What is genetic differentiation,and how should we measure it—GST,D, neither or both?

Estimates of the fixation index, F_ST, have been used as measures of population differentiation for many decades. However, there have been persistent voices in the literature suggesting that these statistics do not measure true differentiation. In particular, the statistics Nei's G_ST and Wier and Cockerham's θ have been criticized for being ‘constrained’ to not equal one in some situations that seem to represent maximal differentiation. Here, we address the issue of how to evaluate exactly how much information a particular statistic contains about the process of differentiation. This criterion can be used to counter most concerns about the performance of G_ST (and related statistics), while also being reconciled with the insights of those who have proposed alternative measures of differentiation. In particular, the likelihood‐based framework that we put forward can justify the use of G_ST as an effective measure of differentiation, but also shows that in some situations G_ST is insufficient on its own and needs supplementing by another measure such as Jost's D or Hedrick's G′_ST. This approach will become increasingly important in the future, as greater emphasis is placed on analysing large data sets.     

Population differentiation of the African cyprinid Barbus neumayeri across dissolved oxygen regimes

Population level response to hypoxia has become an issue of global significance because of increased frequency and intensity of hypoxic events worldwide, and the potential for global warming to exacerbate hypoxic stress. In this study, we sequenced two nuclear intronic regions and a single mitochondrial region across seven populations of the African cyprinid, Barbus neumayeri from two river drainages in Uganda: the Rwembaita Swamp‐Njuguta River System and the Dura River. We then examined two indices of population structure, G_ST and Jost's D, to detect links between oxygen availability and genetic variation and to determine if population genetic structure was associated with (i) dissolved oxygen regime (hypoxia or normoxia), (ii) geographical distance, or (iii) a combination of dissolved oxygen regime and geographical distance. Our results indicate that over a large scale (between drainages), geographical distance significantly affects the genetic structure of populations. However, within a single drainage, dissolved oxygen regime plays a key role in determining the genetic structure of populations. Within the Rwembaita‐Njuguta system, gene flow was high between locations of similar oxygen regimes, but low between areas characterized by divergent oxygen regimes. Interestingly, G_ST analyses appear to yield less realistic measures of population structure than Jost's D, suggesting that caution must be taken when interpreting and comparing the results from different studies. These results support the idea that aquatic dissolved oxygen can act as a selective force limiting gene flow among populations of aquatic species and therefore should be considered when implementing conservation plans and assessing environmental impact of human activities.     

Multilocus genetic analyses and spatial modeling reveal complex population structure and history in a widespread resident North American passerine (Perisoreus canadensis)

An increasing body of studies of widely distributed, high latitude species shows a variety of refugial locations and population genetic patterns. We examined the effects of glaciations and dispersal barriers on the population genetic patterns of a widely distributed, high latitude, resident corvid, the gray jay (Perisoreus canadensis), using the highly variable mitochondrial DNA (mtDNA) control region and microsatellite markers combined with species distribution modeling. We sequenced 914 bp of mtDNA control region for 375 individuals from 37 populations and screened seven loci for 402 individuals from 27 populations across the gray jay range. We used species distribution modeling and a range of phylogeographic analyses (haplotype diversity, Φ_ST, SAMOVA, F_ST, Bayesian clustering analyses) to examine evolutionary history and population genetic structure. MtDNA and microsatellite markers revealed significant genetic differentiation among populations with high concordance between markers. Paleodistribution models supported at least five potential areas of suitable gray jay habitat during the last glacial maximum and revealed distributions similar to the gray jay's contemporary during the last interglacial. Colonization from and prolonged isolation in multiple refugia is evident. Historical climatic fluctuations, the presence of multiple dispersal barriers, and highly restricted gene flow appear to be responsible for strong genetic diversification and differentiation in gray jays.     

Development of genetic diversity,differentiation and structure over 500 years in four ponderosa pine populations

Population history plays an important role in shaping contemporary levels of genetic variation and geographic structure. This is especially true in small, isolated range‐margin populations, where effects of inbreeding, genetic drift and gene flow may be more pronounced than in large continuous populations. Effects of landscape fragmentation and isolation distance may have implications for persistence of range‐margin populations if they are demographic sinks. We studied four small, disjunct populations of ponderosa pine over a 500‐year period. We coupled demographic data obtained through dendroecological methods with microsatellite data to discern how and when contemporary levels of allelic diversity, among and within‐population levels of differentiation, and geographic structure, arose. Alleles accumulated rapidly following initial colonization, demonstrating proportionally high levels of gene flow into the populations. At population sizes of approximately 100 individuals, allele accumulation saturated. Levels of genetic differentiation among populations (F_ST and Jost's D_est) and diversity within populations (F_IS) remained stable through time. There was no evidence of geographic genetic structure at any time in the populations' history. Proportionally, high gene flow in the early stages of population growth resulted in rapid accumulation of alleles and quickly created relatively homogenous genetic patterns among populations. Our study demonstrates that contemporary levels of genetic diversity were formed quickly and early in population development. How contemporary genetic diversity accumulates over time is a key facet of understanding population growth and development. This is especially relevant given the extent and speed at which species ranges are predicted to shift in the coming century.     

Global patterns of population genetic differentiation in seed plants

Evaluating the factors that drive patterns of population differentiation in plants is critical for understanding several biological processes such as local adaptation and incipient speciation. Previous studies have given conflicting results regarding the significance of pollination mode, seed dispersal mode, mating system, growth form and latitudinal region in shaping patterns of genetic structure, as estimated by F_ST values, and no study to date has tested their relative importance together across a broad scale. Here, we assembled a 337‐species data set for seed plants from publications with data on F_ST from nuclear markers and species traits, including variables pertaining to the sampling scheme of each study. We used species traits, while accounting for sampling variables, to perform phylogenetic multiple regressions. Results demonstrated that F_ST values were higher for tropical, mixed‐mating, non‐woody species pollinated by small insects, indicating greater population differentiation, and lower for temperate, outcrossing trees pollinated by wind. Among the factors we tested, latitudinal region explained the largest portion of variance, followed by pollination mode, mating system and growth form, while seed dispersal mode did not significantly relate to F_ST. Our analyses provide the most robust and comprehensive evaluation to date of the main ecological factors predicted to drive population differentiation in seed plants, with important implications for understanding the basis of their genetic divergence. Our study supports previous findings showing greater population differentiation in tropical regions and is the first that we are aware of to robustly demonstrate greater population differentiation in species pollinated by small insects.     

Does GST underestimate genetic differentiation from marker data?

The widely applied genetic differentiation statistics F_ST and G_ST have recently been criticized for underestimating differentiation when applied to highly polymorphic markers such as microsatellites. New statistics claimed to be unaffected by marker polymorphisms have been proposed and advocated to replace the traditional F_ST and G_ST. This study shows that G_ST gives accurate estimates and underestimates of differentiation when demographic factors are more and less important than mutations, respectively. In the former case, all markers, regardless of diversity (H_S), have the same G_ST value in expectation and thus give replicated estimates of differentiation. In the latter case, markers of higher H_S have lower G_ST values, resulting in a negative, roughly linear correlation between G_ST and H_S across loci. I propose that the correlation coefficient between G_ST and H_S across loci, r_GH, can be used to distinguish the two cases and to detect mutational effects on G_ST. A highly negative and significant r_GH, when coupled with highly variable G_ST values among loci, would reveal that marker G_ST values are affected substantially by mutations and marker diversity, underestimate population differentiation, and are not comparable among studies, species and markers. Simulated and empirical data sets are used to check the power and statistical behaviour, and to demonstrate the usefulness of the correlation analysis.     

Spatio‐temporal Genetic Structure of a Tropical Bee Species Suggests High Dispersal Over a Fragmented Landscape

Habitat destruction threatens biodiversity by reducing the amount of available resources and connectivity among geographic areas. For organisms living in fragmented habitats, population persistence may depend on dispersal, which maintains gene flow among fragments and can prevent inbreeding within them. It is centrally important to understand patterns of dispersal for bees living in fragmented areas given the importance of pollination systems and recently documented declines in bee populations. We used population and landscape genetic techniques to characterize patterns of dispersal over a large fragmented area in southern Costa Rica for the orchid bee species Euglossa championi. First, we estimated levels of genetic differentiation among forest fragments as ?_PT, an analog to the traditional summary statistic F_ST, as well as two statistics that may more adequately represent levels of differentiation, G’_ST and D_est. Second, we used a Bayesian approach to determine the number and composition of genetic groups in our sample. Third we investigated how genetic differentiation changes with distance. Fourth, we determined the extent to which deforested areas restrict dispersal. Finally, we estimated the extent to which there were temporal differences in allele frequencies within the same forest fragments. Within years we found low levels of differentiation even over 80 km, and no effect of land use type on level of genetic differentiation. However, we found significant genetic differentiation between years. Taken together our results suggest that there are high levels of gene flow over this geographic area, and that individuals show low site fidelity over time.     

Life history,larval dispersal,and connectivity in coral reef fish among the Scattered Islands of the Mozambique Channel

The Western Indian Ocean harbors one of the world’s most diverse marine biota yet is threatened by exploitation with few conservation measures in place. Primary candidates for conservation in the region are the Scattered Islands (Îles Éparses), a group of relatively pristine and uninhabited islands in the Mozambique Channel. However, while optimal conservation strategies depend on the degree of population connectivity among spatially isolated habitats, very few studies have been conducted in the area. Here, we use highly variable microsatellite markers from two damselfishes (Amphiprion akallopisos and Dascyllus trimaculatus) with differing life history traits [pelagic larval duration (PLD), adult habitat] to compare genetic structure and connectivity among these islands using classic population structure indices as well as Bayesian clustering methods. All classical fixation indexes F _ST, R _ST, G′_ST, and Jost’s D show stronger genetic differentiation among islands for A. akallopisos compared to D. trimaculatus, consistent with the former species’ shorter PLD and stronger adult site attachment, which may restrict larval dispersal potential. In agreement with these results, the Bayesian analysis revealed clear genetic differentiation among the islands in A. akallopisos, separating the southern group (Bassas da India and Europa) from the center (Juan de Nova) and northern (Îles Glorieuses) islands, but not for D. trimaculatus. Local oceanographic patterns such as eddies that occur along the Mozambique Channel appear to parallel the results reported for A. akallopisos, but such features seem to have little effect on the genetic differentiation of D. trimaculatus. The contrasting patterns of genetic differentiation between species within the same family highlight the importance of accounting for diverse life history traits when assessing community-wide connectivity, an increasingly common consideration in conservation planning.

     

Noninvasive sampling reveals population genetic structure in the Royle’s pika,Ochotona roylei,in the western Himalaya

Understanding population genetic structure of climate‐sensitive herbivore species is important as it provides useful insights on how shifts in environmental conditions can alter their distribution and abundance. Herbivore responses to the environment can have a strong indirect cascading effect on community structure. This is particularly important for Royle's pika (Lagomorpha: Ochotona roylei), a herbivorous talus‐dwelling species in alpine ecosystem, which forms a major prey base for many carnivores in the Himalayan arc. In this study, we used seven polymorphic microsatellite loci to detect evidence for recent changes in genetic diversity and population structure in Royle's pika across five locations sampled between 8 and 160 km apart in the western Himalaya. Using four clustering approaches, we found the presence of significant contemporary genetic structure in Royle's pika populations. The detected genetic structure could be primarily attributed to the landscape features in alpine habitat (e.g., wide lowland valleys, rivers) that may act as semipermeable barriers to gene flow and distribution of food plants, which are key determinants in spatial distribution of herbivores. Pika showed low inbreeding coefficients (F_IS) and a high level of pairwise relatedness for individuals within 1 km suggesting low dispersal abilities of talus‐dwelling pikas. We have found evidence of a recent population bottleneck, possibly due to effects of environmental disturbances (e.g., snow melting patterns or thermal stress). Our results reveal significant evidence of isolation by distance in genetic differentiation (F_ST range = 0.04–0.19). This is the first population genetics study on Royle's pika, which helps to address evolutionary consequences of climate change which are expected to significantly affect the distribution and population dynamics in this talus‐dwelling species.     

Hitchhiking the high seas: Global genomics of rafting crabs

Population differentiation and diversification depend in large part on the ability and propensity of organisms to successfully disperse. However, our understanding of these processes in organisms with high dispersal ability is biased by the limited genetic resolution offered by traditional genotypic markers. Many neustonic animals disperse not only as pelagic larvae, but also as juveniles and adults while drifting or rafting at the surface of the open ocean. In theory, the heightened dispersal ability of these animals should limit opportunities for species diversification and population differentiation. To test these predictions, we used next‐generation sequencing of genomewide restriction‐site‐associated DNA tags (RADseq) and traditional mitochondrial DNA sequencing, to investigate the species‐level relationships and global population structure of Planes crabs collected from oceanic flotsam and sea turtles. Our results indicate that species diversity in this clade is low—likely three closely related species—with no evidence of cryptic or undescribed species. Moreover, our results indicate weak population differentiation among widely separated aggregations with genetic indices showing only subtle genetic discontinuities across all oceans of the world (RADseq F_ST = 0.08–0.16). The results of this study provide unprecedented resolution of the systematics and global biogeography of this group and contribute valuable information to our understanding of how theoretical dispersal potential relates to actual population differentiation and diversification among marine organisms. Moreover, these results demonstrate the limitations of single gene analyses and the value of genomic‐level resolution for estimating contemporary population structure in organisms with large, highly connected populations.     

Integrating three comprehensive data sets shows that mitochondrial DNA variation is linked to species traits and paleogeographic events in European butterflies

Understanding the dynamics of biodiversity, including the spatial distribution of genetic diversity, is critical for predicting responses to environmental changes, as well as for effective conservation measures. This task requires tracking changes in biodiversity at large spatial scales and correlating with species functional traits. We provide three comprehensive resources to understand the determinants for mitochondrial DNA differentiation represented by (a) 15,609 COI sequences and (b) 14 traits belonging to 307 butterfly species occurring in Western‐Central Europe and (c) the first multi‐locus phylogenetic tree of all European butterfly species. By applying phylogenetic regressions we show that mitochondrial DNA spatial differentiation (as measured with G_ST, G′_ST, D and D_ST) is negatively correlated with species traits determining dispersal capability and colonization ability. Thanks to the high spatial resolution of the COI data, we also provide the first zoogeographic regionalization maps based on intraspecific genetic variation. The overall pattern obtained by averaging the spatial differentiation of all Western‐Central European butterflies shows that the paradigm of long‐term glacial isolation followed by rapid pulses of post‐glacial expansion has been a pervasive phenomenon in European butterflies. The results and the extensive data sets we provide here constitute the basis for genetically‐informed conservation plans for a charismatic group in a continent where flying insects are under alarming decline.     

Effect of Yangtze River on population genetic structure of the relict plant Parrotia subaequalis in eastern China

Parrotia subaequalis (Hamamelidaceae) is a Tertiary relic species endemic in eastern China. We used inter‐simple sequence repeat (ISSR) markers to access genetic diversity and population genetic structure in natural five populations of P. subaequalis. The levels of genetic diversity were higher at species level (H = 0.2031) but lower at population level (H = 0.1096). The higher genetic diversity at species levels might be attributed to the accumulation of distinctive genotypes which adapted to the different habitats after Quaternary glaciations. Meanwhile, founder effects on the early stage, and subsequent bottleneck of population regeneration due to its biological characteristics, environmental features, and human activities, seemed to explain the low population levels of genetic diversity. The hierarchical AMOVA revealed high levels (42.60%) of among‐population genetic differentiation, which was in congruence with the high levels of Nei's genetic differentiation index (G_ST = 0.4629) and limited gene flow (N_m = 0.5801) among the studied populations. Mantel test showed a significant isolation‐by‐distance, indicating that geographic isolation has a significant effect on genetic structure in this species. Unweighted pair‐group method with arithmetic average clustering, PCoA, and Bayesian analyses uniformly recovered groups that matched the geographical distribution of this species. In particular, our results suggest that Yangtze River has served as a natural barrier to gene flow between populations occurred on both riversides. Concerning the management of P. subaequalis, the high genetic differentiation among populations indicates that preserving all five natural populations in situ and collecting enough individuals from these populations for ex situ conservation are necessary.     

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.     

Partitioning heterozygosity in subdivided populations: Some misuses of Nei's decomposition and an alternative probabilistic approach

Nei's decomposition of total expected heterozygosity in subdivided populations into within‐ and between‐subpopulation components, H_S and D_ST, respectively, is a classical tool in the conservation and management of genetic resources. Reviewing why this is not a decomposition into independent terms of within‐ and between‐subpopulation gene diversity, we illustrate how this approach can be misleading because it overemphasizes the within‐subpopulation component compared to Jost's nonadditive decomposition based on gene diversity indices. Using probabilistic partitioning of the total expected heterozygosity into independent within‐ and between‐subpopulation contributions, we show that the contribution of the within‐subpopulation expected heterozygosity to the total expected heterozygosity is not H_S, as suggested by Nei's decomposition, but H_S/s, with s being the number of subpopulations. Finally, we compare three possible approaches of decomposing total heterozygosity in subdivided populations (i.e., Nei's decomposition, Jost's approach, and probabilistic partitioning) with regard to independence between terms and sensitivity to unequal subpopulation sizes. For the conservation and management of genetic resources, we recommend using probabilistic partitioning and Jost's differentiation parameter rather than Nei's decomposition.     

Parallel speciation or long‐distance dispersal? Lessons from seaweeds (Fucus) in the Baltic Sea

Parallel evolution has been invoked as a forceful mechanism of ecotype and species formation in many animal taxa. However, parallelism may be difficult to separate from recently monophyletically diverged species that are likely to show complex genetic relationships as a result of considerable shared ancestral variation and secondary hybridization in local areas. Thus, species' degrees of reproductive isolation, barriers to dispersal and, in particular, limited capacities for long‐distance dispersal will affect demographical structures underlying mechanisms of divergent evolution. Here, we used nine microsatellite DNA markers to study intra‐ and interspecific genetic diversity of two recently diverged species of brown macroalgae, Fucus radicans (L. Bergström & L. Kautsky) and F. vesiculosus (Linnaeus), in the Baltic Sea. We further performed biophysical modelling to identify likely connectivity patterns influencing the species' genetic structures. For each species, we found intraspecific contrasting patterns of clonality incidence and population structure. In addition, strong genetic differentiation between the two species within each locality supported the existence of two distinct evolutionary lineages (F_ST = 0.15–0.41). However, overall genetic clustering analyses across both species' populations revealed that all populations from one region (Estonia) were more genetically similar to each other than to their own taxon from the other two regions (Sweden and Finland). Our data support a hypothesis of parallel speciation. Alternatively, Estonia may be the ancestral source of both species, but is presently isolated by oceanographic barriers to dispersal. Thus, a limited gene flow in combination with genetic drift could have shaped the seemingly parallel structure.     

mmod: an R library for the calculation of population differentiation statistics

mmod is a library for the R programming language that allows the calculation of the population differentiation measures D_est, G″_ST and φ′_ST. R provides a powerful environment in which to conduct and record population genetic analyses but, at present, no R libraries provide functions for the calculation of these statistics from standard population genetic files. In addition to the calculation of differentiation measures, mmod can produce parametric bootstrap and jackknife samples of data sets for further analysis. By integrating with and complimenting the existing libraries adegenet and pegas , mmod extends the power of R as a population genetic platform.     

Population structure of the invasive forest pathogen Hymenoscyphus pseudoalbidus

Understanding the genetic diversity and structure of invasive pathogens in source and in introduced areas is crucial to the revelation of hidden biological features of an organism, to the reconstruction of the course of invasions and to the establishment of effective control measures. Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) is an invasive and highly destructive fungal pathogen found on common ash Fraxinus excelsior in Europe and is native to East Asia. To gain insights into its dispersal mechanisms and history of invasion, we used microsatellite markers and characterized the genetic structure and diversity of H. pseudoalbidus populations at three spatial levels: (i) between Europe and Japan, (ii) in Europe and (iii) at the epidemic's front in Switzerland. Phylogenetic and network analysis demonstrated that individuals from both regions are conspecific. However, populations from Japan harboured a higher genetic diversity and were genetically differentiated from European ones. No evident population structure was found among the 1208 European strains using Bayesian and multivariate clustering analysis. Only the distribution of genetic diversity in space, pairwise population differentiation (G_ST) and the spatial analysis of principal components revealed a faint geographical pattern around Europe. A significant allele deficiency in most European populations pointed to a recent genetic bottleneck, whereas no pattern of isolation by distance was found. Our data suggest that H. pseudoalbidus was introduced just once by at least two individuals. The potential source region of H. pseudoalbidus is vast, and further investigations are required for a more accurate localization of the source population.     

Habitat fragmentation influences genetic diversity and differentiation: Fine‐scale population structure of Cercis canadensis (eastern redbud)

Forest fragmentation may negatively affect plants through reduced genetic diversity and increased population structure due to habitat isolation, decreased population size, and disturbance of pollen‐seed dispersal mechanisms. However, in the case of tree species, effective pollen‐seed dispersal, mating system, and ecological dynamics may help the species overcome the negative effect of forest fragmentation. A fine‐scale population genetics study can shed light on the postfragmentation genetic diversity and structure of a species. Here, we present the genetic diversity and population structure of Cercis canadensis L. (eastern redbud) wild populations on a fine scale within fragmented areas centered around the borders of Georgia–Tennessee, USA. We hypothesized high genetic diversity among the collections of C. canadensis distributed across smaller geographical ranges. Fifteen microsatellite loci were used to genotype 172 individuals from 18 unmanaged and naturally occurring collection sites. Our results indicated presence of population structure, overall high genetic diversity (H_E = 0.63, H_O = 0.34), and moderate genetic differentiation (F_ST = 0.14) among the collection sites. Two major genetic clusters within the smaller geographical distribution were revealed by STRUCTURE. Our data suggest that native C. canadensis populations in the fragmented area around the Georgia–Tennessee border were able to maintain high levels of genetic diversity, despite the presence of considerable spatial genetic structure. As habitat isolation may negatively affect gene flow of outcrossing species across time, consequences of habitat fragmentation should be regularly monitored for this and other forest species. This study also has important implications for habitat management efforts and future breeding programs.     

A clever solution to a vexing problem

F_ST (as well as related measures such as G_ST) has long been used both as a measure of the relative amount of genetic variation between populations and as an indicator of the amount of gene flow among populations. Unfortunately, F_ST and its clones are also sensitive to mutation, particularly when the mutation rate per locus is greater than the migration rate among populations. Relatively high mutation rates cause estimates of F_ST and G_ST to be much lower than researchers sometimes expect, when migration rates are low in the studied species. Several recent suggestions for dealing with this problem have been unsatisfactory for one reason or another, and no general solution exists (if we are not to abandon these otherwise useful measures of differentiation). In an important article in this issue, Jinliang Wang (2015) shows that it is possible to identify whether the genetic markers in a given study are likely to give estimates of F_ST that are strongly affected by mutation. The proposed test is simple and elegant, and with it, molecular ecologists can determine whether the F_ST from their makers can be depended on for further inference about their species’ genome and the demographic forces which shaped its patterns.     

Unexpected cryptic species diversity in the widespread coral Seriatopora hystrix masks spatial‐genetic patterns of connectivity

Mounting evidence of cryptic species in a wide range of taxa highlights the need for careful analyses of population genetic data sets to unravel within‐species diversity from potential interspecies relationships. Here, we use microsatellite loci and hierarchical clustering analysis to investigate cryptic diversity in sympatric and allopatric (separated by 450 km) populations of the widespread coral Seriatopora hystrix on the Great Barrier Reef. Structure analyses delimited unique genetic clusters that were confirmed by phylogenetic and extensive population‐level analyses. Each of four sympatric yet distinct genetic clusters detected within S. hystrix demonstrated greater genetic cohesion across regional scales than between genetic clusters within regions (<10 km). Moreover, the magnitude of genetic differentiation between different clusters (>0.620 G”_ST) was similar to the difference between S. hystrix clusters and the congener S. caliendrum (mean G”_ST 0.720). Multiple lines of evidence, including differences in habitat specificity, mitochondrial identity, Symbiodinium associations and morphology, corroborate the nuclear genetic evidence that these distinct clusters constitute different species. Hierarchical clustering analysis combined with more traditional population genetic methods provides a powerful approach for delimiting species and should be regularly applied to ensure that ecological and evolutionary patterns interpreted for single species are not confounded by the presence of cryptic species.