High gene flow between populations of Macrotermes michaelseni (Isoptera, Termitidae)

Termite alates are thought to be poor active flyers, and this should lead to considerable genetic differentiation on small spatial scales. However, using four microsatellite loci for the termite Macrotermes michaelseni we found low values of genetic differentiation (F_ST) across a spatial scale of even more than 50 km. Genetic differentiation between populations increased with spatial distance up to 50 km. Furthermore, up to this distance, the scatter around the linear regression of genetic differentiation versus spatial distance increased with spatial distance. This suggests that across such spatial distances gene flow and genetic drift are of about equal importance, and near equilibrium. Using a regional F_ST as well as the distance between populations with non-significant F_ST-values (up to 25 km), gene flow is sufficiently high so that populations may be regarded as panmictic on spatial scales of 25 to 50 km. The apparent contradiction between dispersal distances observed in the field and estimates of gene flow from genetic markers may be due to the masses of swarming alates. Assuming a leptokurtic distribution of dispersal distances, atleast some alates are expected to travel considerable distances, most likely by passive drift. Received 25 January 2005; revised 11 April 2005; accepted 26 April 2005.     

SPATIAL POPULATION STRUCTURE IN THE WHIRLIGIG BEETLE DINEUTUS ASSIMILIS: EVOLUTIONARY INFERENCES BASED ON MITOCHONDRIAL DNA AND FIELD DATA

The spatial population structure of the pond-living water beetle Dineutus assimilis (Coleoptera: Gyrinidae) was investigated through a field study of population dynamics and dispersal, with a concurrent assessment of the spatial distribution of mitochondrial DNA (mtDNA) restriction-fragment-length polymorphism (RFLP). A comprehensive 2-yr survey within a 60-km² study area revealed pronounced fluctuations in local abundances, including extinctions and colonizations. The recapture of marked individuals showed that dispersal among ponds is frequent in both males and females and connects populations on a large geographic scale (maximum observed flight distance: 20 km). The population structure of D. assimilis is thus characterized by both pronounced genetic drift and frequent gene flow. Together, these two forces generate a pattern of very local and transient genetic differentiation. Mitochondrial DNA samples collected within a few kilometers indicate highly significant spatial structure, if newly founded demes or those that experienced recent bottlenecks are included. These results based on four demes within the study area were placed into a regional context by further samples collected at distances of 100 km and 200 km. F_st estimates computed on increasing spatial scales were variable but showed no increasing trend. Thus, gene flow exerts a strong homogenizing force over a wide geographic range but is counteracted locally by genetic drift. These findings highlight the need to supplement estimates of F_st with additional data to arrive at valid interpretations of the genetic information. More generally, this study raises questions about how to capture the relevant features of dynamic, subdivided populations to understand their evolutionary dynamics.     

Linking seed dispersal and genetic structure of trees: a biogeographical approach

Aim Natural and human‐induced differences in frugivore assemblages can influence the seed dispersal distances of trees. An important issue in seed dispersal systems is to understand whether differences in seed dispersal distances also affect the genetic structure of mature trees. One possible approach to test for a relationship between seed dispersal and the genetic structure of mature trees is to compare the genetic structure of two closely related tree species between two biogeographical regions that differ in frugivore assemblages and seed dispersal distances. Previous studies on two Commiphora species revealed that Commiphora guillauminii in Madagascar has a much lower seed dispersal distance than Commiphora harveyi in South Africa. We tested whether the lower seed dispersal distance might have caused decreased gene flow, resulting in a stronger genetic structure in Madagascar than in South Africa. Location Madagascar and South Africa. Methods Using amplified fragment length polymorphism markers we investigated the genetic structure of 134 trees in Madagascar and 158 trees in South Africa at a local and a regional spatial scale. Results In concordance with our hypothesis, kinship analysis suggests that gene flow was restricted mostly to 3 km in Madagascar and to 30 km in South Africa. At the local spatial scale, the genetic differentiation among groups of trees within sample sites was marginally significantly higher in Madagascar (F_ST = 0.069) than in South Africa (F_ST = 0.021). However, at a regional spatial scale genetic differentiation was lower in Madagascar (F_ST = 0.053) than in South Africa (F_ST = 0.163). Main conclusions Our results show that lower seed dispersal distances of trees were linked to higher genetic differentiation of trees only at a local spatial scale. This suggests that seed dispersal affects the genetic population structure of trees at a local, but not at a regional, spatial scale.     

HIERARCHICAL GENETIC STRUCTURE AND GENE FLOW IN MACROGEOGRAPHIC POPULATIONS OF THE EASTERN TENT CATERPILLAR (MALACOSOMA AMERICANUM)

Genetic structure and inferred rates of gene flow in macrogeographic populations of the eastern tent caterpillar Malacosoma americanum were analyzed at two hierarchical scales: local demes and regional subpopulations. Wright's F-statistics were used to estimate population genetic structure using multilocus genotypic data generated electrophoretically. Estimated values of F_ST and the distribution of private alleles were then used to obtain indirect estimates of gene flow. We found modest, though significant, genetic structure at both spatial scales, a pattern consistent with high rates of gene flow over the large distances involved. Modest values obtained for Nei's genetic distance also suggested high levels of gene flow across the range of this species, although some gene-flow restriction resulting from isolation by distance was suggested by a positive regression of genetic distance on geographic distance. The observed homogeneity at enzyme loci across the range of M. americanum parallels the reported uniformity in morphology, suggesting a general absence of local genetic differentiation in this widely distributed species. The genetic homogeneity observed in this wide-ranging insect is discussed in terms of organism-specific environmental experience at different spatial scales. Some organisms occupying apparently heterogeneous environments may ameliorate unsuitable local conditions through microhabitat selection or behavioral modification of their microenvironment. This may be accomplished in M. americanum through group shelter construction and behavioral thermoregulation, closely tying thermoregulation to social biology in this species. If in this way the tent helps produce an effectively homogeneous environment for this species across its extensive range, this system may provide a unique example of how social behavior can influence the distribution of genetic variation in a population.     

Microgeographic population structure of green swordail fish: genetic differentiation despite abundant migration

Swordtails (Xiphophorus; Poeciliidae) have figured prominently in research on fish mating behaviours, sexual selection, and carcinogenesis, but their population structures and dispersal patterns have been relatively neglected. Using nine microsatellite loci, we estimated genetic differentiation in Xiphophorus helleri within and between adjacent streams in Belize. The genetic data were complemented by a tagging study of movement within one stream. In the absence of physical dispersal barriers (waterfalls), population structure followed an isolation by distance (IBD) pattern. Genetic differentiation (F_ST up to 0.07) was significant between and within creeks, despite high dispersal in the latter as judged by the tagging data. Such heterogeneity apparently was a result of genetic drift in local demes, due to small population sizes and highly skewed paternity. The IBD pattern was interrupted by waterfalls, boosting F_ST above 0.30 between adjacent samples across these barriers. Overall, our results are helpful in understanding the interplay of evolutionary forces and population dynamics in a small fish living in a changeable habitat.     

Gene flow in malaria vector Anopheles stephensi (Diptera: Culicidae) across the Aravalli Hills in North-west India

The population structure of An. stephensi in North-west India was studied to assess the impact of the Aravalli Hills, as a barrier to gene flow using microsatellite markers. Large and significant genetic differentiation was found along the sides of, as well as across, the Aravalli Hills as the mean F_ST and R_ST on west vs. east of the Aravalli Hills were 0.213, 0.112 and 0.179, 0.056, respectively. Similarly, across the hills, mean values of F_ST and R_ST were 0.100 and 0.094, respectively. Genetic diversity on both sides did not vary significantly. The F_ST values were more sensitive than R_ST values, indicating that genetic drift might have caused genetic differentiation between populations. A positive correlation (r = 0.0149 and 0.157, respective to F_ST and R_ST) was found between genetic differentiations and geographic distances irrespective of the hills. Low level of gene flow was found along both sides (Nm = 0.92 and 0.14; west vs. east of Aravalli Hills, respectively) as compared to across the Aravalli Hills (Nm = 2.25). It was found that the Aravalli Hills are not working as an effective barrier to gene flow for An. Stephensi, maybe because of the low average height and discontinuous hills, however, the distance is playing a major role for differentiation between populations due to active mode of dispersal of An. stephensi mosquitoes which have a short flight range. All this information should help draw the strategies for genetic control of mosquitoes using transgenic mosquitoes.     

A MULTISPECIES APPROACH TO THE ANALYSIS OF GENE FLOW IN MARINE SHORE FISHES

Ten species of marine shore fishes with a wide range of life-history strategies were collected from four areas in southern California, U.S.A., and Baja California, Mexico, and examined for patterns of genetic differentiation. Multilocus D and F_ST values (based on 32–42 presumptive gene loci in each species) were both negatively correlated with estimated dispersal capability. These results were robust to variations in the number and type of loci used in the analysis and are compatible with the hypothesis that levels of genetic differentiation in these shore fishes are determined primarily by gene flow and genetic drift. There is no a priori reason to expect the observed correlation to result from natural selection or historical factors. The findings thus suggest that populations of these shore fishes are in at least a quasi-equilibrium with respect to migration, mutation, and genetic drift. Present data were also used to compare estimates of mN_e obtained by three different methods. Estimates based on F_ST values calculated by the methods of Nei and Chesser (F_ST(N)) and Weir and Cockerham (F_ST(W)) were highly correlated, but F_ST(N) ≤ F_ST(W) for every species, leading to generally higher mN_e estimates for Nei and Chesser's method. Estimates of mN_e based on the frequency of private alleles (Slatkin, 1985a) were not as strongly correlated with dispersal capability as were F_ST and D values. A low incidence of private alleles in many species may be responsible for this relatively weak correlation and may limit the general usefulness of Slatkin's method. In spite of their sensitivity to natural selection, F_ST and D may be better indicators of relative gene flow levels for high gene flow species.     

Spatial patterns of genetic structure among populations of a stone‐cased caddis (Trichoptera: Tasimiidae) in south‐east Queensland,Australia

1. Gene flow and dispersal among populations of a stone‐cased caddis (Tasimiidae: Tasimia palpata) were estimated indirectly using a 460 bp region of the cytochrome oxidase I gene of mitochondrial DNA. 2. There was no significant differentiation at the largest spatial scale (between catchments) and no correlation between genetic distance and geographic distance. These results are consistent with widespread adult dispersal. 3. Conversely, significant genetic differentiation was detected at the smallest spatial scale examined (among reaches within streams). This pattern was primarily because of significant F_ST values in a single stream (Bundaroo Creek). 4. Bundaroo Creek also had the lowest mean number of haplotypes per population (n = 7) suggesting that a limited number of females may be responsible for recruitment at these sites. Significant F_ST's at the reach scale may be a result of this ‘patchy’ recruitment. However, additional evidence regarding the long‐range dispersal ability and fecundity of T. palpata females is needed to test this hypothesis fully.     

CORRELATION OF PAIRWISE GENETIC AND GEOGRAPHIC DISTANCE MEASURES: INFERRING THE RELATIVE INFLUENCES OF GENE FLOW AND DRIFT ON THE DISTRIBUTION OF GENETIC VARIABILITY

Attempts to relate estimates of regional F_ST to gene flow and drift via Wright's (1931) equation F_ST ≈ 1/ (4Nm + 1) are often inappropriate because most natural sets of populations probably are not at equilibrium (McCauley 1993), as assumed by the island model upon which the equation is based, or ineffective because the influences of gene flow and drift are confounded in the product Nm. Evaluations of the association between genetic (F_ST) and geographic distances separating all pairwise populations combinations in a region allows one to test for regional equilibrium, to evaluate the relative influences of gene flow and drift on population structure both within and between regions, and to visualize the behavior of the association across all degrees of geographic separation. Tests of the model using microsatellite data from 51 populations of eastern collared lizards (Crotaphytus collaris collaris) collected from four distinct geographical regions gave results highly consistent with predicted patterns of association based on regional differences in various historical and ecological factors that affect the amount of drift and gene flow. The model provides a prerequisite for and an alternative to regional F_ST analyses, which often simply assume regional equilibrium, thus potentially leading to erroneous and misleading inferences regarding regional population structure.     

Spatial Scales of Pollen and Seed-Mediated Gene Flow in Tropical Rain Forest Trees

Gene flow via seed and pollen is a primary determinant of genetic and species diversity in plant communities at different spatial scales. This paper reviews studies of gene flow and population genetic structure in tropical rain forest trees and places them in ecological and biogeographic context. Although much pollination is among nearest neighbors, an increasing number of genetic studies report pollination ranging from 0.5–14 km for canopy tree species, resulting in extensive breeding areas in disturbed and undisturbed rain forest. Direct genetic measures of seed dispersal are still rare; however, studies of fine scale spatial genetic structure (SGS) indicate that the bulk of effective seed dispersal occurs at local scales, and we found no difference in SGS (Sp statistic) between temperate (N?=?24 species) and tropical forest trees (N?=?15). Our analysis did find significantly higher genetic differentiation in tropical trees (F _ST?=?0.177; N?=?42) than in temperate forest trees (F _ST?=?0.116; N?=?82). This may be due to the fact that tropical trees experience low but significant rates of self-fertilization and bi-parental inbreeding, whereas half of the temperate tree species in our survey are wind pollinated and are more strictly allogamous. Genetic drift may also be more pronounced in tropical trees due to the low population densities of most species.     

GENETIC DIFFERENTIATION AMONG CHEWING LOUSE POPULATIONS (MALLOPHAGA: TRICHODECTIDAE) IN A POCKET GOPHER CONTACT ZONE (RODENTIA: GEOMYIDAE)

Genetic variation among populations of chewing lice (Geomydoecus actuosi) was examined in relation to chromosomal and electrophoretic variation among populations of their hosts (Thomomys bottae) at a contact zone. Louse demes were characterized by low levels of genetic heterozygosity (H? = 0.039) that may result from founder effects during primary infestation of hosts, compounded by seasonal reductions in louse population size. Louse populations sampled from different hosts showed high levels of genetic structuring both within and among host localities. Microgeographic differentiation of louse populations is high (mean F_ST = 0.092) suggesting that properties of this host–parasite system promote differentiation of louse populations living on different individual hosts. Among-population differentiation in lice (F_ST = 0.240) was similar to that measured among host populations (F_ST = 0.236), suggesting a close association between gene flow in pocket gophers and gene flow in their lice.     

Genetic structure is influenced by environmental barriers: empirical evidence from the common voleMicrotus arvalis populations

One of the major challenges in population biology is the identification of barriers to gene flow and/or secondary contacts between differentiated entities. The level of genetic differentiation among eight populations of the common voleMicrotus arvalis (Pallas, 1779) around the Biebrza Wetlands, NE Poland was examined by analyzing seven microsatellite loci for 140 voles and testing for the presence of barriers to gene flow. Overall population differentiation was moderate and significant (F _ST = 0.081,p < 0.001) and there was no correlation between geographical and genetic distances among populations. We found a relatively high level of genetic variability within the populations studied. This could be explained by male bias in dispersal, a phenomenon recently found inM. arvalis. Patterns of genetic structure visualized in synthetic genetic maps showed clear gradients along a southeast-northwest axis across the study area, as well as the presence of a potential barrier to dispersal. The position of a barrier to gene flow identified using Monmonier’s maximum difference algorithm likely corresponds to humid habitats of the Biebrza Wetlands. These results suggest that the presence of environmental barriers to gene flow and drift may be responsible for the observed spatial genetic structure ofM. arvalis in the Biebrza Valley. Institute of Biology, University of Białystok, OEwierkowa 20 B, 15-950 Białystok, Poland,     

Genetic Diversity and Population Structure of Sitodiplosis mosellana in Northern China

The wheat midge, Sitodiplosis mosellana, is an important pest in Northern China. We tested the hypothesis that the population structure of this species arises during a range expansion over the past 30 years. This study used microsatellite and mitochondrial loci to conduct population genetic analysis of S. mosellana across its distribution range in China. We found strong genetic structure among the 16 studied populations, including two genetically distinct groups (the eastern and western groups), broadly consistent with the geography and habitat fragmentation. These results underline the importance of natural barriers in impeding dispersal and gene flow of S. mosellana populations. Low to moderate genetic diversity among the populations and moderate genetic differentiation (F _ST = 0.117) between the two groups were also found. The populations in the western group had lower genetic diversity, higher genetic differentiation and lower gene flow (F _ST = 0.116, Nm = 1.89) than those in the eastern group (F _ST = 0.049, Nm = 4.91). Genetic distance between populations was positively and significantly correlated with geographic distance (r = 0.56, P<0.001). The population history of this species provided no evidence for population expansion or bottlenecks in any of these populations. Our data suggest that the distribution of genetic diversity, genetic differentiation and population structure of S. mosellana have resulted from a historical event, reflecting its adaptation to diverse habitats and forming two different gene pools. These results may be the outcome of a combination of restricted gene flow due to geographical and environmental factors, population history, random processes of genetic drift and individual dispersal patterns. Given the current risk status of this species in China, this study can offer useful information for forecasting outbreaks and designing effective pest management programs.     

ESTIMATES OF INTRAGAMETOPHYTIC SELFING AND INTERPOPULATIONAL GENE FLOW IN HOMOSPOROUS FERNS

Relatively little information is available on mating systems and interpopulational gene flow in species of homosporous pteridophytes. Because of the proximity of antheridia and archegonia on the same thallus, it has long been maintained that intragametophytic selling is the predominant mode of reproduction in natural populations of homosporous ferns and other homosporous plants. Furthermore, quantitative estimates of interpopulational gene flow via spore dispersal are lacking. In this paper, we examine five species of homosporous ferns (Botrychium virginianum, Polystichum munitum, P. imbricans, Blechnum spicant, and Dryopteris expansa) and present estimates of 1) rates of intragametophytic selling, 2) levels of interpopulational gene flow, and 3) interpopulational genetic differentiation (F_ST). Our data demonstrate that mating systems vary among species of ferns, just as they do among species of seed plants. The data also suggest that levels of interpopulational gene flow are generally high. The F_ST values indicate little genetic divergence among populations for all species except Dryopteris expansa, which exhibits significant levels of interpopulational genetic differentiation. Patterns of genetic diversity in the five species examined are related to the mating system and rate of interpopulational gene flow in each species. The F_ST values for all species except Botrychium virginianum are in close agreement with those predicted for an island model of population structure.     

Gene flow rates in Yugoslavian populations of the smooth newt Triturus vulgaris

Allozymic variation in 22 loci in several Yugoslavian populations of four subspecies of the smooth newt Triturus vulgaris, has been analyzed. The frequency of private alleles and the coefficient of genetic differentiation, F_ST, give very different indirect estimates of the effective number of migrants per generation, Nm. However, such Nm estimates, in most cases higher than 1, imply that gene flow between populations is large enough as to prevent differentiation by random drift. In the case of T.v. vulgaris, of which sixteen populations amply distributed through Yugoslavia were sampled, there is evidence that frequent extinction and recolonization processes might be responsible for the observed genetic structure. This conclusion has been reached after testing the correlation between genetic, environmental and geographical matrices.     

Evidence for adaptive phenotypic differentiation in Baltic Sea sticklebacks

The evidence for adaptive phenotypic differentiation in mobile marine species remains scarce, partly due to the difficulty of obtaining quantitative genetic data to demonstrate the genetic basis of the observed phenotypic differentiation. Using a combination of phenotypic and molecular genetic approaches, we elucidated the relative roles of natural selection and genetic drift in explaining lateral plate number differentiation in threespine sticklebacks (Gasterosteus aculeatus) across the entire Baltic Sea basin (approximately 392 000 km²). We found that phenotypic differentiation (P_ST = 0.213) in plate number exceeded that in neutral markers (F_ST = 0.008), suggesting an adaptive basis for the observed differentiation. Because a close correspondence was found between plate phenotype and genotype at a quantitative trait loci (QTL; STN381) tightly linked to the gene (Ectodysplasin) underlying plate variation, the evidence for adaptive differentiation was confirmed by comparison of F_ST at the QTL (F_STQ = 0.089) with F_ST at neutral marker loci. Hence, the results provide a comprehensive demonstration of adaptive phenotypic differentiation in a high‐gene‐flow marine environment with direct, rather than inferred, verification for the genetic basis of this differentiation. In general, the results illustrate the utility of P_ST–F_ST–F_STQ comparisons in uncovering footprints of natural selection and evolution and add to the growing evidence for adaptive genetic differentiation in high‐gene‐flow marine environments, including that of the relatively young Baltic Sea.     

Contrasting patterns of morphological and neutral genetic divergence among geographically proximate populations of sockeye salmon Oncorhynchus nerka in Lake Aleknagik,Alaska

Levels of differentiation in morphological traits (age at maturity, body length at age, egg mass and body depth) and spawning time were examined in sockeye salmon Oncorhynchus nerka from three geographically proximate but physically distinct creeks in Lake Aleknagik, Alaska. Happy Creek fish had significantly greater values for most measured morphological traits, and Eagle Creek fish spawned significantly later than fish in the other creeks. Phenotypic differentiation between creeks, measured as P_ST, was then compared with microsatellite marker differentiation between creeks, measured as F_ST. No correlations were apparent between P_ST and F_ST values, and P_ST values were generally significantly larger than zero (P_ST= 0·0018–0·31) whereas F_ST values were not (F_ST=?0·0004 to 0·0016). The insignificant pair‐wise F_ST values between creek samples indicated that gene flow occurs between creeks, assuming the creek populations have reached migration–drift equilibrium. However, the strong homing behaviour of sockeye salmon precludes a scenario in which fish from the three creeks constitute a single population that segregates by body size. Rather, significant phenotypic differentiation suggests that strong divergent selection occurs on the phenotypic traits despite the homogenizing effects of gene flow.     

Genetic diversity in fragmented populations of the critically endangered spider orchid Caladenia huegelii: implications for conservation

The Orchidaceae is characterised by a diverse range of life histories, reproductive strategies and geographic distribution, reflected in a variety of patterns in the population genetic structure of different species. In this study, the genetic diversity and structure was assessed within and among remnant populations of the critically endangered sexually deceptive orchid, Caladenia huegelii. This species has experienced severe recent habitat loss in a landscape marked by ancient patterns of population fragmentation within the Southwest Australian Floristic Region, a global biodiversity hotspot. Using seven polymorphic microsatellite loci, high levels of within-population diversity (mean alleles/locus = 6.73; mean H _E = 0.690), weak genetic structuring among 13 remnant populations (F _ST = 0.047) and a consistent deficit of heterozygotes from Hardy–Weinberg expectation were found across all populations (mean F _IS = 0.22). Positive inbreeding coefficients are most likely due to Wahlund effects and/or inbreeding effects from highly correlated paternity and typically low fruit set. Indirect estimates of gene flow (Nm = 5.09 using F _ST; Nm = 3.12 using the private alleles method) among populations reflects a historical capacity for gene flow through long distance pollen dispersal by sexually deceived wasp pollinators and/or long range dispersal of dust-like orchid seed. However, current levels of gene flow may be impacted by habitat destruction, fragmentation and reduced population size. A genetically divergent population was identified, which should be a high priority for conservation managers. Very weak genetic differentiation indicates that the movement and mixing of seeds from different populations for reintroduction programs should result in minimal negative genetic effects.     

Genetic Diversity and Population Structure of Alnus hirsuta (Betulaceae) in Korea

Alnus hirsuta in Korea was measured to estimate the amount and pattern of genetic diversity and population structure. The mean genetic diversity within populations was 0.166. Korean alder populations have slightly high levels of genetic diversity compared to those of two Canadian alder species. The genetic differentiation among populations accounted for 9% of the total variation. The rate of gene flow was estimated high (Nm=2.63). Analysis of inbreeding coefficient, calculated for all polymorphic loci in each population, showed a substantial heterozygote deficiency relative to Hardy-Weinberg expectations. The mean G _ST value of A. hirsuta in Korea was 0.087. The low value of G _ST in this species, reflecting little spatial genetic differentiation, may indicate extensive gene flow. A relationship between the mean heterozygosity and annual rainfall showed a positive relationship (r ²=0.54, F=4.67). Received 8 August 1998/ Accepted in revised form 7 July 1999     

Walk,swim or fly? Locomotor mode predicts genetic differentiation in vertebrates

Limited dispersal is commonly used to explain differences in diversification rates. An obvious but unexplored factor affecting dispersal is the mode of locomotion used by animals. Whether individuals walk, swim or fly can dictate the type and severity of geographical barriers to dispersal, and determine the general range over which genetic differentiation might occur. We collated information on locomotion mode and genetic differentiation (F_ST) among vertebrate populations from over 400 published articles. Our results showed that vertebrate species that walk tend to have higher genetic differentiation among populations than species that swim or fly. Within species that swim, vertebrates in freshwater systems have higher genetic differentiation than those in marine systems, which is consistent with the higher number of species in freshwater environments. These results show that locomotion mode can impact gene flow among populations, supporting at a broad‐scale what has previously been proposed at smaller taxonomical scales.