The Paradox of Predictability Provides a Bridge Between Micro- and Macroevolution.

The relationship between the evolutionary dynamics observed in contemporary populations (microevolution) and evolution on timescales of millions of years (macroevolution) has been a topic of considerable debate. Historically, this debate centers on inconsistencies between microevolutionary processes and macroevolutionary patterns. Here, we characterize a striking exception: emerging evidence indicates that standing variation in contemporary populations and macroevolutionary rates of phenotypic divergence are often positively correlated. This apparent consistency between micro- and macroevolution is paradoxical because it contradicts our previous understanding of phenotypic evolution and is so far unexplained. Here, we explore the prospects for bridging evolutionary timescales through an examination of this "paradox of predictability." We begin by explaining why the divergence-variance correlation is a paradox, followed by data analysis to show that the correlation is a general phenomenon across a broad range of temporal scales, from a few generations to tens of millions of years. Then we review complementary approaches from quantitative-genetics, comparative morphology, evo-devo, and paleontology to argue that they can help to address the paradox from the shared vantage point of recent work on evolvability. In conclusion, we recommend a methodological orientation that combines different kinds of short-term and long-term data using multiple analytical frameworks in an interdisciplinary research program. Such a program will increase our general understanding about how evolution works within and across timescales.

Meta-analytical evidence for frequency-dependent selection across the tree of life.

Explaining the maintenance of genetic variation in fitness-related traits within populations is a fundamental challenge in ecology and evolutionary biology. Frequency-dependent selection (FDS) is one mechanism that can maintain such variation, especially when selection favours rare variants (negative FDS). However, our general knowledge about the occurrence of FDS, its strength and direction remain fragmented, limiting general inferences about this important evolutionary process. We systematically reviewed the published literature on FDS and assembled a database of 747 effect sizes from 101 studies to analyse the occurrence, strength, and direction of FDS, and the factors that could explain heterogeneity in FDS. Using a meta-analysis, we found that overall, FDS is more commonly negative, although not significantly when accounting for phylogeny. An analysis of absolute values of effect sizes, however, revealed the widespread occurrence of modest FDS. However, negative FDS was only significant in laboratory experiments and non-significant in mesocosms and field-based studies. Moreover, negative FDS was stronger in studies measuring fecundity and involving resource competition over studies using other fitness components or focused on other ecological interactions. Our study unveils key general patterns of FDS and points in future promising research directions that can help us understand a long-standing fundamental problem in evolutionary biology and its consequences for demography and ecological dynamics.

Incidence and clinical significance of non-tuberculous mycobacteria among migrants in Denmark: A nationwide register-based cohort study from 1991 through 2021.

BACKGROUND: There is limited knowledge about non-tuberculous mycobacteria (NTM) infections in migrants. We aimed to assess the incidence and clinical significance of NTM among migrants in Denmark. METHOD: Nationwide register-based cohort study of migrants with a positive NTM culture in Denmark from 1991 through 2021, stratified by patient demographics, disease localisation, species, and clinical significance. RESULTS: 433 migrants had a positive NTM culture, resulting in an overall incidence rate (IR) of 3.7 (95%CI 3.3-4.0) per 100,000 migrants. Overall NTM IRs for definite disease were 1.0 (95%CI 0.9-1.2), possible disease 1.0 (95%CI 0.8-1.2), and isolation 1.7 (95%CI 1.4-1.9) per 100,000 migrants. Migrants had considerably higher age- and sex-adjusted NTM IRs of positive cultures (incidence rate ratio [IRR] = 2.1, 95%CI 1.9-2.3, p < 0.001), possible disease (IRR = 2.4, 95%CI 2.0-3.0, p < 0.001), and isolation (IRR = 4.6, 95%CI 3.9-5.4, p < 0.001) compared to Danish-born, but not of definite disease (IRR = 1.1, 95%CI 0.9-1.3, p = 0.562). IRs of migrants with positive NTM cultures did not increase over time (-0.8 %/year, p = 0.133). CONCLUSIONS: Migrants have a higher, but stable, burden of NTM compared with Danish-born. The higher rates likely reflect that more specimens are examined for Mycobacterium tuberculosis. Microbiologically classified definite NTM disease is not substantially more common among migrants.

Applying whole genome sequencing to predict phenotypic drug resistance in Mycobacterium tuberculosis: leveraging 20 years of nationwide data from Denmark.

Infection with Mycobacterium tuberculosis remains one of the biggest causes of death from a single microorganism worldwide, and the continuous emergence of drug resistance aggravates our ability to cure the disease. New improved resistance detection methods are needed to provide adequate treatment, such as whole genome sequencing (WGS), which has been used increasingly to identify resistance-conferring mutations over the last decade. The steadily increasing knowledge of resistance-conferring mutations increases our ability to predict resistance based on genomic data alone. This study evaluates the performance of WGS to predict M. tuberculosis complex resistance. It compares WGS predictions with the phenotypic (culture-based) drug susceptibility results based on 20 years of nationwide Danish data. Analyzing 6,230 WGS-sequenced samples, the sensitivities for isoniazid, rifampicin, ethambutol, and pyrazinamide were 82.5% [78.0%-86.5%, 95% confidence interval (CI)], 97.3% (90.6%-99.7%, 95% CI), 58.0% (43.2%-71.8%, 95% CI), and 60.5% (49.0%-71.2%, 95% CI), respectively, and specificities were 99.8% (99.7%-99.9%, 95% CI), 99.8% (99.7%-99.9%, 95% CI), 99.4% (99.2%-99.6%, 95% CI), and 99.9% (99.7%-99.9%, 95% CI), respectively. A broader range of both sensitivities and specificities was observed for second-line drugs. The results conform with previously reported values and indicate that WGS is reliable for routine resistance detection in resource-rich tuberculosis low-incidence and low-resistance settings such as Denmark.

Why Do Some Lineages Radiate While Others Do Not? Perspectives for Future Research on Adaptive Radiations.

Understanding the processes that drive phenotypic diversification and underpin speciation is key to elucidating how biodiversity has evolved. Although these processes have been studied across a wide array of clades, adaptive radiations (ARs), which are systems with multiple closely related species and broad phenotypic diversity, have been particularly fruitful for teasing apart the factors that drive and constrain diversification. As such, ARs have become popular candidate study systems for determining the extent to which ecological features, including aspects of organisms and the environment, and inter- and intraspecific interactions, led to evolutionary diversification. Despite substantial past empirical and theoretical work, understanding mechanistically how ARs evolve remains a major challenge. Here, we highlight a number of understudied components of the environment and of lineages themselves, which may help further our understanding of speciation and AR. We also outline some substantial remaining challenges to achieving a detailed understanding of adaptation, speciation, and the role of ecology in these processes. These major challenges include identifying factors that have a causative impact in promoting or constraining ARs, gaining a more holistic understanding of features of organisms and their environment that interact resulting in adaptation and speciation, and understanding whether the role of these organismal and environmental features varies throughout the radiation process. We conclude by providing perspectives on how future investigations into the AR process can overcome these challenges, allowing us to glean mechanistic insights into adaptation and speciation.

Dual-centre evaluation of the FluoroType MTBDR version 2 assay for detection of Mycobacterium tuberculosis complex and resistance-conferring mutations in pulmonary and extrapulmonary samples from Denmark, Germany and Sierra Leone.

OBJECTIVES: We evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a second-step real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures. METHODS: FTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing. RESULTS: Of the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and specificity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and specificities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and specificity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively. DISCUSSION: FTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes.

Heritable variation in thermal profiles is associated with reproductive success in the world's largest bird.

Organisms inhabiting extreme thermal environments, such as desert birds, have evolved spectacular adaptations to thermoregulate during hot and cold conditions. However, our knowledge of selection for thermoregulation and the potential for evolutionary responses is limited, particularly for large organisms experiencing extreme temperature fluctuations. Here we use thermal imaging to quantify selection and genetic variation in thermoregulation in ostriches (Struthio camelus), the world's largest bird species that is experiencing increasingly volatile temperatures. We found that females who are better at regulating their head temperatures ("thermoregulatory capacity") had higher egg-laying rates under hotter conditions. Thermoregulatory capacity was both heritable and showed signatures of local adaptation: females originating from more unpredictable climates were better at regulating their head temperatures in response to temperature fluctuations. Together these results reveal that past and present evolutionary processes have shaped genetic variation in thermoregulatory capacity, which appears to protect critical organs, such as the brain, from extreme temperatures during reproduction.

Tropical Origin, Global Diversification, and Dispersal in the Pond Damselflies (Coenagrionoidea) Revealed by a New Molecular Phylogeny.

The processes responsible for the formation of Earth's most conspicuous diversity pattern, the latitudinal diversity gradient (LDG), remain unexplored for many clades in the Tree of Life. Here, we present a densely sampled and dated molecular phylogeny for the most speciose clade of damselflies worldwide (Odonata: Coenagrionoidea) and investigate the role of time, macroevolutionary processes, and biome-shift dynamics in shaping the LDG in this ancient insect superfamily. We used process-based biogeographic models to jointly infer ancestral ranges and speciation times and to characterize within-biome dispersal and biome-shift dynamics across the cosmopolitan distribution of Coenagrionoidea. We also investigated temporal and biome-dependent variation in diversification rates. Our results uncover a tropical origin of pond damselflies and featherlegs ~105 Ma, while highlighting the uncertainty of ancestral ranges within the tropics in deep time. Even though diversification rates have declined since the origin of this clade, global climate change and biome-shifts have slowly increased diversity in warm- and cold-temperate areas, where lineage turnover rates have been relatively higher. This study underscores the importance of biogeographic origin and time to diversify as important drivers of the LDG in pond damselflies and their relatives, while diversification dynamics have instead resulted in the formation of ephemeral species in temperate regions. Biome-shifts, although limited by tropical niche conservatism, have been the main factor reducing the steepness of the LDG in the last 30 Myr. With ongoing climate change and increasing northward range expansions of many damselfly taxa, the LDG may become less pronounced. Our results support recent calls to unify biogeographic and macroevolutionary approaches to improve our understanding of how latitudinal diversity gradients are formed and why they vary across time and among taxa.

The genomics and evolution of inter-sexual mimicry and female-limited polymorphisms in damselflies.

Sex-limited morphs can provide profound insights into the evolution and genomic architecture of complex phenotypes. Inter-sexual mimicry is one particular type of sex-limited polymorphism in which a novel morph resembles the opposite sex. While inter-sexual mimics are known in both sexes and a diverse range of animals, their evolutionary origin is poorly understood. Here, we investigated the genomic basis of female-limited morphs and male mimicry in the common bluetail damselfly. Differential gene expression between morphs has been documented in damselflies, but no causal locus has been previously identified. We found that male mimicry originated in an ancestrally sexually dimorphic lineage in association with multiple structural changes, probably driven by transposable element activity. These changes resulted in ~900 kb of novel genomic content that is partly shared by male mimics in a close relative, indicating that male mimicry is a trans-species polymorphism. More recently, a third morph originated following the translocation of part of the male-mimicry sequence into a genomic position ~3.5 mb apart. We provide evidence of balancing selection maintaining male mimicry, in line with previous field population studies. Our results underscore how structural variants affecting a handful of potentially regulatory genes and morph-specific genes can give rise to novel and complex phenotypic polymorphisms.

Immunogenicity of Bacillus Calmette-Guérin in pigs: potential as a translational model of non-specific effects of BCG.

Background: Clinical and immunological studies in humans show that the live attenuated Bacillus Calmette-Guérin (BCG) vaccine has beneficial non-specific effects, increasing resistance against diseases other than tuberculosis. The underlying mechanisms are currently being explored. The pig exhibits considerable physiological similarity to humans in anatomy and physiology, suggesting that similar responses to BCG could be expected. Studies of the non-specific effects of BCG in pigs are scarce. We investigated the feasibility of using pigs as a large animal model to investigate the non-specific immunological effects of BCG. Methods: In a series of experiments, we randomized newborn or young piglets from conventional farms to receiving BCG or placebo and investigated the persistence of live BCG bacteria in various tissues, the immunogenicity of BCG in ex vivo blood and in vitro stimulation assays, and the acute phase protein and clinical responses to heterologous infectious challenge with influenza A virus or Actinobacillus pleuropneumoniae. Results: The BCG vaccine was generally well tolerated. In contrast to humans, no skin reaction in the form of abscesses, ulcers, or scars was observed. Live BCG was recovered from draining lymph nodes in 2/13 animals 20 weeks after vaccination. Specific in vitro responses of IFN-γ to antigen-specific re-stimulation with mycobacterial antigen were increased but not TNF-responses to TLR2 or TLR4 agonists. A few genes were differentially expressed in blood after vaccination, including the antiviral genes RIG-I and CSF1, although the effect disappeared after correction for multiple testing. Clinical symptoms after heterologous bacterial or viral respiratory infections did not differ, nor did virus copies in nasopharyngeal samples after the challenge. However, the acute phase protein response was significantly reduced in BCG-vaccinated animals after influenza challenge but not after A. pleuropneumoniae challenge. Discussion: BCG was safe in pigs, inducing specific immunological responses, but our model did not corroborate the innate immunological responsiveness to BCG seen in humans. The dose of BCG or the bacterial and viral challenges may have been sub-optimal. Even so, the acute phase protein response to influenza infection was significantly reduced in BCG-vaccinated animals.

Phenotypic selection in natural populations: what have we learned in 40 years?

In 1983, Russell Lande and Stevan Arnold published "The measurement of selection on correlated characters," which became a highly influential citation classic in evolutionary biology. This paper stimulated a cottage industry of field studies of natural and sexual selection in nature and resulted in several large-scale meta-analyses, statistical developments, and method papers. The statistical tools they suggested contributed to a breakdown of the traditional dichotomy between ecological and evolutionary time scales and stimulated later developments such as "eco-evolutionary dynamics". However, regression-based selection analyses also became criticized from philosophical, methodological, and statistical viewpoints and stimulated some still ongoing debates about causality in evolutionary biology. Here I return to this landmark paper by Lande and Arnold, analyze the controversies and debates it gave rise to and discuss the past, present, and future of selection analyses in natural populations. A remaining legacy of Lande & Arnold, 1983 is that studies of selection and inheritance can fruitfully be decoupled and be studied separately, since selection acts on phenotypes regardless of their genetic basis, and hence selection and evolutionary responses to selection are distinct processes.

Towards clinical breakpoints for non-tuberculous mycobacteria - Determination of epidemiological cut off values for the Mycobacterium avium complex and Mycobacterium abscessus using broth microdilution.

OBJECTIVE: For non-tuberculous mycobacteria (NTM), minimum inhibitory concentration (MIC) distributions of wild-type isolates have not been systematically evaluated despite their importance for establishing antimicrobial susceptibility testing (AST) breakpoints. METHODS: We gathered MIC distributions for drugs used against the Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) obtained by commercial broth microdilution (SLOMYCOI and RAPMYCOI) from 12 laboratories. Epidemiological cut-off values (ECOFFs) and tentative ECOFFs (TECOFFs) were determined by EUCAST methodology including quality control (QC) strains. RESULTS: The clarithromycin ECOFF was 16 mg/L for M. avium (n = 1271) whereas TECOFFs were 8 mg/L for M. intracellulare (n = 415) and 1 mg/L for MAB (n = 1014) confirmed by analysing MAB subspecies without inducible macrolide resistance (n = 235). For amikacin, the ECOFFs were 64 mg/L for MAC and MAB. For moxifloxacin, the WT spanned >8 mg/L for both MAC and MAB. For linezolid, the ECOFF and TECOFF were 64 mg/L for M. avium and M. intracellulare, respectively. Current CLSI breakpoints for amikacin (16 mg/L), moxifloxacin (1 mg/L) and linezolid (8 mg/L) divided the corresponding WT distributions. For QC M. avium and M. peregrinum, ≥95% of MIC values were well within recommended QC ranges. CONCLUSION: As a first step towards clinical breakpoints for NTM, (T)ECOFFs were defined for several antimicrobials against MAC and MAB. Broad wild-type MIC distributions indicate a need for further method refinement which is now under development within the EUCAST subcommittee for anti-mycobacterial drug susceptibility testing. In addition, we showed that several CLSI NTM breakpoints are not consistent in relation to the (T)ECOFFs.

Successful Direct Whole Genome Sequencing and Revivification of Freeze-Dried Nontuberculous Mycobacteria after More than Half a Century of Storage.

In this study, 28 "historical" clinical freeze-dried nontuberculous mycobacterial isolates collected from 1948 to 1957, were analyzed by investigating their viability and performing whole genome sequencing (WGS) on DNA extracted (i) directly from freeze-dried cells versus (ii) after culturing, to determine cell properties and DNA quality after centuries of freeze-dried storage. The isolated DNA was sequenced on the Illumina MiSeq platform and data quality evaluated analyzing the per-base quality scores of paired-end sequencing reads as well as the overall contiguity of resulting de novo assemblies. After 72 years in storage, all freeze-dried isolates were viable, and showed no signs of cell damage and limited signs of contamination when reculturing. They were recultured without problems and identified through WGS with only four of 13 parameters showing statistical significance based on sequence data obtained directly from the freeze-dried cells versus after reculturing, indicating no DNA degradation. Thus, mycobacteria can be whole genome sequenced successfully directly from freeze-dried material without prior recultivation, saving laboratory time and resources, and emphasizing the value of freeze-drying for long-term storage. Our study lays the groundwork for further genomic investigations of freeze-dried bacterial isolates, and the approximately 4,000 historical isolates in our collection will provide a unique opportunity to investigate mycobacterial DNA from a variety of NTM species unexposed to antimicrobials, some maybe still undescribed species. IMPORTANCE The genus Mycobacterium was described more than a century ago and new species are continuously identified and described. There is an ongoing discussion about an increase in the incidence of disease caused by nontuberculous mycobacteria (NTM). How the different bacteria looked before exposure to antibiotics can only be investigated by looking at strains from before the antibiotic era. Strains from that era will be stored in different ways, for example by freeze-drying. The question is how to investigate these strains, and if they are still viable, whether they need to be cultured, and if that changes the DNA. Here, we test all these parameters on freeze-dried strains and show that NGS can be applied directly without culturing.

Evolutionary trade-offs between heat and cold tolerance limit responses to fluctuating climates.

The evolutionary potential of species to cope with short-term temperature fluctuations during reproduction is critical to predicting responses to future climate change. Despite this, vertebrate research has focused on reproduction under high or low temperatures in relatively stable temperate climates. Here, we characterize the genetic basis of reproductive thermal tolerance to temperature fluctuations in the ostrich, which lives in variable environments in tropical and subtropical Africa. Both heat and cold tolerance were under selection and heritable, indicating the potential for evolutionary responses to mean temperature change. However, we found evidence for a negative, genetic correlation between heat and cold tolerance that should limit the potential for adaptation to fluctuating temperatures. Genetic constraints between heat and cold tolerance appear a crucial, yet underappreciated, factor influencing responses to climate change.

Phenotypic plasticity is aligned with phenological adaptation on both micro- and macroevolutionary timescales.

In seasonally variable environments, phenotypic plasticity in phenology may be critical for adaptation to fluctuating environmental conditions. Using an 18-generation longitudinal dataset from natural damselfly populations, we show that phenology has strongly advanced. Individual fitness data suggest this is likely an adaptive response towards a temperature-dependent optimum. A laboratory experiment revealed that developmental plasticity qualitatively matches the temperature dependence of selection, partially explaining observed advance in phenology. Expanding our analysis to the macroevolutionary level, we use a database of over 1-million occurrence records and spatiotemporally matched temperature data from 49 Swedish Odonate species to infer macroevolutionary dynamics of phenology. Phenological plasticity was more closely aligned with adaptation for species that have recently colonised northern latitudes, but with higher phenological mismatch at lower latitudes. Our results show that phenological plasticity plays a key role in microevolutionary dynamics within a single species, and such plasticity may have facilitated post-Pleistocene range expansion in this insect clade.

Latitudinal clines in sexual selection, sexual size dimorphism and sex-specific genetic dispersal during a poleward range expansion.

Range expansions can be shaped by sex differences in behaviours and other phenotypic traits affecting dispersal and reproduction. Here, we investigate sex differences in morphology, behaviour and genomic population differentiation along a climate-mediated range expansion in the common bluetail damselfly (Ischnura elegans) in northern Europe. We sampled 65 sites along a 583-km gradient spanning the I. elegans range in Sweden and quantified latitudinal gradients in site relative abundance, sex ratio and sex-specific shifts in body size and mating status (a measure of sexual selection). Using single nucleotide polymorphism (SNP) data for 426 individuals from 25 sites, we further investigated sex-specific landscape and climatic effects on neutral genetic connectivity and migration patterns. We found evidence for sex differences associated with the I. elegans range expansion, namely (a) increased male body size with latitude, but no latitudinal effect on female body size, resulting in reduced sexual dimorphism towards the range limit, (b) a steeper decline in male genetic similarity with increasing geographic distance than in females, (c) male-biased genetic migration propensity and (d) a latitudinal cline in migration distance (increasing migratory distances towards the range margin), which was stronger in males. Cooler mean annual temperatures towards the range limit were associated with increased resistance to gene flow in both sexes. Sex ratios became increasingly male biased towards the range limit, and there was evidence for a changed sexual selection regime shifting from favouring larger males in the south to favouring smaller males in the north. Our findings suggest sex-specific spatial phenotype sorting at the range limit, where larger males disperse more under higher landscape resistance associated with cooler climates. The combination of latitudinal gradients in sex-biased dispersal, increasing male body size and (reduced) sexual size dimorphism should have emergent consequences for sexual selection dynamics and the mating system at the expanding range front. Our study illustrates the importance of considering sex differences in the study of range expansions driven by ongoing climate change.

The importance of pre- and postcopulatory sexual selection promoting adaptation to increasing temperatures.

Global temperatures are increasing rapidly affecting species globally. Understanding if and how different species can adapt fast enough to keep up with increasing temperatures is of vital importance. One mechanism that can accelerate adaptation and promote evolutionary rescue is sexual selection. Two different mechanisms by which sexual selection can facilitate adaptation are pre- and postcopulatory sexual selection. However, the relative effects of these different forms of sexual selection in promoting adaptation are unknown. Here, we present the results from an experimental study in which we exposed fruit flies Drosophila melanogaster to either no mate choice or 1 of 2 different sexual selection regimes (pre- and postcopulatory sexual selection) for 6 generations, under different thermal regimes. Populations showed evidence of thermal adaptation under precopulatory sexual selection, but this effect was not detected in the postcopulatory sexual selection and the no choice mating regime. We further demonstrate that sexual dimorphism decreased when flies evolved under increasing temperatures, consistent with recent theory predicting more sexually concordant selection under environmental stress. Our results suggest an important role for precopulatory sexual selection in promoting thermal adaptation and evolutionary rescue.