Weanling gut microbiota composition of a mouse model selectively bred for high voluntary wheel-running behavior.

We compared the fecal microbial community composition and diversity of four replicate lines of mice selectively bred for high wheel-running activity over 81 generations (HR lines) and four non-selected control lines. We performed 16S rRNA gene sequencing on fecal samples taken 24 h after weaning, identifying a total of 2074 bacterial operational taxonomic units. HR and control mice did not significantly differ for measures of alpha diversity, but HR mice had a higher relative abundance of the family Clostridiaceae. These results differ from a study of rats, where a line bred for high forced-treadmill endurance and that also ran more on wheels had lower relative abundance of Clostridiaceae, as compared with a line bred for low endurance that ran less on wheels. Within the HR and control groups, replicate lines had unique microbiomes based on unweighted UniFrac beta diversity, indicating random genetic drift and/or multiple adaptive responses to selection.

Natural selection drives genome-wide evolution via chance genetic associations.

Understanding selection's impact on the genome is a major theme in biology. Functionally neutral genetic regions can be affected indirectly by natural selection, via their statistical association with genes under direct selection. The genomic extent of such indirect selection, particularly across loci not physically linked to those under direct selection, remains poorly understood, as does the time scale at which indirect selection occurs. Here, we use field experiments and genomic data in stick insects, deer mice and stickleback fish to show that widespread statistical associations with genes known to affect fitness cause many genetic loci across the genome to be impacted indirectly by selection. This includes regions physically distant from those directly under selection. Then, focusing on the stick insect system, we show that statistical associations between SNPs and other unknown, causal variants result in additional indirect selection in general and specifically within genomic regions of physically linked loci. This widespread indirect selection necessarily makes aspects of evolution more predictable. Thus, natural selection combines with chance genetic associations to affect genome-wide evolution across linked and unlinked loci and even in modest-sized populations. This process has implications for the application of evolutionary principles in basic and applied science.

The short-term, genome-wide effects of indirect selection deserve study: A response to Charlesworth and Jensen (2022).

We recently published a paper quantifying the genome-wide consequences of natural selection, including the effects of indirect selection due to the correlation of genetic regions (neutral or selected) with directly selected regions (Gompert et al., 2022). In their critique of our paper, Charlesworth and Jensen (2022) make two main points: (i) indirect selection is equivalent to hitchhiking and thus well documented (i.e., our results are not novel) and (ii) that we do not demonstrate the source of linkage disequilibrium (LD) between SNPs and the Mel-Stripe locus in the Timema cristinae experiment we analyse. As we discuss in detail below, neither of these are substantial criticisms of our work.

Early-life effects of juvenile Western diet and exercise on adult gut microbiome composition in mice.

Alterations to the gut microbiome caused by changes in diet, consumption of antibiotics, etc., can affect host function. Moreover, perturbation of the microbiome during critical developmental periods potentially has long-lasting impacts on hosts. Using four selectively bred high runner and four non-selected control lines of mice, we examined the effects of early-life diet and exercise manipulations on the adult microbiome by sequencing the hypervariable internal transcribed spacer region of the bacterial gut community. Mice from high runner lines run ∼3-fold more on wheels than do controls, and have several other phenotypic differences (e.g. higher food consumption and body temperature) that could alter the microbiome, either acutely or in terms of coevolution. Males from generation 76 were given wheels and/or a Western diet from weaning until sexual maturity at 6 weeks of age, then housed individually without wheels on standard diet until 14 weeks of age, when fecal samples were taken. Juvenile Western diet reduced bacterial richness and diversity after the 8-week washout period (equivalent to ∼6 human years). We also found interactive effects of genetic line type, juvenile diet and/or juvenile exercise on microbiome composition and diversity. Microbial community structure clustered significantly in relation to both line type and diet. Western diet also reduced the relative abundance of Muribaculum intestinale These results constitute one of the first reports of juvenile diet having long-lasting effects on the adult microbiome after a substantial washout period. Moreover, we found interactive effects of diet with early-life exercise exposure, and a dependence of these effects on genetic background.

Selection of Laboratory Mice for the Cognitive Task Successful Solution and for the Inability to Solve It.

Using the selected mouse strain EX as the founding population (selection for extrapolation ability) three selection generations of mice were obtained, which were selected for successful solution of object permanence test (plus-sub-strain) and for lack of such solution (minus-sub-strain). The successful solution required not only the ability to operate the object permanence rule (by J. Piajet), but the performance of complicated action (executive function) which was significantly higher in plus-substrain, and this is the unique example of successful selection for cognitive trait.

Rapid genomic and phenotypic change in response to climate warming in a widespread plant invader.

Predicting plant distributions under climate change is constrained by our limited understanding of potential rapid adaptive evolution. In an experimental evolution study with the invasive common ragweed (Ambrosia artemisiifolia L.) we subjected replicated populations of the same initial genetic composition to simulated climate warming. Pooled DNA sequencing of parental and offspring populations showed that warming populations experienced greater genetic divergence from their parents, than control populations. In a common environment, offspring from warming populations showed more convergent phenotypes in seven out of nine plant traits, with later flowering and larger biomass, than plants from control populations. For both traits, we also found a significantly higher ratio of phenotypic to genetic differentiation across generations for warming than for control populations, indicating stronger response to selection under warming conditions. As a measure for evolutionary rate, the phenotypic and sequence divergence between generations were assessed using the Haldane metric. Our approach combining comparisons between generations (allochronic) and between treatments (synchronic) in an experimental evolutionary field study, and linking population genomic data with phenotyping analyses provided a powerful test to detect rapid responses to selection. Our findings demonstrate that ragweed populations can rapidly evolve in response to climate change within a single generation. Short-term evolutionary responses to climate change may aggravate the impact of some plant invaders in the future and should be considered when making predictions about future distributions and impacts of plant invaders.

Endosymbionts facilitate rapid evolution in a polyphagous herbivore.

Maternally transmitted bacterial symbionts can be important mediators of the interactions between insect herbivores and their foodplants. These symbionts are often facultative (present in some host individuals but not others) and can have large effects on their host's phenotype, thus giving rise to heritable variation upon which selection can act. In the cowpea aphid (Aphis craccivora), it has been established that the facultative endosymbiont Arsenophonus improves aphid performance on black locust trees (Robinia pseudoacacia) but not on fava (Vicia faba). Here, we tested whether this fitness differential translated into contemporaneous evolution of aphid populations associated with the different plants. In a laboratory study lasting 16 weeks, we found that the frequency of Arsenophonus-infected individuals significantly increased over time for aphid populations on black locust but declined for aphid populations on fava. By the end of the experiment, Arsenophonus infection was >3× more common on black locust than fava, which is comparable to previously described infection frequencies in natural field populations. Our results clearly demonstrate that aphid populations with mixed facultative symbiont infection status can rapidly evolve in response to the selective environments imposed by different host plants. This selection differential may be a sufficient explanation for the global association between Arsenophonus-infected cowpea aphids and black locust trees, without invoking additional assortative mechanisms. Because the aphid and plant originate from different parts of the world, we further hypothesize that Arsenophonus infection may have acted as a preadaptation that has promoted functional specialization of infected aphids on a novel host plant.

Lagging Adaptation to Climate Supersedes Local Adaptation to Herbivory in an Annual Monkeyflower.

While native populations are often adapted to historical biotic and abiotic conditions at their home site, populations from other locations in the range may be better adapted to current conditions due to changing climates or extreme conditions in a single year. We examine whether local populations of a widespread species maintain a relative advantage over distant populations that have evolved at sites better matching the current climate. Specifically, we grew lines derived from low- and high-elevation annual populations in California and Oregon of the common monkeyflower (Erythranthe guttata) and conducted phenotypic selection analyses in low- and high-elevation common gardens in Oregon to examine relative fitness and the traits mediating relative fitness. Californian low-elevation populations have the highest relative fitness at the low-elevation site, and Californian high-elevation populations have the highest relative fitness at the high-elevation site. Relative fitness differences are mediated by selection for properly timed transitions to flowering, with selection favoring more rapid growth rates at the low-elevation site and greater vegetative biomass prior to flowering at the high-elevation site. Fitness advantages for Californian plants occur despite incurring higher herbivory at both sites than the native Oregonian plants. Our findings suggest that a lag in adaptation causes maladaptation in extreme years that may be more prevalent in future climates, but local populations still have high growth rates and thus are not yet threatened.

Arousal from Tonic Immobility by Vibration Stimulus.

Tonic immobility (TI) is an effective anti-predator strategy. However, long immobility status on the ground increases the risk of being eaten by predators, and thus insects must rouse themselves when appropriate stimulation is provided. Here, the strength of vibration causing arousal from the state of TI was examined in strains artificially selected for longer duration of TI (L-strains: long sleeper) in a beetle. We provided different strengths of vibration stimuli to the long sleepers in Tribolium castaneum. Although immobilized beetles were never awakened by the stimuli from 0.01 to 0.12 mm in amplitude, almost of the beetles were aroused from immobilized status by the stimulus at 0.21 mm. There was a difference in sensitivity of individuals when the stimuli of 0.14 mm and 0.18 mm were provided. F2 individuals were also bred by crossing experiments of the strains selected for shorter and longer duration of TI. The arousal sensitivity to vibration was well separated in the F2 individuals. A positive relationship was observed between the duration of TI and the vibration amplitude, suggesting that immobilized beetles are difficult to arouse from a deep sleep, while light sleepers are easily aroused by even small vibrations. The results indicate a genetic basis for sensitivity to arousal from TI.

Genomic Response to Selection for Predatory Behavior in a Mammalian Model of Adaptive Radiation.

If genetic architectures of various quantitative traits are similar, as studies on model organisms suggest, comparable selection pressures should produce similar molecular patterns for various traits. To test this prediction, we used a laboratory model of vertebrate adaptive radiation to investigate the genetic basis of the response to selection for predatory behavior and compare it with evolution of aerobic capacity reported in an earlier work. After 13 generations of selection, the proportion of bank voles (Myodes [=Clethrionomys] glareolus) showing predatory behavior was five times higher in selected lines than in controls. We analyzed the hippocampus and liver transcriptomes and found repeatable changes in allele frequencies and gene expression. Genes with the largest differences between predatory and control lines are associated with hunger, aggression, biological rhythms, and functioning of the nervous system. Evolution of predatory behavior could be meaningfully compared with evolution of high aerobic capacity, because the experiments and analyses were performed in the same methodological framework. The number of genes that changed expression was much smaller in predatory lines, and allele frequencies changed repeatably in predatory but not in aerobic lines. This suggests that more variants of smaller effects underlie variation in aerobic performance, whereas fewer variants of larger effects underlie variation in predatory behavior. Our results thus contradict the view that comparable selection pressures for different quantitative traits produce similar molecular patterns. Therefore, to gain knowledge about molecular-level response to selection for complex traits, we need to investigate not only multiple replicate populations but also multiple quantitative traits.

A comparative analysis of experimental selection on the stickleback pelvis.

Mechanisms of natural selection can be identified using experimental approaches. However, such experiments often yield nonsignificant effects and imprecise estimates of selection due to low power and small sample sizes. Combining results from multiple experimental studies might produce an aggregate estimate of selection that is more revealing than individual studies. For example, bony pelvic armour varies conspicuously among stickleback populations, and predation by vertebrate and insect predators has been hypothesized to be the main driver of this variation. Yet experimental selection studies testing these hypotheses frequently fail to find a significant effect. We experimentally manipulated length of threespine stickleback (Gasterosteus aculeatus) pelvic spines in a mesocosm experiment to test whether prickly sculpin (Cottus asper), an intraguild predator of stickleback, favours longer spines. The probability of survival was greater for stickleback with unclipped pelvic spines, but this effect was noisy and not significant. We used meta-analysis to combine the results of our mesocosm experiment with previously published experimental studies of selection on pelvic armour. We found evidence that fish predation indeed favours increased pelvic armour, with a moderate effect size. The same approach found little evidence that insect predation favours reduced pelvic armour. The causes of reduced pelvic armour in many stickleback populations remain uncertain.

Evidence that a herbivore tolerance response affects selection on floral traits and inflorescence architecture in purple loosestrife (Lythrum salicaria).

Background and Aims: The study of the evolution of floral traits has generally focused on pollination as the primary driver of selection. However, herbivores can also impose selection on floral traits through a variety of mechanisms, including florivory and parasitism. Less well understood is whether floral and inflorescence architecture traits that influence a plant's tolerance to herbivory, such as compensatory regrowth, alter pollinator-mediated selection. Methods: Because herbivore damage to Lythrum salicaria meristems typically leads to an increase in the number of inflorescences and the size of the floral display, an experiment was conducted to test whether simulated herbivory (i.e. clipping the developing meristem) could alter the magnitude or direction of pollinator-mediated selection on a suite of floral and inflorescence architecture traits. Using a pollen supplementation protocol, pollen limitation was compared in the presence and absence of meristem damage in order to quantify any interaction between pollinator and herbivore-mediated selection on floral traits. Key Results: Surprisingly, in spite of an obvious impact on floral display and architecture, with clipped plants producing more inflorescences and more flowers, there was no difference in pollen limitation between clipped and unclipped plants. Correspondingly, there was no evidence that imposing herbivore damage altered pollinator-mediated selection in this system. Rather, the herbivory treatment alone was found to alter direct selection on floral display, with clipped plants experiencing greater selection for earlier flowering and weaker selection for number of inflorescences when compared with unclipped plants. Conclusions: These findings imply that herbivory on its own can drive selection on plant floral traits and inflorescence architecture in this species, even more so than pollinators. Specifically, herbivory can impose selection on floral traits if such traits influence a plant's tolerance to herbivory, such as through the timing of flowering and/or the compensatory regrowth response.

Initial Molecular-Level Response to Artificial Selection for Increased Aerobic Metabolism Occurs Primarily through Changes in Gene Expression.

Experimental evolution combined with genome or transcriptome resequencing (Evolve and Resequence) represents a promising approach for advancing our understanding of the genetic basis of adaptation. Here, we applied this strategy to investigate the effect of selection on a complex trait in lines derived from a natural population of a small mammal. We analyzed the liver and heart transcriptomes of bank voles (Myodes [=Clethrionomys] glareolus) that had been selected for increased aerobic metabolism. The organs were sampled from 13th generation voles; at that point, the voles from four replicate selected lines had 48% higher maximum rates of oxygen consumption than those from four control lines. At the molecular level, the response to selection was primarily observed in gene expression: Over 300 genes were found to be differentially expressed between the selected and control lines and the transcriptome-wide pattern of expression distinguished selected lines from controls. No evidence for selection-driven changes of allele frequencies at coding sites was found: No single nucleotide polymorphism (SNP) changed frequency more than expected under drift alone and frequency changes aggregated over all SNPs did not separate selected and control lines. Nevertheless, among genes which showed highest differentiation in allele frequencies between selected and control lines we identified, using information about gene functions and the biology of the selected phenotype, plausible targets of selection; these genes, together with those identified in expression analysis, have been prioritized for further studies. Because our selection lines were derived from a natural population, the amount and the spectrum of variation available for selection probably closely approximated that typically found in populations of small mammals. Therefore, our results are relevant to the understanding of the molecular basis of complex adaptations occurring in natural vertebrate populations.

Can selection on a male mating character result in evolutionary change? A selection experiment on California wild radish, Raphanus sativus.

PREMISE OF THE STUDY: Whenever more pollen grains arrive on stigmas than necessary to fertilize ovules, sexual selection is possible. However, the role of sexual selection remains controversial, in part because of lack of evidence on genetic bases of traits and the response of relevant characters to selection. METHODS: In an experiment with Raphanus sativus, we selected on tendency to sire seeds in the stylar or basal regions of fruits. This character is likely related to pollen tube growth rate, and seed position affects rates of abortion and seed predation. We measured differences among families in seed siring and related characters and evaluated responses to selection. KEY RESULTS: All replicates showed strong effects of pollen donor family on proportion of seeds sired per fruit in mixed pollinations. Most also showed effects of pollen donor family on number of pollen grains per flower and pollen diameter. Two of four replicates showed a response to selection on position of seeds sired. In responding replicates, we found trade-offs in pollen grain size and number; plants with larger pollen grains sired more seeds in the basal region. CONCLUSIONS: Our data suggest a genetic basis for pollen donor ability to sire seeds in competition. The significant response to selection in two replicates shows that position of seeds sired can respond to selection. Thus, all components for sexual selection to occur and affect traits are present. Variation in results among replicates might be due to changes in greenhouse conditions. Environmental effects may contribute to the maintenance of variation in these fitness-related characters.

Genetic control of the environmental variance for birth weight in seven generations of a divergent selection experiment in mice.

Data from seven generations of a divergent selection experiment designed for environmental variability of birth weight were analysed to estimate genetic parameters and to explore signs of selection response. A total of 10 783 birth weight records from 638 females and 1127 litters in combination with 10 007 pedigree records were used. Each record of birth weight was assigned to the mother of the pup in a heteroscedastic model, and after seven generations of selection, evidence of success in the selection process was shown. A Bayesian analysis showed that success of the selection process started from the first generation for birth weight and from the second generation for its environmental variability. Genetic parameters were estimated across generations. However, only from the third generation onwards were the records useful to consider the results to be reliable. The results showed a consistent positive and low genetic correlation between the birth weight trait and its environmental variability, which could allow an independent selection process. This study has demonstrated that the genetic control of the birth weight environmental variability is possible in mice. Nevertheless, before the results are applied directly in farm animals, it would be worth confirming any other implications on other important traits, such as robustness, longevity and welfare.

Testing the role of genetic background in parallel evolution using the comparative experimental evolution of antibiotic resistance.

Parallel evolution is the independent evolution of the same phenotype or genotype in response to the same selection pressure. There are examples of parallel molecular evolution across divergent genetic backgrounds, suggesting that genetic background may not play an important role in determining the outcome of adaptation. Here, we measure the influence of genetic background on phenotypic and molecular adaptation by combining experimental evolution with comparative analysis. We selected for resistance to the antibiotic rifampicin in eight strains of bacteria from the genus Pseudomonas using a short term selection experiment. Adaptation occurred by 47 mutations at conserved sites in rpoB, the target of rifampicin, and due to the high diversity of possible mutations the probability of within-strain parallel evolution was low. The probability of between-strain parallel evolution was only marginally lower, because different strains substituted similar rpoB mutations. In contrast, we found that more than 30% of the phenotypic variation in the growth rate of evolved clones was attributable to among-strain differences. Parallel molecular evolution across strains resulted in divergent phenotypic evolution because rpoB mutations had different effects on growth rate in different strains. This study shows that genetic divergence between strains constrains parallel phenotypic evolution, but had little detectable impact on the molecular basis of adaptation in this system.

On being the right size: increased body size is associated with reduced telomere length under natural conditions.

Evolution of body size is likely to involve trade-offs between body size, growth rate and longevity. Within species, larger body size is associated with faster growth and ageing, and reduced longevity, but the cellular processes driving these relationships are poorly understood. One mechanism that might play a key role in determining optimal body size is the relationship between body size and telomere dynamics. However, we know little about how telomere length is affected when selection for larger size is imposed in natural populations. We report here on the relationship between structural body size and telomere length in wild house sparrows at the beginning and end of a selection regime for larger parent size that was imposed for 4 years in an isolated population of house sparrows. A negative relationship between fledgling size and telomere length was present at the start of the selection; this was extended when fledgling size increased under the selection regime, demonstrating a persistent covariance between structural size and telomere length. Changes in telomere dynamics, either as a correlated trait or a consequence of larger size, could reduce potential longevity and the consequent trade-offs could thereby play an important role in the evolution of optimal body size.

Milk output and composition in mice divergently selected for basal metabolic rate.

From an evolutionary perspective, the high basal metabolic rate (BMR) of homeotherms is hypothesised to be a by-product of natural selection for effective parental care. We estimated daily milk output during two consecutive lactation bouts in mice divergently selected for high/low BMR and applied a cross-fostered design to control for potential differences in the between-line suckling abilities of nursed juveniles. Additionally, to remedy the potential limitation imposed by the ability of mother mice to dissipate excess heat, we exposed them to an ambient temperature of 17°C during the most energetically demanding second week of lactation. We found that the mice selected for high BMR produced significantly more milk in a 24 h period in both reproductive bouts. The milk samples obtained from the high BMR females had lower protein concentration and did not differ with respect to fat. However, the concentration of the primary milk carbohydrate ­ lactose ­ was higher. Although all the above between-line differences were statistically significant, their magnitude was too small to unambiguously ascribe them as stemming from a positive genetic correlation between the physiological traits underlying BMR and lactation performance. Nevertheless, our study lends such support at least at the level of phenotypic variation.

Effects of food restriction on voluntary wheel-running behavior and body mass in selectively bred High Runner lines of mice.

Food restriction can have profound effects on various aspects of behavior, physiology, and morphology. Such effects might be amplified in animals that are highly active, given that physical activity can represent a substantial fraction of the total daily energy budget. More specifically, some effects of food restriction could be associated with intrinsic, genetically based differences in the propensity or ability to perform physical activity. To address this possibility, we studied the effects of food restriction in four replicate lines of High Runner (HR) mice that have been selectively bred for high levels of voluntary wheel running. We hypothesized that HR mice would respond differently than mice from four non-selected Control (C) lines. Healthy adult females from generation 65 were housed individually with wheels and provided access to food and water ad libitum for experimental days 1-19 (Phase 1), which allowed mice to attain a plateau in daily running distances. Ad libitum food intake of each mouse was measured on days 20-22 (Phase 2). After this, each mouse experienced a 20 % food restriction for 7 days (days 24-30; Phase 3), and then a 40 % food restriction for 7 additional days (days 31-37; Phase 4). Mice were weighed on experimental days 1, 8, 9, 15, 20, and 23-37 and wheel-running activity was recorded continuously, in 1-minute bins, during the entire experiment. Repeated-measures ANOVA of daily wheel-running distance during Phases 2-4 indicated that HR mice always ran much more than C, with values being 3.29-fold higher during the ad libitum feeding trial, 3.58-fold higher with -20 % food, and 3.06-fold higher with -40 % food. Seven days of food restriction at -20 % did not significantly reduce wheel-running distance of either HR (-5.8 %, P = 0.0773) or C mice (-13.3 %, P = 0.2122). With 40 % restriction, HR mice showed a further decrease in daily wheel-running distance (P = 0.0797 vs. values at 20 % restriction), whereas C mice did not (P = 0.4068 vs. values at 20 % restriction) and recovered to levels similar to those on ad libitum food (P = 0.3634). For HR mice, daily running distances averaged 11.4 % lower at -40 % food versus baseline values (P = 0.0086), whereas for C mice no statistical difference existed (-4.8 %, P = 0.7004). Repeated-measures ANOVA of body mass during Phases 2-4 indicated a highly significant effect of food restriction (P = 0.0001), but no significant effect of linetype (P = 0.1764) and no interaction (P = 0.8524). Both HR and C mice had a significant reduction in body mass only when food rations were reduced by 40 % relative to ad libitum feeding, and even then the reductions averaged only -0.60 g for HR mice (-2.6 %) and -0.49 g (-2.0 %) for C mice. Overall, our results indicate a surprising insensitivity of body mass to food restriction in both high-activity (HR) and ordinary (C) mice, and also insensitivity of wheel running in the C lines of mice, thus calling for studies of compensatory mechanisms that allow this insensitivity.

Clinal variation in the temperature and photoperiodic control of reproductive diapause in Drosophila montana females.

Insect adaptation to climatic conditions at different latitudes has required changes in life-history traits linked with survival and reproduction. Several species, including Drosophila montana, show robust latitudinal variation in the critical day length (CDL), below which more than half of the emerging females enter reproductive diapause at a given temperature. Here we used a novel approach to find out whether D. montana also shows latitudinal variation in the critical temperature (CTemp), above which the photoperiodic regulation of diapause is disturbed so that the females develop ovaries in daylengths that are far below their CDL. We estimated CTemp for 53 strains from different latitudes on 3 continents after measuring their diapause proportions at a range of temperatures in 12 h daylength (for 29 of the strains also in continuous darkness). In 12 h daylength, CTemp increased towards high latitudes alongside an increase in CDL, and in 3 high-latitude strains diapause proportion exceeded 50% in all temperatures. In continuous darkness, the diapause proportion was above 50% in the lowest temperature(s) in only 9 strains, all of which came from high latitudes. In the second part of the study, we measured changes in CTemp and CDL in a selection experiment favouring reproduction in short daylength (photoperiodic selection) and by exercising selection for females that reproduce in LD12:12 at low temperature (photoperiodic and temperature selection). In both experiments selection induced parallel changes in CDL and CTemp, confirming correlations seen between these traits along latitudinal clines. Overall, our findings suggest that selection towards strong photoperiodic diapause and long CDL at high latitudes has decreased the dependency of D. montana diapause on environmental temperature. Accordingly, the prevalence and timing of the diapause of D. montana is likely to be less vulnerable to climate warming in high- than low-latitude populations.