate windowFig. 2.Adult wing uppersides and undersides. Eumaeus childrenae (two Upper Left images), E. atala (two Upper Right images), Theorema eumenia (two Lower Left images), and Mithras nautes (two Lower Right images). Scale bar, 1 cm.Eumaeus has been classified as a separate family (12–14), a genus in the Riodinidae (15‒16), or a monotypic subfamily or tribe of the Lycaenidae (17–20). Alternatively, others called it a typical member of the Neotropical Lycaenidae (21‒22). The evolutionary question behind this discordant taxonomic history is whether Eumaeus is a phylogenetically isolated lineage long associated with cycads (3) or an embedded clade in which a recent food plant shift to cycads resulted in the rapid evolution of aposematism. Recent molecular evidence for a limited number of taxa suggested the latter (23). To answer this question definitively, we analyzed genomic sequences of Eumaeus and its relatives.To trace the evolution of cycad feeding, we report the caterpillar food plants of the genera most closely related to Eumaeus and illustrate their immature stages (Fig. 1 and SI Appendix). This natural history information combined with analyses of genome sequences is the foundation for investigating the subsequent evolutionary impact on the Eumaeus genome of the switch to eating cycads.     

From the Cover: Evidence from South Africa for a protracted end-Permian extinction on land

Earth’s largest biotic crisis occurred during the Permo–Triassic Transition (PTT). On land, this event witnessed a turnover from synapsid- to archosauromorph-dominated assemblages and a restructuring of terrestrial ecosystems. However, understanding extinction patterns has been limited by a lack of high-precision fossil occurrence data to resolve events on submillion-year timescales. We analyzed a unique database of 588 fossil tetrapod specimens from South Africa’s Karoo Basin, spanning ∼4 My, and 13 stratigraphic bin intervals averaging 300,000 y each. Using sample-standardized methods, we characterized faunal assemblage dynamics during the PTT. High regional extinction rates occurred through a protracted interval of ∼1 Ma, initially co-occurring with low origination rates. This resulted in declining diversity up to the acme of extinction near the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary. Regional origination rates increased abruptly above this boundary, co-occurring with high extinction rates to drive rapid turnover and an assemblage of short-lived species symptomatic of ecosystem instability. The “disaster taxon” Lystrosaurus shows a long-term trend of increasing abundance initiated in the latest Permian. Lystrosaurus comprised 54% of all specimens by the onset of mass extinction and 70% in the extinction aftermath. This early Lystrosaurus abundance suggests its expansion was facilitated by environmental changes rather than by ecological opportunity following the extinctions of other species as commonly assumed for disaster taxa. Our findings conservatively place the Karoo extinction interval closer in time, but not coeval with, the more rapid marine event and reveal key differences between the PTT extinctions on land and in the oceans.

Mass extinctions are major perturbations of the biosphere resulting from a wide range of different causes including glaciations and sea level fall (1), large igneous provinces (2), and bolide impacts (3, 4). These events caused permanent changes to Earth’s ecosystems, altering the evolutionary trajectory of life (5). However, links between the broad causal factors of mass extinctions and the biological and ecological disturbances that lead to species extinctions have been difficult to characterize. This is because ecological disturbances unfold on timescales much shorter than the typical resolution of paleontological studies (6), particularly in the terrestrial record (6–8). Coarse-resolution studies have demonstrated key mass extinction phenomena including high extinction rates and lineage turnover (7, 9), changes in species richness (10), ecosystem instability (11), and the occurrence of disaster taxa (12). However, finer time resolutions are central to determining the association and relative timings of these effects, their potential causal factors, and their interrelationships. Achieving these goals represents a key advance in understanding the ecological mechanisms of mass extinctions.The end-Permian mass extinction (ca. 251.9 Ma) was Earth’s largest biotic crisis as measured by taxon last occurrences (13–15). Large outpourings from Siberian Trap volcanism (2) are the likely trigger of calamitous climatic changes, including a runaway greenhouse effect and ocean acidification, which had profound consequences for life on land and in the oceans (16–18). An estimated 81% of marine species (19) and 89% of tetrapod genera became extinct as established Permian ecosystems gave way to those of the Triassic. In the ocean, this included the complete extinction of reef-forming tabulate and rugose corals (20, 21) and significant losses in previously diverse ammonoid, brachiopod, and crinoid families (22). On land, many nonmammalian synapsids became extinct (16), and the glossopterid-dominated floras of Gondwana also disappeared (23). Stratigraphic sequences document a global “coral gap” and “coal gap” (24, 25), suggesting reef and forest ecosystems were rare or absent for up to 5 My after the event (26). Continuous fossil-bearing deposits documenting patterns of turnover across the Permian–Triassic transition (PTT) on land (27) and in the oceans (28) are geographically widespread (29, 30), including marine and continental successions that are known from China (31, 32) and India (33). Continental successions are known from Russia (34), Australia (35), Antarctica (36), and South Africa’s Karoo Basin (Fig. 1 and 37–40), the latter providing arguably the most densely sampled and taxonomically scrutinized (41–43) continental record of the PTT. The main extinction has been proposed to occur at the boundary between two biostratigraphic zones with distinctive faunal assemblages, the Daptocephalus and Lystrosaurus declivis assemblage zones (Fig. 1), which marks the traditional placement of the Permian–Triassic geologic boundary [(37) but see ref. 44]. Considerable research has attempted to understand the anatomy of the PTT in South Africa (38, 39, 45–52) and to place it in the context of biodiversity changes across southern Gondwana (53, 54) and globally (29, 31, 32, 44, 47, 55).Open in a separate windowFig. 1.Map of South Africa depicting the distribution of the four tetrapod fossil assemblage zones (Cistecephalus, Daptocephalus, Lystrosaurus declivis, Cynognathus) and our two study sites where fossils were collected in this study (sites A and B). Regional lithostratigraphy and biostratigraphy within the study interval are shown alongside isotope dilution–thermal ionization mass spectrometry dates retrieved by Rubidge et al., Botha et al., and Gastaldo et al. (37, 44, 80). The traditional (dashed red line) and associated PTB hypotheses for the Karoo Basin (37, 44) are also shown. Although traditionally associated with the PTB, the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary is defined by first appearances of co-occurring tetrapod assemblages, so its position relative to the three PTB hypotheses is unchanged. The Ripplemead member (*) has yet to be formalized by the South African Committee for Stratigraphy.Decades of research have demonstrated the richness of South Africa’s Karoo Basin fossil record, resulting in hundreds of stratigraphically well-documented tetrapod fossils across the PTT (37, 39, 56). This wealth of data has been used qualitatively to identify three extinction phases and an apparent early postextinction recovery phase (39, 45, 51). Furthermore, studies of Karoo community structure and function have elucidated the potential role of the extinction and subsequent recovery in breaking the incumbency of previously dominant clades, including synapsids (11, 57). Nevertheless, understanding patterns of faunal turnover and recovery during the PTT has been limited by the scarcity of quantitative investigations. Previous quantitative studies used coarsely sampled data (i.e., assemblage zone scale, 2 to 3 Ma time intervals) to identify low species richness immediately after the main extinction, potentially associated with multiple “boom and bust” cycles of primary productivity based on δ¹³C variation during the first 5 My of the Triassic (41, 58). However, many details of faunal dynamics in this interval remain unknown. Here, we investigate the dynamics of this major tetrapod extinction at an unprecedented time resolution (on the order of hundreds of thousands of years), using sample-standardized methods to quantify multiple aspects of regional change across the Cistecephalus, Daptocephalus, and Lystrosaurus declivis assemblage zones.     

The evolution of red color vision is linked to coordinated rhodopsin tuning in lycaenid butterflies

Color vision has evolved multiple times in both vertebrates and invertebrates and is largely determined by the number and variation in spectral sensitivities of distinct opsin subclasses. However, because of the difficulty of expressing long-wavelength (LW) invertebrate opsins in vitro, our understanding of the molecular basis of functional shifts in opsin spectral sensitivities has been biased toward research primarily in vertebrates. This has restricted our ability to address whether invertebrate G_q protein-coupled opsins function in a novel or convergent way compared to vertebrate G_t opsins. Here we develop a robust heterologous expression system to purify invertebrate rhodopsins, identify specific amino acid changes responsible for adaptive spectral tuning, and pinpoint how molecular variation in invertebrate opsins underlie wavelength sensitivity shifts that enhance visual perception. By combining functional and optophysiological approaches, we disentangle the relative contributions of lateral filtering pigments from red-shifted LW and blue short-wavelength opsins expressed in distinct photoreceptor cells of individual ommatidia. We use in situ hybridization to visualize six ommatidial classes in the compound eye of a lycaenid butterfly with a four-opsin visual system. We show experimentally that certain key tuning residues underlying green spectral shifts in blue opsin paralogs have evolved repeatedly among short-wavelength opsin lineages. Taken together, our results demonstrate the interplay between regulatory and adaptive evolution at multiple G_q opsin loci, as well as how coordinated spectral shifts in LW and blue opsins can act together to enhance insect spectral sensitivity at blue and red wavelengths for visual performance adaptation.

Opsins belong to a diverse multigene family of G protein-coupled receptors that bind to a small nonprotein retinal moiety to form photosensitive rhodopsins and enable vision across animals (1–4). The tight relationship between opsin genotypes and spectral sensitivity phenotypes offers an ideal framework to analyze how specific molecular changes give rise to adaptations in visual behaviors (5). Notably, independent opsin gene gains and losses (6–13), genetic variation across opsins (14–16), spectral tuning mutations within opsins (17–21), and alterations in visual regulatory networks (22, 23) have contributed to opsin adaptation. Yet, the molecular and structural changes underlying the remarkable diversification of spectral sensitivity phenotypes identified in some invertebrates, including crustaceans and insects (24–27), are far less understood than those in vertebrate lineages (28–32).The diversity of opsin-based photoreceptors observed across animal visual systems is produced by distinct ciliary vertebrate c-opsin and invertebrate rhabdomeric based r-opsin subfamilies that mediate separate phototransduction cascades (31, 33–35). Vertebrate c-opsins function through the G protein transducing (G_t) signaling pathway, which activates cyclic nucleotide phosphodiesterase, ultimately resulting in a hyperpolarization response in photoreceptor cells through the opening of selective K⁺ channels (31, 36). By contrast, insect opsins transmit light stimuli through a G_q-type G protein (33, 37) with phosphoinositol (PLCβ) acting as an effector enzyme to achieve TRP channel depolarization in the invertebrate photoreceptor cell (34, 38).All vertebrate visual cone opsins derive from four gene families: short-wavelength-sensitive opsins SWS1 (or ultraviolet [UV]) with λ_max 344 to 445 nm and SWS2 with λ_max 400 to 470 nm, and longer-wavelength-sensitive opsins that specify the green MWS (or Rh2) pigments with λ_max 480 to 530 nm and red-sensitive LWS pigments with λ_max 500 to 570 nm (5, 30). Most birds and fish have retained the four ancestral opsin genes (39), with notable opsin expansions in cichlid fish opsins (23, 40), whereas SWS1 is extinct in monotremes, and SWS2 and M opsins are lost in marsupials and eutherian mammals (41). In primates, trichromatic vision is conferred through SWS1 (λ_max = 414 nm) and recent duplicate MWS (λ_max = 530 nm) and LWS opsins (λ_max = 560 nm) (42–44). In vertebrates, molecular evolutionary approaches and well-established in vitro opsin purification have identified the complex interplay between opsin duplications, regulatory and protein-coding mutations controlling opsin gene tuning, and spectral phenotypes notably in birds, fish, and mammals (45–47).Insect opsins are phylogenetically distinct but functionally analogous to those of vertebrates, and the ancestral opsin repertoire consists of three types of light-absorbing rhabdomeric G_q-type opsin specifying UV (350 nm), short-wavelength (blue, 440 nm) and long-wavelength pigments (LW, 530 nm) (48). Given the importance of color-guided behaviors and the remarkable photoreceptor spectral diversity observed in insects (26, 27), the dynamic opsin gene diversification found across lineages (Fig. 1) highlights their potentially central role in adaptation (27, 49, 50), yet the molecular basis of opsin functionality of rhabdomeric invertebrate G_q opsins remains understudied.Open in a separate windowFig. 1.Visual opsin gene evolution and spectral tuning mechanisms in insects. Visual opsin genes of the Atala hairstreak (E. atala, Lepidoptera, Lycaenidae) in comparison with those encoded in the genomes of diverse insects. The opsin types are highlighted in gray for UV, in blue for short wavelength (SW), and in green for long wavelength (LW). Numbers indicate multiple opsins, whereas no dot indicates gene loss. Colored circles indicate instances of shifted spectral sensitivities in at least one of the encoded opsins. The direction of shift is inferred from the opsin lambda max that departs from the typical range of absorbance in the opsin subfamily using wavelength boundaries for the various colors: UV <380 nm, violet 380 to 435 nm, blue 435 to 492 nm, green 492 to 530 nm, and red shifted >530 nm. Coleopteran lineages, and some hemipterans, lost the blue opsin locus and compensated for the loss of blue sensitivity via UV and/or LW gene duplications across lineages (11, 12). In butterflies, extended photosensitivity at short wavelengths is observed in Heliconius erato with two UV opsins at λ_max = 355 nm and 398 nm (10) and in P. rapae with two blue opsins with λ_max = 420 and 450 nm (17). A blue opsin duplication occurred independently in lycaenid butterflies (61). LW opsin duplications occurred independently in most major insect lineages (6, 16, 55) and confer a variable range of LW sensitivities with or without additional contributions from lateral filtering. In order to extend spectral sensitivity at longer wavelengths while sharpening blue acuity, some lycaenid butterflies have evolved a new color vision mechanism combining spectral shifts at a duplicate blue opsin and at the LW opsin. Images credit: Christopher Adams (illustrator).The recurrent evolution of red receptors in insects in particular suggests that perception of longer wavelengths can play an important role in the context of foraging, oviposition, and/or conspecific recognition (6, 27, 51–54). In butterflies, several mechanisms are likely to have provided extended spectral sensitivity to longer wavelengths. LW opsin duplications along with the evolution of lateral filtering between ommatidia has been demonstrated in two papilionids, Papilio xuthus (27) and Graphium sarpedon (55), as well as in a riodinid (Apodemia mormo) (6, 54). Lateral filtering pigments are relatively widespread across butterfly lineages, e.g., Heliconius (56), Pieris (57), Colias erate (58), and some moths [Adoxophyes orana (59) and Paysandisia archon (60)]. These pigments absorb short wavelengths and aid in shifting the sensitivity peak of green LW photoreceptors to longer wavelengths (27, 51, 56, 57, 61, 62). Despite creating distinct spectral types that can contribute to color vision, as identified in nymphalid (56), pierid (57), and lycaenid (62) species, all of which lack duplicated LW opsins (61, 63), lateral filtering alone cannot extend photoreceptor sensitivity toward the far red (700 to 750 nm) beyond the exponentially decaying long-wavelength rhodopsin absorbance spectrum (51). Thus, molecular variation of ancestral LW opsin genes is likely to have contributed an as yet underexplored mechanism to the diversification of long-wavelength photoreceptor spectral sensitivity. However, disentangling the relative contributions of lateral filtering and pure LW opsin properties has remained technically challenging using classical electrophysiological approaches (14, 64, although see, e.g., refs. 65, 66, 67) and has been limited by the lack of in vitro expression systems suitable for LW opsins.While opsin duplicates have been identified in numerous organisms, the spectral tuning mechanisms and interplay between new opsin photoreceptors in invertebrate visual system evolution are less well understood. Here we combine physiological, molecular, and heterologous approaches to start closing this gap in our knowledge of invertebrate G_q opsin evolution by investigating the functions, spectral tuning, and implications of evolving new combinations of short- and long-wavelength opsin types in lycaenid species. This butterfly group, comprising the famous blues, coppers, and hairstreaks, is the second largest family with about 5,200 (28%) of the some 18,770 described butterfly species (68). In light of their remarkable behavioral, ecological, and morphological diversity (69, 70), as well as pioneer studies in the Lycaena and Polyommatus genera supporting the rapid evolution of color vision in certain lineages (56, 61, 62), lycaenids provide an ideal candidate system for investigating opsin evolution and visual adaptations. Using the Atala hairstreak, Eumaeus atala, as a molecular and ecological model, we find coordinated spectral shifts at short- and long-wavelength G_q opsin loci and demonstrate that the combination of six ommatidial classes of photoreceptors in the compound eye uniquely extend spectral sensitivity at long wavelengths toward the far-red while concurrently sharpening acuity of multiple blue wavelengths. Together, these findings link the evolution of four-opsin visual systems to adaptation in the context of finely tuned color perception critical to the behavior of these butterflies.     

Competitive history shapes rapid evolution in a seasonal climate

Eco-evolutionary dynamics will play a critical role in determining species’ fates as climatic conditions change. Unfortunately, we have little understanding of how rapid evolutionary responses to climate play out when species are embedded in the competitive communities that they inhabit in nature. We tested the effects of rapid evolution in response to interspecific competition on subsequent ecological and evolutionary trajectories in a seasonally changing climate using a field-based evolution experiment with Drosophila melanogaster. Populations of D. melanogaster were either exposed, or not exposed, to interspecific competition with an invasive competitor, Zaprionus indianus, over the summer. We then quantified these populations’ ecological trajectories (abundances) and evolutionary trajectories (heritable phenotypic change) when exposed to a cooling fall climate. We found that competition with Z. indianus in the summer affected the subsequent evolutionary trajectory of D. melanogaster populations in the fall, after all interspecific competition had ceased. Specifically, flies with a history of interspecific competition evolved under fall conditions to be larger and have lower cold fecundity and faster development than flies without a history of interspecific competition. Surprisingly, this divergent fall evolutionary trajectory occurred in the absence of any detectible effect of the summer competitive environment on phenotypic evolution over the summer or population dynamics in the fall. This study demonstrates that competitive interactions can leave a legacy that shapes evolutionary responses to climate even after competition has ceased, and more broadly, that evolution in response to one selective pressure can fundamentally alter evolution in response to subsequent agents of selection.

Although ecological and evolutionary dynamics have traditionally been studied as independent processes assumed to proceed on fundamentally different timescales, it is now widely recognized that evolution often occurs rapidly enough to shape ecological outcomes (1–3). There is a growing interest in understanding the eco-evolutionary dynamics that result (4, 5), motivated in part by their potential importance in determining species’ fates under global environmental change (6, 7).Climate is a principal abiotic pressure that species face in the wild that can exert strong selection capable of driving rapid ecological and evolutionary change (8, 9). Understanding species’ evolutionary responses to climatic conditions has become essential, as temperature, its variability, and the frequency of extreme weather events increase under global change (10). Unfortunately, this understanding remains limited by a lack of experimental tests that place species in the complex and competitive environments in which ecology and evolution actually occur (11, 12). This represents a critical knowledge gap, as species confronted with changing climatic regimes not only face native competitors, but may also face novel competitors in the form of invasive species and species migrating in response to climate change (13, 14).We have several reasons to expect that selection imposed by competitors could shape species’ ecological and evolutionary responses to climate. First, most species live embedded in communities of competitors, rendering these interactions a likely source of selection in nature. Second, interspecific competition is widely recognized as a key driver of ecological (15, 16) and macroevolutionary dynamics (17, 18). Finally, a handful of experiments have demonstrated that species can rapidly adapt to interspecific competition (2, 19–21). Nonetheless, given that experimental evaluations of rapid evolution tend to focus on single-species populations (22, 23) or selection imposed by consumers or disease (24, 25), we have little understanding of how evolution in response to interspecific competition affects species’ abilities to persist in or adapt to new thermal regimes.Through changes in the genetic composition and phenotypic traits of populations, rapid evolution in response to competition could alter a species’ ecological trajectory, evolutionary trajectory, or both. We would expect rapid adaptation to competition to influence ecological trajectories under a shifting climate if competition drives the evolution of a phenotype, such as body size, that also influences individual performance and therefore population dynamics as temperatures change (26, 27). Selection from competition could be exerted directly via aggressive interactions with a competitor or indirectly through changes in the availability of shared resources. Studies that have experimentally demonstrated the effects of rapid evolution in response to interspecific competition have identified shifts in phenotypic traits (19, 28) that can affect population dynamics by altering birth and death rates (2, 29). Moreover, adaptive responses to competition have been shown to alter species’ population trajectories when they are also faced with changing environmental conditions, including CO₂ enrichment (23, 30).In addition to these ecological consequences of adapting to competitors, such adaptation could also alter species’ evolutionary trajectories when faced with shifting climatic conditions (31). This could arise through several mechanisms. First, theory indicates that a reduction in population size and strong selection caused by competition can reduce standing genetic variation, which could hinder adaptation to a changing climate (31–33). Second, by altering the genetic composition of populations (2, 34), adaptation to interspecific competition could influence both the magnitude and the direction of evolutionary change when organisms are exposed to novel climatic conditions (31). Traits that link genetic change and competitive performance are likely to be complex and polygenic (35–38), and, as such, the evolution of these traits may be particularly affected by epistasis and pleiotropy (39, 40). As a result, adaptation to interspecific competition could have cryptic but far-reaching consequences for subsequent evolutionary trajectories in response to changing climate if competition drives changes in allele frequencies at loci underlying variation in climate-relevant traits, or if genetic correlations link phenotypes selected under competition with those that affect fitness in a changing climate (41, 42). However, theory examining how evolutionary responses to competition can affect subsequent evolutionary responses to a changing climate remains scarce (27, 43), and the more general links between rapid adaptation in response to the changing selective agents described above have yet to be tested in a natural context.We tested how rapid evolution in response to interspecific competition influences ecological and evolutionary dynamics in a seasonal climate using a large-scale field-based experimental evolution study with the vinegar fly Drosophila melanogaster and its invasive competitor Zaprionus indianus. The interactions between D. melanogaster and Z. indianus in the seasonal climate of the northeastern United States provide an excellent natural context in which to evaluate the eco-evolutionary interactions between competition and climate. D. melanogaster maintains resident populations throughout the year in temperate North American orchards (35, 44). After emerging from diapause each spring, populations expand and rapidly evolve under warm summer conditions while feeding and laying eggs on fallen fruit (36, 37, 45). Then in fall and early winter, populations gradually decline and evolve under cooling conditions (35, 36, 46).In contrast, Z. indianus has invaded tropical regions across the globe and now seasonally invades the northeastern United States from more southern latitudes (47). Compared to D. melanogaster, Z. indianus is larger-bodied, less cold-tolerant, and slower to develop (48). In both its native and invasive range, it competes with D. melanogaster adults for food and oviposition space on rotting fruit and with D. melanogaster larvae for food during development (48). Because of its cold intolerance, Z. indianus suffers high mortality and reproductive arrest as temperatures drop in the fall (49, 50), leaving fall D. melanogaster populations to continue to reproduce and adapt to fall conditions in the absence of their interspecific competitor. It is not known how selection imposed by competition with Z. indianus over the summer affects D. melanogaster and shapes its ecology and evolution in the cooler fall.We conducted an experimental evolution study with replicate fly populations in an experimental orchard that mimics our focal species’ primary northeastern US habitat. The field mesocosms that we used experience natural temperature fluctuations and contain many of the predators and microbes that co-occur with local natural populations of D. melanogaster (37, 45). To examine the consequences of rapid evolution in response to interspecific competition on ecological and evolutionary dynamics in the fall, we first allowed replicate populations of D. melanogaster to grow and evolve in the presence or absence of Z. indianus for approximately six generations over the summer (Fig. 1). At the end of summer, we removed Z. indianus, equalized abundances of D. melanogaster across populations, and allowed the populations to continue their ecological and evolutionary dynamics through the fall (approximately three generations). We quantified the ecological (population dynamic) consequences of our treatments with weekly censuses of relative fly abundances throughout the summer and fall. We quantified the evolutionary consequences of our treatments by measuring 10 key phenotypes of D. melanogaster collected at the end of summer and end of the fall and then reared for two generations in a common garden to remove plastic responses to treatments or field conditions.Open in a separate windowFig. 1.Experimental design to determine the effect of rapid evolution in response to interspecific competition on the ecological and evolutionary trajectory of D. melanogaster in a cool fall climate. Each replicate population consisted of a large outdoor cage containing thousands (up to 100,000) of genetically diverse flies. At each “phenotyping” time point,10 fly phenotypes were measured on each replicate population after two generations in a common garden environment. In evolving populations, eggs laid in the field experiment were allowed to develop into adult flies, whereas in replacement populations, eggs laid in the field experiment were replaced by eggs laid by laboratory populations in order to prevent intergenerational adaptation to fall conditions. Colors and dashing of lines to distinguish treatments are also used in Figs. 2–4.The mechanisms that drive ecological and evolutionary patterns can be difficult to untangle in cases where ecological and evolutionary dynamics occur simultaneously (1, 51), and this is further complicated by the polygenic and multiphenotypic nature of D. melanogaster’s adaptive responses to climatic and biotic conditions (35, 36, 45, 52, 53). We therefore implemented an additional treatment in the fall phase of the experiment to provide insight into the mechanisms underlying the effects of competition on ecological and evolutionary responses to fall climate. In the fall, we effectively stopped intergenerational adaptation to fall conditions in half of our populations by replacing all eggs laid in field mesocosms with eggs laid by populations of flies collected from the experiment at the end of the summer and maintained in a nonseasonal laboratory environment (hereafter called “replacement” populations) (2, 45) (Fig. 1 and Methods). From an ecological perspective, this replacement treatment allowed us to determine the effect of adaptation to competition on fall population dynamics in both the presence and absence of further intergenerational adaptation to fall conditions. From an evolutionary perspective, it allowed us to evaluate the extent to which responses depended on intergenerational genetic change (e.g., recombination reducing negative epistatic or pleiotropic effects of adaptation or cumulative effects of selection across generations) versus recurrent selection of standing genetic variation within individual cohorts.We predicted that if competition with Z. indianus and cold fall temperatures exert opposing selection on D. melanogaster (e.g., opposing effects on body size or development time), evolution to interspecific competition would accelerate fall population decline. This could arise if, for example, the presence of slower-developing Z. indianus exerts selection for faster larval development that allows D. melanogaster to avoid larval competition but is detrimental under cold conditions (54, 55). If, instead, competition and climate were to select in the same direction, evolution to interspecific competition could slow fall population decline. This could occur if, for example, competition with the large-bodied Z. indianus for oviposition space selects for large adult body size in D. melanogaster that is beneficial under cold conditions. These expectations, of course, depend on simple relationships between genetic change, trait change, and success under competition and climate. Because the complex genetic architecture underlying fitness-associated traits is likely to generate complex links between adaptation to different selective pressures, we also predicted more generally that any divergent phenotypic and genetic changes resulting from adaptation to the summer competitive environment would shape the outcome of adaptation to subsequent fall conditions.     

The role of lateral erosion in the evolution of nondendritic drainage networks to dendricity and the persistence of dynamic networks

Dendritic, i.e., tree-like, river networks are ubiquitous features on Earth’s landscapes; however, how and why river networks organize themselves into this form are incompletely understood. A branching pattern has been argued to be an optimal state. Therefore, we should expect models of river evolution to drastically reorganize (suboptimal) purely nondendritic networks into (more optimal) dendritic networks. To date, current physically based models of river basin evolution are incapable of achieving this result without substantial allogenic forcing. Here, we present a model that does indeed accomplish massive drainage reorganization. The key feature in our model is basin-wide lateral incision of bedrock channels. The addition of this submodel allows for channels to laterally migrate, which generates river capture events and drainage migration. An important factor in the model that dictates the rate and frequency of drainage network reorganization is the ratio of two parameters, the lateral and vertical rock erodibility constants. In addition, our model is unique from others because its simulations approach a dynamic steady state. At a dynamic steady state, drainage networks persistently reorganize instead of approaching a stable configuration. Our model results suggest that lateral bedrock incision processes can drive major drainage reorganization and explain apparent long-lived transience in landscapes on Earth.

What should a drainage network look like? Fig. 1A shows a single channel, winding its way through the catchment so as to have access to water and sediment from unchannelized zones in the same manner as the dendritic (tree-like) network of Fig. 1B. It appears straightforward that the dendritic pattern is a model for nature, and the single channel is not. Dendritic drainage networks are called such because of their similarity to branching trees, and their patterns are “characterized by irregular branching in all directions” (1) with “tributaries joining at acute angles” (2). Drainage networks can also take on other forms such as parallel, pinnate, rectangular, and trellis in nature (2). However, drainage networks in their most basic form without topographic, lithologic, and tectonic constraints should tend toward a dendritic form (2). In addition, drainage networks that take a branching, tree-like form have been argued to be “optimal channel networks” that minimize total energy dissipation (3, 4). Therefore, we would expect that models simulating river network formation, named landscape evolution models (LEMs), that use the nondendritic pattern of Fig. 1A as an initial condition to massively reorganize and approach the dendritic steady state of Fig. 1B. To date, no numerical LEM has shown the ability to do this. Here, we present a LEM that can indeed accomplish such a reorganization. A corollary of this ability is the result that landscapes approach a dynamic, rather than static steady state.Open in a separate windowFig. 1.Schematic diagram of a nondendritic and a dendritic drainage network. This figure shows the Wolman Run Basin in Baltimore County, MD (A) drained by a single channel winding across the topography and (B) drained by a dendritic network of channels. Both networks have similar drainage densities (53, 54), but there is a stark difference between their stream ordering (53–56). This figure invites discussion as to how a drainage system might evolve from the configuration of A to that of B.There is indeed debate as to whether landscapes tend toward an equilibrium that is frozen or highly dynamic (5). Hack (6) hypothesized that erosional landscapes attain a steady state where “all elements of the topography are downwasting at the same rate.” This hypothesis has been tested in numerical models and small-scale experiments. Researchers found that numerical LEMs create static topographies (7, 8). In this state, erosion and uplift are in balance in all locations in the landscape, resulting in landscapes that are dissected by stable drainage networks in geometric equilibrium (9). The landscape has achieved geometric equilibrium in planform when a proxy for steady-state river elevation, named χ (10), has equal values across all drainage divides. In contrast, experimental landscapes (7, 11) develop drainage networks that persistently reorganize. Recent research on field landscapes suggests that drainage divides migrate until reaching geometric equilibrium (9), but other field-based research suggests that landscapes may never attain geometric equilibrium (12).The dynamism of the equilibrium state determines the persistence of initial conditions in experimental and model landscapes. It is important to understand initial condition effects (13) to better constrain uncertainty in LEM predictions. Kwang and Parker (7) demonstrate that numerical LEMs exhibit “extreme memory,” where small topographic perturbations in initial conditions are amplified and preserved during a landscape’s evolution (Fig. 2A). Extreme memory in the numerical models is closely related to the feasible optimality phenomenon found within the research on optimal channel networks (4). These researchers suggest that nature’s search for the most “stable” river network configuration is “myopic” and unable to find configurations that completely ignore their initial condition. In contrast to numerical models, experimental landscapes (7, 11) reach a highly dynamic state where all traces of initial surface conditions are erased by drainage network reorganization. It has been hypothesized that lateral erosion processes are responsible for drainage network reorganization in landscapes (7, 14); these processes are not included in most LEMs.Open in a separate windowFig. 2.A comparison of LEM-woLE (A) and LEM-wLE (B). Both models utilize the same initial condition, i.e., an initially flat topography with an embedded sinusoidal channel (1.27 m deep) without added topographic perturbations. Without perturbations, the landscape produces angular tributaries that are attached to the main sinusoidal channel (compare with SI Appendix, Fig. S7). Here, LEM-wLE quickly shreds the signal of the initial condition over time, removing the angular tributaries. By 10 RUs eroded the sinusoidal signal is mostly erased. After 100 RUs, the drainage network continues to reorganize itself (i.e., dynamic steady state). The landscape continues to reorganize as shown in Movies S1.Most widely used LEMs simulate incision into bedrock solely in the vertical direction. However, there is growing recognition that bedrock channels also shape the landscape by incising laterally (15, 16). Lateral migration into bedrock is important for the creation of strath terraces (17, 18) and the morphology of wide bedrock valleys (19–21). Recently, Langston and Tucker (22) developed a formulation for lateral bedrock erosion in LEMs. Here, we implement their submodel to explore the long-term behavior of LEMs that incorporate lateral erosion.The LEM submodel of Langston and Tucker (22) allows for channels to migrate laterally. By including this autogenic mechanism, we hypothesize that lateral bedrock erosion creates instabilities that 1) shred (23) the memory of initial conditions such as the unrealistic configurations of Fig. 1A and 2) produce landscapes that achieve a statistical steady state instead of a static one. By incorporating the lateral incision component (22) into a LEM, we aim to answer the following: 1) What controls the rate of decay of signals from initial conditions? 2) What are the frequency and magnitude of drainage reorganization in an equilibrium landscape? 3) What roles do model boundary conditions play in landscape reorganization?     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Radiation with reticulation marks the origin of a major malaria vector

Advances in genomics have led to an appreciation that introgression is common, but its evolutionary consequences are poorly understood. In recent species radiations the sharing of genetic variation across porous species boundaries can facilitate adaptation to new environments and generate novel phenotypes, which may contribute to further diversification. Most Anopheles mosquito species that are of major importance as human malaria vectors have evolved within recent and rapid radiations of largely nonvector species. Here, we focus on one of the most medically important yet understudied anopheline radiations, the Afrotropical Anopheles funestus complex (AFC), to investigate the role of introgression in its diversification and the possible link between introgression and vector potential. The AFC comprises at least seven morphologically similar species, yet only An. funestus sensu stricto is a highly efficient malaria vector with a pan-African distribution. Based on de novo genome assemblies and additional whole-genome resequencing, we use phylogenomic and population genomic analyses to establish species relationships. We show that extensive interspecific gene flow involving multiple species pairs has shaped the evolutionary history of the AFC since its diversification. The most recent introgression event involved a massive and asymmetrical movement of genes from a distantly related AFC lineage into An. funestus, an event that predated and plausibly facilitated its subsequent dramatic geographic range expansion across most of tropical Africa. We propose that introgression may be a common mechanism facilitating adaptation to new environments and enhancing vectorial capacity in Anopheles mosquitoes.

Once considered a rare anthropogenic aberration in animals, interspecific hybridization is now recognized to be both taxonomically widespread and pervasive, particularly in rapidly diversifying groups (1–3). Moreover, mounting genome-scale evidence suggests that introgression, the genetic exchange between species through hybridization and backcrossing, is also prevalent and may be consequential for evolution. Examples from fish, birds, mammals, and insects—including Anopheles mosquitoes—have shown that introgressed variation favored by natural selection can facilitate adaptation, enhance fitness, and drive evolutionary innovation and diversification (4–7). It has been postulated that introgressive hybridization is most prevalent in species-rich and rapidly diversifying radiations (2, 3, 8). Introgression in these groups may solely be opportunistic, given the multiplicity of young species in geographic proximity, but the process may also favor adaptive radiation through the generation of completely novel phenotypes (6, 9, 10).There are three to four dozen Anopheles mosquito species that are of major importance as human malaria vectors, and all have evolved within recent and rapid radiations of morphologically cryptic species (informally classified as species complexes) (11, 12). Most members of these species complexes play no or very minor roles in disease transmission. The repeated de novo origin of major malaria vectors across these independent species radiations therefore holds clues about the nature of key evolutionary innovations that confer the ability to transmit disease widely and efficiently. However, most Anopheles species complexes are understudied. This is especially true of the secondary or nonvector species for which genomic resources are lacking, and basic knowledge of distribution, ecology, and behavior is scant.Until now, the single best-studied group has been the Anopheles gambiae complex, composed of at least eight morphologically indistinguishable species that diversified rapidly and recently, likely within the last half-million years (7, 13, 14). Phylogenomic analysis revealed widespread genealogical discordance (7). Some discordance was due to incomplete lineage sorting as a result of both rapid radiation and large effective population sizes (7), but the majority was caused by massive introgression between the main vector species, involving both the autosomes and the centromere-proximal region of the X chromosome. So extensive was its impact that the inferred species branching order was evident in only 2% of the genome—mostly on the distal portion of the X chromosome, which is protected from introgression by a succession of fixed chromosomal inversion differences.One of the most medically important of the understudied Anopheles species complexes is the Afrotropical Anopheles funestus complex (AFC). The AFC comprises at least seven morphologically similar species (15–18), yet only An. funestus sensu stricto (hereafter, An. funestus) is a highly efficient malaria vector, rivaled in importance solely by An. gambiae and its sister species Anopheles coluzzii in the An. gambiae complex (19–22). Comparative genomics of these two complexes may therefore be instructive with regard to malaria vectorial capacity. Both groups diversified in sub-Saharan Africa and may have experienced common geographic, ecoclimatic, and anthropogenic forces that shaped their history. In addition, the primary vector An. funestus broadly shares several characteristics with primary vectors in the An. gambiae complex: a geographic range that encompasses most of tropical Africa (Fig. 1A), high levels of chromosomal inversion polymorphism (23–25), large effective population size, and little population genetic structure across the continent (26, 27). Furthermore, the discovery of two very distantly related mitochondrial DNA (mtDNA) haplotypes (clades 1 and 2) segregating in An. funestus (27) raises the prospect of historical introgression analogous to that documented for An. gambiae, prompting an intriguing question: Can introgression be a source of evolutionary novelty leading to augmented vectoral capacity?Open in a separate windowFig. 1.Distribution and genetic variation in the AFC. Color coding of species is consistent across panels. (A) Location and distribution of sampled species, adapted from ref. 21. Approximate sample locations for An. funestus are indicated by a black star. For full sample information, see SI Appendix, Table S1. (B) Phylogeny of complete mtDNA genomes constructed using BEAST2 indicating divergent clades of An. funestus (red shading) and An. funestus-like (green shading) (see SI Appendix, Fig. S12 for phylogeny with outgroup). (C) Neighbor-joining phylogeny averaged over the complete nuclear genome. (D) Summary evolutionary history displaying three introgression events as inferred by the methods described in the main text. Introgression events shown as green horizontal arrows between pairs of species indicate the majority direction of introgression. Median divergence and introgression times are displayed in millions of years ago (Mya). See SI Appendix, Table S11 for details. An. funestus (Fun), An. funestus-like (Lik), An. longipalpis C (Lon), An. parensis (Par), and An. vaneedeni (Van), An. rivulorum (Riv).Here, we examine the role of introgression in the evolution of the AFC, using recent methods of phylogenetic network reconstruction that allow for divergence and reticulation to be inferred jointly. We use a combination of phylogenomic and population genomic analyses, based on de novo genome assemblies and additional whole genome resequencing, to: 1) establish species relationships, 2) determine the direction, extent, and genomic architecture of introgression across the complex, and 3) assess the role of introgression in the evolution of the primary vector An. funestus. We show that extensive interspecific gene flow involving multiple species pairs has shaped the evolutionary history of the AFC since its diversification ∼216 thousand years ago (Kya). The most recent introgression event ∼13 Kya involved a massive and asymmetrical movement of genes from a distantly related AFC lineage into An. funestus, an event that predated and plausibly facilitated its subsequent dramatic geographic range expansion across most of tropical Africa. We propose that introgression may be a common mechanism facilitating adaptation to new environments and enhancing vectorial capacity in Anopheles mosquitoes.     

Three genomes in the algal genus Volvox reveal the fate of a haploid sex-determining region after a transition to homothallism

Transitions between separate sexes (dioecy) and other mating systems are common across eukaryotes. Here, we study a change in a haploid dioecious green algal species with male- and female-determining chromosomes (U and V). The genus Volvox is an oogamous (with large, immotile female gametes and small, motile male gametes) and includes both heterothallic species (with distinct male and female genotypes, associated with a mating-type system that prevents fusion of gametes of the same sex) and homothallic species (bisexual, with the ability to self-fertilize). We date the origin of an expanded sex-determining region (SDR) in Volvox to at least 75 Mya, suggesting that homothallism represents a breakdown of dioecy (heterothallism). We investigated the involvement of the SDR of the U and V chromosomes in this transition. Using de novo whole-genome sequences, we identified a heteromorphic SDR of ca 1 Mbp in male and female genotypes of the heterothallic species Volvox reticuliferus and a homologous region (SDLR) in the closely related homothallic species Volvox africanus, which retained several different hallmark features of an SDR. The V. africanus SDLR includes a large region resembling the female SDR of the presumptive heterothallic ancestor, whereas most genes from the male SDR are absent. However, we found a multicopy array of the male-determining gene, MID, in a different genomic location from the SDLR. Thus, in V. africanus, an ancestrally female genotype may have acquired MID and thereby gained male traits.

As first noted by Darwin when studying plant sexuality, self-fertilization can lead to inbreeding depression, though this potential disadvantage relative to outcrossing can be offset by higher probability of fertilization success (1, 2). Thus, transitions between inbreeding and outbreeding mating systems attract the attention of evolutionary biologists and have been documented in sexual systems across a broad range of taxa including animals, land plants, algae (Fig. 1), protists, and fungi (3–9). Hermaphroditic mating systems in diploid animals and plants are fairly common, and the evolution of hermaphroditism in animals has been extensively studied (10–12). The molecular genetic bases of hermaphroditism have also been recently studied in several flowering plants (13–15).Open in a separate windowFig. 1.Schematic representation of life cycles of two closely related species of Volvox sect. Merrillosphaera (SI Appendix, Fig. S1) showing different sexual systems (heterothallism and homothallism), based on Nozaki et al. (29). Note gonidia (g) in asexual spheroids and sperm packets (sp) and eggs (e) in sexual spheroids. (A) NIES-3781. (B) NIES-3783. (C and D) NIES-3782. (E and H) NIES-3784. (F and G) NIES-3780. For materials and methods of light micrographs, refer to Nozaki et al. (29). All photographs are original.In haploid organisms, two basic types of mating systems are recognized: heterothallic (with two or more self-incompatible mating types in isogamous species or with males and females in anisogamous/oogamous species) and homothallic (self-compatible with isogamy or bisexual with anisogamy/oogamy) (SI Appendix, Table S1). In heterothallic species, gamete compatibility in isogamy or maleness versus femaleness is usually determined by a single complex locus on a chromosome (9). The process by which this genetic system breaks down to allow a single genotype to acquire functions of both sexes and mating-types in the homothallic species is poorly understood outside of fungi (8).Organisms with a heterothallic haploid generation such as algae and early diverging land plants like mosses and liverworts have sex-determining regions (SDRs) that are differentiated between the two sexes and located on male and female determining (U and V) chromosomes (9). SDRs exhibit suppressed recombination and harbor fully sex-linked genes including sex-specific genes (found in only one of the two sexes) and gametologs (genes with alleles in both haplotypes of the SDR) (9). A recent study has characterized genes involved in a change from sexual reproduction to parthenogenesis in a heterothallic brown alga with UV chromosome (16). However, genomics of transitions between heterothallism and homothallism have not been previously studied in the context of haploid SDRs, and the fates of sex-determining and sex-related genes present on ancestral SDRs in UV chromosomes after transitions to homothallism are unknown.The volvocine green algal lineage is an especially well-studied evolutionary model for investigating the origins of sexes and transitions in sexuality, because it includes extant organisms with a graded range of sexual or mating phenotypes from unicellular isogamous Chlamydomonas through genera with increasing degrees of sexual dimorphism, such as multicellular, isogamous Gonium, and oogamous Volvox (Fig. 2) (17–22). In heterothallic species of the isogamous volvocine genera such as Chlamydomonas, there are two mating-types, plus and minus, both of which are needed for sexual reproduction (9). Ferris and Goodenough (23) characterized the first mating-type locus (MT) in Chlamydomonas reinhardtii, which was discovered to have features similar to those of a typical SDR, including large size and suppressed recombination. Subsequent studies of heterothallic species of multicellular volvocine algae revealed conservation of orthologs of the mating-type determining gene MID in the mating type minus genotype of the isogamous genera Gonium and Yamagishiella and in the SDR of males of anisogamous Eudorina and the oogamous Volvox (19, 21, 24). This indicates that the evolution of anisogamy has involved genetic changes that are closely associated with the MT, presumably because this ensures the ability of male and female gametes to fuse with each other as proposed on theoretical grounds (25). The MT or SDR haplotypes in heterothallic volvocine algae range from 7 kbp to 1 Mbp and are structurally heteromorphic with chromosome rearrangements that distinguish the haplotypes and various degrees of genetic differentiation between them (21) (Fig. 2). The ca 1 Mbp, highly differentiated Volvox carteri SDR has expanded in size about fivefold relative to that in Chlamydomonas (19), and this was likely to have occurred after the transition from isogamy to anisogamy/oogamy (21). However, the timing of the SDR expansion in the genus Volvox and its significance with respect to the evolution of sexual dimorphism remain unknown.Open in a separate windowFig. 2.Volvocine green algal phylogeny and SDR or MT evolution. Asexual or vegetative phase, sexual phase, MTM and MTF, or SDLR and phylogenetic positions of V. reticuliferus and V. africanus are illustrated with those of five other species previously studied [C. reinhardtii, Gonium pectorale, Yamagishiella unicocca, Eudorina sp., and V. carteri (19, 21, 23, 24)]. Note that all three of these Volvox species belongs to the section Merrillosphaera (SI Appendix, Fig. S1).To date, genomic studies of volvocine green algae have focused on heterothallic species (Fig. 2), but most genera within this lineage have members that also underwent transitions from heterothallism to homothallism (7). These algae are found in freshwater habitats and form a dormant diploid zygote with a resistant wall that allows it to survive under unfavorable conditions; diploidy in the zygote could be of selective value for survival when chromosomal damage and mutation may be occurring at increased frequencies (26). Thus, transitions from heterothallism to homothallism in the volvocine algae may be favored for production of the resistant diploid cells (zygotes) by self-fertilization. We previously noted the presence of a MID gene in two homothallic species of the genus Volvox (27), and an artificial homothallic phenotype was shown in the male genotype of V. carteri (28). However, it remains unknown how a naturally occurring homothallic mating system could arise from an ancestral SDR in UV chromosome system (or vice versa).In most members of the genus Volvox, the transition from vegetative to sexual reproduction involves modified embryogenesis and the production of morphologically distinct male and/or female sexual spheroids (7). Recently, we characterized two species of Volvox that are closely related but have different mating systems: heterothallic Volvox reticuliferus, which upon sexual induction makes differentiated male or female, and homothallic Volvox africanus, which produces both male spheroids and bisexual spheroids from single clonal cultures (27, 29) (Figs. 1 and ​and22 and SI Appendix, Table S1). These two species diverged ca 11 MYA; together with the more distantly related V. carteri, they represent a monophyletic group, the “section Merrillosphaera,” which originated ca 75 MYA and is an infrageneric taxon of the polyphyletic genus Volvox (SI Appendix, Fig. S1). While ancestral heterothallism in Merrillosphaera is more likely than homothallism, statistical support for this inference is not strong, and the directionality of transitions between these two types of sexuality within this clade remains somewhat inconclusive (7).Here, we performed de novo whole-genome sequencing of male and female genotypes of heterothallic V. reticuliferus and of homothallic V. africanus in order to characterize their SDR and SD-like regions (SDLRs), respectively. We used this sequence information to infer the likely ancestral state of heterothallism in this clade and to reconstruct the SDR changes which occurred during the evolution of homothallism in V. africanus, including tracking the fates of male- and female-specific genes and male versus female gametologs derived from the putative heterothallic ancestral species.     

Structured sequences emerge from random pool when replicated by templated ligation

The central question in the origin of life is to understand how structure can emerge from randomness. The Eigen theory of replication states, for sequences that are copied one base at a time, that the replication fidelity has to surpass an error threshold to avoid that replicated specific sequences become random because of the incorporated replication errors [M. Eigen, Naturwissenschaften 58 (10), 465–523 (1971)]. Here, we showed that linking short oligomers from a random sequence pool in a templated ligation reaction reduced the sequence space of product strands. We started from 12-mer oligonucleotides with two bases in all possible combinations and triggered enzymatic ligation under temperature cycles. Surprisingly, we found the robust creation of long, highly structured sequences with low entropy. At the ligation site, complementary and alternating sequence patterns developed. However, between the ligation sites, we found either an A-rich or a T-rich sequence within a single oligonucleotide. Our modeling suggests that avoidance of hairpins was the likely cause for these two complementary sequence pools. What emerged was a network of complementary sequences that acted both as templates and substrates of the reaction. This self-selecting ligation reaction could be restarted by only a few majority sequences. The findings showed that replication by random templated ligation from a random sequence input will lead to a highly structured, long, and nonrandom sequence pool. This is a favorable starting point for a subsequent Darwinian evolution searching for higher catalytic functions in an RNA world scenario.

One of the dominant hypotheses to explain the origin of life (1–3) is the concept of the RNA world. It is built on the fact that catalytically active RNA molecules can enzymatically promote their own replication (4–6) via active sites in their three-dimensional structures (7–9). These so-called ribozymes have a minimal length of 30 to 41 base pairs (9, 10) and, thus, a sequence space of more than 4³⁰ ∼ 10¹⁸. The subset of functional, catalytically active sequences in this vast sequence space is vanishingly small (11), making spontaneous assembly of ribozymes from monomers or oligomers all but impossible. Therefore, prebiotic evolution has likely provided some form of selection guiding single nucleotides to form functional sequences and thereby lowering the sequence entropy of this system.The problem of nonenzymatic formation of single base nucleotides and short oligomers in settings reminiscent of the primordial soup has been studied before (12–17). However, the continuation of this evolutionary path toward early replication networks would require a preselection mechanism of oligonucleotides (see Fig. 1A), lowering the information entropy of the resulting sequence pool (18–22). In principle, such selection modes include optimization for information storage, local oligomer enrichment (e.g., in hydrogels or in catalytically functional sites).Open in a separate windowFig. 1.Templated ligation of random sequence DNA 12-mers. (A) Before cells evolved, the first ribozymes were thought to perform basic cell functions. In the exponentially vast sequence space, spontaneous emergence of a functional ribozyme is highly unlikely, therefore preselection mechanisms were likely necessary. (B) In our experiment, DNA strands hybridize at low temperatures to form three-dimensional complexes that can be ligated and preserved in the high temperature dissociation steps. The system self-selects for sequences with specific ligation site motifs as well as for strands that continue acting as templates. Hairpin sequences are therefore suppressed. (C) Concentration analysis shows progressively longer strands emerging after multiple temperature cycles. The inset (A-red, T-blue) shows that, although 12-mers (88,009 strands) have essentially random sequences (white), various sequence patterns emerge in longer strands (60-mers, 235,913 strands analyzed). (D) Samples subjected to different number (0 to 1,000) of temperature cycles between 75 °C and 33 °C. Concentration quantification is done on PAGE with SYBR poststained DNA.An important aspect of a selection mechanism is its nonequilibrium driving force. Today’s highly evolved cells function through multistep and multicomponent metabolic pathways like glycolysis in the Warburg effect (23) or by specialized enzymes like adenosine triphosphate (ATP) synthase which provide energy-rich ATP (24). In contrast, it is widely assumed (3, 4, 25–28) that selection mechanisms for molecular evolution at the dawn of life must have been much simpler (e.g., mediated by random binding between biomolecules subject to nonequilibrium driving forces such as fluid flow and cyclic changes in temperature).Here, we explored the possibility of a significant reduction of sequence entropy driven by templated ligation (19) and mediated by Watson–Crick base pairing (29). Starting from a random pool of oligonucleotides, we observed a gradual formation of longer chains showing reproducible sequence landscape inhibiting self-folding and promoting templated ligation. Here, we argue that base pairing combined with ligation chemistry can trigger processes that have many features of the Darwinian evolution.As a model oligomer, we decided to use DNA instead of RNA since the focus of our study is on base pairing, which is very similar for both (30). We started our experiments with a random pool of 12-mers formed of bases A (adenine) and T (thymine). This binary code facilitates binding between molecules and allows us to sample the whole sequence space in microliter volumes (2¹² << 10 µM × 20 µL × N_A = 10¹⁴).Formation of progressively longer oligomers from shorter ones requires ligation reactions, a method commonly employed in hairpin-mediated RNA and DNA replication (31, 32). At the origin of life, this might have been achieved by activated oligomers (33, 34) or activation agents (35–37), whereas later on the formation of simple ribozyme ligases seemed possible (38). Our study is focused on inherent properties of self-assembly by base pairing in random pools of oligomers and not on chemical mechanisms of ligation. Hence, we decided to use TAQ DNA ligase—an evolved enzyme for templated ligation of DNA (21) that is known for its ligation site sequence specificity (39, 40) and lack of sequence-dependent ligation rate (compare SI Appendix, section 21). This allowed for fast turnovers of ligation and enabled the observation of sequence dynamics.     

Plant species richness at archaeological sites suggests ecological legacy of Indigenous subsistence on the Colorado Plateau

Humans have both intentional and unintentional impacts on their environment, yet identifying the enduring ecological legacies of past small-scale societies remains difficult, and as such, evidence is sparse. The present study found evidence of an ecological legacy that persists today within an semiarid ecosystem of western North America. Specifically, the richness of ethnographically important plant species is strongly associated with archaeological complexity and ecological diversity at Puebloan sites in a region known as Bears Ears on the Colorado Plateau. A multivariate model including both environmental and archaeological predictors explains 88% of the variation in ethnographic species richness (ESR), with growing degree days and archaeological site complexity having the strongest effects. At least 31 plant species important to five tribal groups (Navajo, Hopi, Zuni, Ute Mountain Ute, and Apache), including the Four Corners potato (Solanum jamesii), goosefoot (Chenopodium sp.), wolfberry (Lycium pallidum), and sumac (Rhus trilobata), occurred at archaeological sites, despite being uncommon across the wider landscape. Our results reveal a clear ecological legacy of past human behavior: even when holding environmental variables constant, ESR increases significantly as a function of past investment in habitation and subsistence. Consequently, we suggest that propagules of some species were transported and cultivated, intentionally or not, establishing populations that persist to this day. Ensuring persistence will require tribal input for conserving and restoring archaeo-ecosystems containing “high-priority” plant species, especially those held sacred as lifeway medicines. This transdisciplinary approach has important implications for resource management planning, especially in areas such as Bears Ears that will experience greater visitation and associated impacts in the near future.

Local resource abundance is important for determining where in a given landscape humans decide to live. Nearby water, game, soil, and plants provide readily available wild resources for foraging and conditions that allow for cultivation (1–5). However, humans also modify their surrounding environments in order to increase the abundance and diversity of local plant (6–11) and animal (12–15) resources. Such “human niche construction” is a hallmark of ancient and modern societies (16, 17), having positive and negative impacts on global biodiversity while possibly creating enduring ecological legacies (18–21). This may be especially true for more sedentary and dense populations (22, 4) that are more likely to find investment worthwhile (23) and to produce unintentional impacts. Thus, variation in contemporary ecological diversity may in part reflect past land use dynamics and, therefore, be revealed through coupled archaeological and ecological research (24–33).Coupled ecological and archaeological research has led to the discovery of altered patterns of succession resulting from 1) forest clearing and changes in canopy light regime (34, 35), 2) alterations of soil especially linked to food refuse (36, 37), 3) changes in fire regimes (38, 39), and, more rarely, 4) the importation of plant propagules from distant sites of collection (40, 41). Identifying such long-lost dynamics between humans and landscapes can inform conservation aimed at restoring site-specific artifacts, features, and the associated resource base past and present, here termed “archaeo-ecosystems” (42, 43). This would greatly facilitate cross-cultural management of public lands (44) in ways that promote Indigenous health, cultural reclamation, and sovereignty (7, 45). The linkages, however, between ecological legacies, archaeo-ecosystem restoration and cross-cultural management have yet to be systematically tested or practically applied.Here, we offer a formal evaluation of this archaeo-ecosystem approach by using paired archaeological and ecological survey data focused on Puebloan occupation of a region known as Bears Ears on the Colorado Plateau in southeastern Utah (Fig. 1). Puebloan populations modified their environment by constructing terraces and check dams, developing blinds and wing traps, importing exogenous species, and setting fires (4, 22, 46), but investments were not uniform across the region. We test the hypothesis that locations with greater investment indicated by larger and more complex archaeological sites should today have higher richness of culturally significant plant species, here termed ethnographic species richness (ESR), as an enduring legacy of past investment. Our study expands previous work on ecological legacies by using field surveys to develop an explanatory model applied to 265 sites across one million acres of semiarid public lands. It documents the occurrence of uncommon and ethnographically significant plant species associated with those sites and infuses traditional ecological knowledge into proposed management actions for conserving these archaeo-ecosystems. Controlling for underlying environmental variation, our results indicate that past human habitation increases the diversity of plant species important for Indigenous subsistence.Open in a separate windowFig. 1.Location of Bears Ears National Monument in southeastern Utah. The predicted ESR at 265 known archaeological sites across the original and reduced monument boundaries and surrounding region are shown.     

A solution to a sex ratio puzzle in Melittobia wasps

The puzzling sex ratio behavior of Melittobia wasps has long posed one of the greatest questions in the field of sex allocation. Laboratory experiments have found that, in contrast to the predictions of theory and the behavior of numerous other organisms, Melittobia females do not produce fewer female-biased offspring sex ratios when more females lay eggs on a patch. We solve this puzzle by showing that, in nature, females of Melittobia australica have a sophisticated sex ratio behavior, in which their strategy also depends on whether they have dispersed from the patch where they emerged. When females have not dispersed, they lay eggs with close relatives, which keeps local mate competition high even with multiple females, and therefore, they are selected to produce consistently female-biased sex ratios. Laboratory experiments mimic these conditions. In contrast, when females disperse, they interact with nonrelatives, and thus adjust their sex ratio depending on the number of females laying eggs. Consequently, females appear to use dispersal status as an indirect cue of relatedness and whether they should adjust their sex ratio in response to the number of females laying eggs on the patch.

Sex allocation has produced many of the greatest success stories in the study of social behaviors (1–4). Time and time again, relatively simple theory has explained variation in how individuals allocate resources to male and female reproduction. Hamilton’s local mate competition (LMC) theory predicts that when n diploid females lay eggs on a patch and the offspring mate before the females disperse, the evolutionary stable proportion of male offspring (sex ratio) is (n − 1)/2n (Fig. 1) (5). A female-biased sex ratio is favored to reduce competition between sons (brothers) for mates and to provide more mates (daughters) for those sons (6–8). Consistent with this prediction, females of >40 species produce female-biased sex ratios and reduce this female bias when multiple females lay eggs on the same patch (higher n; Fig. 1) (9). The fit of data to theory is so good that the sex ratio under LMC has been exploited as a “model trait” to study the factors that can constrain “perfect adaptation” (4, 10–13).Open in a separate windowFig. 1.LMC. The sex ratio (proportion of sons) is plotted versus the number of females laying eggs on a patch. The bright green dashed line shows the LMC theory prediction for the haplodiploid species (5, 39). A more female-biased sex ratio is favored in haplodiploids because inbreeding increases the relative relatedness of mothers to their daughters (7, 32). Females of many species adjust their offspring sex ratio as predicted by theory, such as the parasitoid Nasonia vitripennis (green diamonds) (82). In contrast, the females of several Melittobia species, such as M. australica, continue to produce extremely female-biased sex ratios, irrespective of the number of females laying eggs on a patch (blue squares) (15).In stark contrast, the sex ratio behavior of Melittobia wasps has long been seen as one of the greatest problems for the field of sex allocation (3, 4, 14–21). The life cycle of Melittobia wasps matches the assumptions of Hamilton’s LMC theory (5, 15, 19, 21). Females lay eggs in the larvae or pupae of solitary wasps and bees, and then after emergence, female offspring mate with the short-winged males, who do not disperse. However, laboratory experiments on four Melittobia species have found that females lay extremely female-biased sex ratios (1 to 5% males) and that these extremely female-biased sex ratios change little with increasing number of females laying eggs on a patch (higher n; Fig. 1) (15, 17–20, 22). A number of hypotheses to explain this lack of sex ratio adjustment have been investigated and rejected, including sex ratio distorters, sex differential mortality, asymmetrical male competition, and reciprocal cooperation (15–18, 20, 22–26).We tested whether Melittobia’s unusual sex ratio behavior can be explained by females being related to the other females laying eggs on the same patch. After mating, some females disperse to find new patches, while some may stay at the natal patch to lay eggs on previously unexploited hosts (Fig. 2). If females do not disperse, they can be related to the other females laying eggs on the same host (27–31). If females laying eggs on a host are related, this increases the extent to which relatives are competing for mates and so can favor an even more female-biased sex ratio (28, 32–35). Although most parasitoid species appear unable to directly assess relatedness, dispersal behavior could provide an indirect cue of whether females are with close relatives (36–38). Consequently, we predict that when females do not disperse and so are more likely to be with closer relatives, they should maintain extremely female-biased sex ratios, even when multiple females lay eggs on a patch (28, 35).Open in a separate windowFig. 2.Host nest and dispersal manners of Melittobia. (A) Photograph of the prepupae of the leaf-cutter bee C. sculpturalis nested in a bamboo cane and (B) a diagram showing two ways that Melittobia females find new hosts. The mothers of C. sculpturalis build nursing nests with pine resin consisting of individual cells in which their offspring develop. If Melittobia wasps parasitize a host in a cell, female offspring that mate with males inside the cell find a different host on the same patch (bamboo cane) or disperse by flying to other patches.We tested whether the sex ratio of Melittobia australica can be explained by dispersal status in a natural population. We examined how the sex ratio produced by females varies with the number of females laying eggs on a patch and whether or not they have dispersed before laying eggs. To match our data to the predictions of theory, we developed a mathematical model tailored to the unique population structure of Melittobia, where dispersal can be a cue of relatedness. We then conducted a laboratory experiment to test whether Melittobia females are able to directly access the relatedness to other females and adjust their sex ratio behavior accordingly. Our results suggest that females are adjusting their sex ratio in response to both the number of females laying eggs on a patch and their relatedness to the other females. However, relatedness is assessed indirectly by whether or not they have dispersed. Consequently, the solution to the puzzling behavior reflects a more-refined sex ratio strategy.     

Structure,self-assembly,and properties of a truncated reflectin variant

Naturally occurring and recombinant protein-based materials are frequently employed for the study of fundamental biological processes and are often leveraged for applications in areas as diverse as electronics, optics, bioengineering, medicine, and even fashion. Within this context, unique structural proteins known as reflectins have recently attracted substantial attention due to their key roles in the fascinating color-changing capabilities of cephalopods and their technological potential as biophotonic and bioelectronic materials. However, progress toward understanding reflectins has been hindered by their atypical aromatic and charged residue-enriched sequences, extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for aggregation. Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a straightforward mechanical agitation-based methodology for controlling this variant’s hierarchical assembly, and establish a direct correlation between the protein’s structural characteristics and intrinsic optical properties. Altogether, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells’ color-changing functionalities, and may inform new research directions across biochemistry, cellular biology, bioengineering, and optics.

Materials from naturally occurring and recombinant proteins are frequently employed for the study of fundamental biological processes and leveraged for applications in fields as diverse as electronics, optics, bioengineering, medicine, and fashion (1–13). Such broad utility is enabled by the numerous advantageous characteristics of protein-based materials, which include sequence modularity, controllable self-assembly, stimuli-responsiveness, straightforward processability, inherent biological compatibility, and customizable functionality (1–13). Within this context, unique structural proteins known as reflectins have recently attracted substantial attention because of their key roles in the fascinating color-changing capabilities of cephalopods, such as the squid shown in Fig. 1A, and have furthermore demonstrated their utility for unconventional biophotonic and bioelectronic technologies (11–40). For example, in vivo, Bragg stack-like ultrastructures from reflectin-based high refractive index lamellae (membrane-enclosed platelets) are responsible for the angle-dependent narrowband reflectance (iridescence) of squid iridophores, as shown in Fig. 1B (15–20). Analogously, folded membranes containing distributed reflectin-based particle arrangements within sheath cells lead to the mechanically actuated iridescence of squid chromatophore organs, as shown in Fig. 1C (15, 16, 21, 22). Moreover, in vitro, films processed from squid reflectins not only exhibit proton conductivities on par with some state-of-the-art artificial materials (23–27) but also support the growth of murine and human neural stem cells (28, 29). Additionally, morphologically variable coatings assembled from different reflectin isoforms can enable the functionality of chemically and electrically actuated color-changing devices, dynamic near-infrared camouflage platforms, and stimuli-responsive photonic architectures (27, 30–34). When considered together, these discoveries and demonstrations constitute compelling motivation for the continued exploration of reflectins as model biomaterials.Open in a separate windowFig. 1.(A) A camera image of a D. pealeii squid for which the skin contains light-reflecting cells called iridophores (bright spots) and pigmented organs called chromatophores (colored spots). Image credit: Roger T. Hanlon (photographer). (B) An illustration of an iridophore (Left), which shows internal Bragg stack-like ultrastructures from reflectin-based lamellae (i.e., membrane-enclosed platelets) (Inset). (C) An illustration of a chromatophore organ (Left), which shows arrangements of reflectin-based particles within the sheath cells (Inset). (D) The logo of the 28-residue-long N-terminal motif (RMN), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (E) The logo of the 28-residue-long internal motif (RMI), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (F) The logo of the 21-residue-long C-terminal motif (RMC), which depicts the constituent amino acids (Upper) and their predicted secondary structures (Lower). (G) The amino acid sequence of full-length D. pealeii reflectin A1, which contains a single RMN motif (gray oval) and five RMI motifs (orange ovals). (H) An illustration of the selection of the prototypical truncated reflectin variant (denoted as RfA1TV) from full-length D. pealeii reflectin A1.Given reflectins’ demonstrated significance from both fundamental biology and applications perspectives, some research effort has been devoted to resolving their three-dimensional (3D) structures (30, 31, 35–39). For example, fibers drawn from full-length Euprymna scolopes reflectin 1a and films processed from truncated E. scolopes reflectin 1a were shown to possess secondary structural elements (i.e., α-helices or β-sheets) (30, 31). In addition, precipitated nanoparticles and drop-cast films from full-length Doryteuthis pealeii reflectin A1 have exhibited β-character, which was seemingly associated with their conserved motifs (35, 36). Moreover, nanoparticles assembled from both full-length and truncated Sepia officinalis reflectin 2 variants have demonstrated signatures consistent with β-sheet or α-helical secondary structure, albeit in the presence of surfactants (38). However, such studies were made exceedingly challenging by reflectins’ atypical primary sequences enriched in aromatic and charged residues, documented extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for poorly controlled aggregation (12, 14, 15, 30–32, 34–39). Consequently, the reported efforts have all suffered from multiple drawbacks, including the need for organic solvents or denaturants, the evaluation of only polydisperse or aggregated (rather than monomeric) proteins, a lack of consensus among different experimental techniques, inadequate resolution that precluded molecular-level insight, imperfect agreement between computational predictions and experimental observations, and/or the absence of conclusive correlations between structure and optical functionality. As such, there has emerged an exciting opportunity for investigating reflectins’ molecular structures, which remain poorly understood and the subject of some debate.Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a robust methodology for controlling this variant’s hierarchical assembly, and establish a direct correlation between its structural characteristics and optical properties. We first rationally select a prototypical reflectin variant expected to recapitulate the behavior of its parent protein by using a bioinformatics-guided approach. We next map the conformational and energetic landscape accessible to our selected protein by means of all-atom molecular dynamics (MD) simulations. We in turn produce our truncated reflectin variant with and without isotopic labeling, develop solution conditions that maintain the protein in a monomeric state, and characterize the variant’s size and shape with small-angle X-ray scattering (SAXS). We subsequently resolve our protein’s dynamic secondary and tertiary structures and evaluate its backbone conformational fluctuations with NMR spectroscopy. Finally, we demonstrate a straightforward mechanical agitation-based approach to controlling our truncated reflectin variant’s secondary structure, hierarchical self-assembly, and bulk refractive index distribution. Overall, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells’ color-changing functionalities, and appear poised to inform new directions across biochemistry, cellular biology, bioengineering, and optics.     

Herbivory and warming interact in opposing patterns of covariation between arctic shrub species at large and local scales

A major challenge in predicting species’ distributional responses to climate change involves resolving interactions between abiotic and biotic factors in structuring ecological communities. This challenge reflects the classical conceptualization of species’ regional distributions as simultaneously constrained by climatic conditions, while by necessity emerging from local biotic interactions. A ubiquitous pattern in nature illustrates this dichotomy: potentially competing species covary positively at large scales but negatively at local scales. Recent theory poses a resolution to this conundrum by predicting roles of both abiotic and biotic factors in covariation of species at both scales, but empirical tests have lagged such developments. We conducted a 15-y warming and herbivore-exclusion experiment to investigate drivers of opposing patterns of covariation between two codominant arctic shrub species at large and local scales. Climatic conditions and biotic exploitation mediated both positive covariation between these species at the landscape scale and negative covariation between them locally. Furthermore, covariation between the two species conferred resilience in ecosystem carbon uptake. This study thus lends empirical support to developing theoretical solutions to a long-standing ecological puzzle, while highlighting its relevance to understanding community compositional responses to climate change.

A readily observable phenomenon in nature is the tendency for the distributions of potentially competing species to covary positively at large spatial scales but negatively at small scales (1, 2). This scale dependence in patterns of species covariation is a defining phenomenon in ecology (3), and a classic illustration of it derives from MacArthur’s observations of Dendroica sp. warblers in mixed forests of the northeastern United States (1) and related theoretical work (4, 5). However, while opposing patterns of species covariation at large and local scales are ubiquitous, assigning causality to interacting drivers of such patterns in natural systems is challenging. Originally, theory explained this phenomenon as a product of distinct types of drivers of species abundance and distribution at large versus local scales. According to this framework, regional factors, such as climate, determine species’ distributions over large scales, while biotic interactions such as exploitation and interference determine presence, absence, and relative abundances of species at local scales (5–10). Hence, species with similar resource demands should, and often do, overlap spatially (covary positively) at broad scales as their distributions track abiotic niche requirements such as favorable climatic conditions (11). Meanwhile, the same species should, and often do, covary negatively at smaller spatial scales, where local biotic interactions such as competition, interference, niche complementarity, or exploitation by consumers or pathogens promote exclusion or segregation (5, 12–14). More recent theoretical developments have, however, highlighted the potential for roles of both types of drivers in patterns at both scales (7, 15, 16). Understanding whether, and how, climate and biotic interactions simultaneously influence species’ covariation at large and local scales has been repeatedly identified as a key challenge in improving predictions of species’ distributional and biodiversity responses to climate change (15, 17, 18).In contrast to progress in theory, field experimental tests of such potential interactions between biotic and abiotic factors in opposing patterns of species covariation at large and local scales have been lacking (14), in part because of the challenges inherent in conducting sufficiently controlled field experiments over suitably long time scales (19, 20). Consequently, novel empirical support for the role of, for example, biotic interactions in large scale patterns of species covariation has been strictly observational (21). Application of more robust empirical tests of predictions deriving from recent theory on this topic may also improve understanding of the consequences of patterns of species covariation at opposing spatial scales for important aspects of ecosystem function (22), including carbon exchange (23–26). Here, we present results of a 15-y warming and herbivore-exclusion experiment conducted at a remote arctic field site aimed at investigating influences of both drivers on patterns of covariation between two dominant shrub species at local and large spatial scales. The experimental design targets temperature as the abiotic limiting factor and herbivory (and associated ancillary effects) as the biotic limiting factor (Methods).The two focal shrub species in this study, dwarf birch (Betula nana) and gray willow (Salix glauca), hereafter “birch” and “willow,” respectively, are the most abundant plant species at our study site in low-arctic Greenland (27), and their functional role in ecosystem CO₂ exchange far exceeds that of any other vascular plant species at the site (28, 29). Furthermore, the two species are codominant across much of the Arctic (Fig. 1) (30, 31), but some experimental evidence indicates that Betula has the capacity to outcompete Salix at local scales in the Arctic due to its greater developmental plasticity and ability to invest rapidly in stem growth (32). Hence, although annual sampling throughout the duration of our experiment has assessed aboveground dynamics of all components of the plant community (Methods), our focus here is on patterns of covariation between birch and willow. Although birch is generally more common than willow across the study site (SI Appendix), the two species share similar distributions across the site, occur mainly on low to mid elevation slopes and plateaus, and predictably avoid arid steep slopes and stagnant mesic or saturated lowlands and fens (Fig. 1B). Each of the two species readily forms monospecific “shrub islands” at the local scale (Fig. 1C and SI Appendix, Fig. S3).Open in a separate windowFig. 1.(A) Circum-Arctic distributions of the two focal shrub species, dwarf birch (B. nana) and gray willow (S. glauca). Shaded polygons were derived from published range maps (30, 65). Point locations were derived from occurrence records (66–68) and the GBIF data portal (www.gbif.org). (B and C) Landscape and local scale views of patterns of covariation between the two species at the study site near Kangerlussuaq, Greenland. (B) South-facing hillside and lowland plains at the study site illustrating cooccurrence of dwarf birch (B. nana) and gray willow (S. glauca) at the landscape scale. (C) Monospecific shrub islands of each species are evident at smaller plot scales at the study site. In both photographs, birch appears dark or olive green, while willow appears lighter green. Image credit: E.P.