Among‐year variation in selection during early life stages and the genetic basis of fitness in Arabidopsis thaliana

Incomplete information regarding both selection regimes and the genetic basis of fitness limits our understanding of adaptive evolution. Among‐year variation in the genetic basis of fitness is rarely quantified, and estimates of selection are typically based on single components of fitness, thus potentially missing conflicting selection acting during other life‐history stages. Here, we examined among‐year variation in selection on a key life‐history trait and the genetic basis of fitness covering the whole life cycle in the annual plant Arabidopsis thaliana. We planted freshly matured seeds of >200 recombinant inbred lines (RILs) derived from a cross between two locally adapted populations (Italy and Sweden), and both parental genotypes at the native site of the Swedish population in three consecutive years. We quantified selection against the nonlocal Italian genotype, mapped quantitative trait loci (QTL) for fitness and its components, and quantified selection on timing of germination during different life stages. In all 3 years, the local Swedish genotype outperformed the nonlocal Italian genotype. However, both the contribution of early life stages to relative fitness, and the effects of fitness QTL varied among years. Timing of germination was under conflicting selection through seedling establishment vs. adult survival and fecundity, and both the direction and magnitude of net selection varied among years. Our results demonstrate that selection during early life stages and the genetic basis of fitness can vary markedly among years, emphasizing the need for multiyear studies considering the whole life cycle for a full understanding of natural selection and mechanisms maintaining local adaptation.     

Population differentiation in an annual legume: local adaptation

Abstract. Studies of many plants species have demonstrated adaptive genetic differentiation to local environmental conditions. Typically these studies are conducted to evaluate adaptation to contrasting environments. As a consequence, although local adaptation has been frequently demonstrated, we have little information as to the spatial scale of adaptive evolution. We evaluated adaptive differentiation between populations of the annual legume Chamaecrista fasciculata using a replicated common-garden design. Study sites were established in three field locations that are home to native populations of C. fasciculata . Each location was planted for two years with seed from the population native to the study site (home population) and populations located six distances (0.1-2000 km) from each site (transplanted populations). Seeds were planted into the study sites with minimum disturbance to determine the scale of local adaptation, as measured by a home-site fitness advantage, for five fitness components: germination, survival, vegetative biomass, fruit production, and the number of fruit produced per seed planted (an estimate of cumulative fitness). For all characters there was little evidence for local adaptation, except at the furthest spatial scales. Patterns of adaptive differentiation were fairly consistent in two of the three sites, but varied between years. Little genetic variation was expressed at the third site. These results, combined with previous estimates of limited gene flow, suggest that metapopulation processes and temporal environmental variation act together to reduce local adaptation, except over long distances.     

Local adaptation in European populations of Arabidopsis lyrata (Brassicaceae)

We studied local adaptation to contrasting environments using an organism that is emerging as a model for evolutionary plant biology-the outcrossing, perennial herb Arabidopsis lyrata subsp. petraea (Brassicaceae). With reciprocal transplant experiments, we found variation in cumulative fitness, indicating adaptive differentiation among populations. Nonlocal populations did not have significantly higher fitness than the local population. Experimental sites were located in Norway (alpine), Sweden (coastal), and Germany (continental). At all sites after one year, the local population had higher cumulative fitness, as quantified by survival combined with rosette area, than at least one of the nonlocal populations. At the Norwegian site, measurements were done for two additional years, and fitness differences persisted. The fitness components that contributed most to differences in cumulative fitness varied among sites. Relatively small rosette area combined with a large number of inflorescences produced by German plants may reflect differentiation in life history. The results of the current study demonstrate adaptive population differentiation in A. lyrata along a climatic gradient in Europe. The studied populations harbor considerable variation in several characters contributing to adaptive population differentiation. The wealth of genetic information available makes A. lyrata a highly attractive system also for examining the functional and genetic basis of local adaptation in plants.     

Strong genetic differentiation but not local adaptation toward the range limit of a coastal dune plant

All species have limited geographic distributions; but the ecological and evolutionary mechanisms causing range limits are largely unknown. That many species’ geographic range limits are coincident with niche limits suggests limited evolutionary potential of marginal populations to adapt to conditions experienced beyond the range. We provide a test of range limit theory by combining population genetic analysis of microsatellite polymorphisms with a transplant experiment within, at the edge of, and 60 km beyond the northern range of a coastal dune plant. Contrary to expectations, lifetime fitness increased toward the range limit with highest fitness achieved by most populations at and beyond the range edge. Genetic differentiation among populations was strong, with very low, nondirectional gene flow suggesting range limitation via constraints to dispersal. In contrast, however, local adaptation was negligible, and a distance‐dependent decline in fitness only occurred for those populations furthest from home when planted beyond the range limit. These results challenge a commonly held assumption that stable range limits match niche limits, but also raise questions about the unique value of peripheral populations in expanding species’ geographical ranges.     

The genetic basis of adaptive population differentiation: a quantitative trait locus analysis of fitness traits in two wild barley populations from contrasting habitats

We used a quantitative trait locus (QTL) approach to study the genetic basis of population differentiation in wild barley, Hordeum spontaneum. Several ecotypes are recognized in this model species, and population genetic studies and reciprocal transplant experiments have indicated the role of local adaptation in shaping population differences. We derived a mapping population from a cross between a coastal Mediterranean population and a steppe inland population from Israel and assessed F3 progeny fitness in the natural growing environments of the two parental populations. Dilution of the local gene pool, estimated as the proportion of native alleles at 96 marker loci in the recombinant lines, negatively affected fitness traits at both sites. QTLs for fitness traits tended to differ in the magnitude but not in the direction of their effects across sites, with beneficial alleles generally conferring a greater fitness advantage at their native site. Several QTLs showed fitness effects at one site only, but no opposite selection on individual QTLs was observed across the sites. In a common-garden experiment, we explored the hypothesis that the two populations have adapted to divergent nutrient availabilities. In the different nutrient environments of this experiment, but not under field conditions, fitness of the F3 progeny lines increased with the number of heterozygous marker loci. Comparison of QTL-effects that underlie genotype x nutrient interaction in the common-garden experiment and genotype x site interaction in the field suggested that population differentiation at the field sites may have been driven by divergent nutrient availabilities to a limited extent. Also in this experiment no QTLs were observed with opposite fitness effects in contrasting environments. Our data are consistent with the view that adaptive differentiation can be based on selection on multiple traits changing gradually along ecological gradients. This can occur without QTLs showing opposite fitness effects in the different environments, that is, in the absence of genetic trade-offs in performance between environments.     

QTL mapping of freezing tolerance: links to fitness and adaptive trade‐offs

Local adaptation, defined as higher fitness of local vs. nonlocal genotypes, is commonly identified in reciprocal transplant experiments. Reciprocally adapted populations display fitness trade‐offs across environments, but little is known about the traits and genes underlying fitness trade‐offs in reciprocally adapted populations. We investigated the genetic basis and adaptive significance of freezing tolerance using locally adapted populations of Arabidopsis thaliana from Italy and Sweden. Previous reciprocal transplant studies of these populations indicated that subfreezing temperature is a major selective agent in Sweden. We used quantitative trait locus (QTL) mapping to identify the contribution of freezing tolerance to previously demonstrated local adaptation and genetic trade‐offs. First, we compared the genomic locations of freezing tolerance QTL to those for previously published QTL for survival in Sweden, and overall fitness in the field. Then, we estimated the contributions to survival and fitness across both field sites of genotypes at locally adaptive freezing tolerance QTL. In growth chamber studies, we found seven QTL for freezing tolerance, and the Swedish genotype increased freezing tolerance for five of these QTL. Three of these colocalized with locally adaptive survival QTL in Sweden and with trade‐off QTL for overall fitness. Two freezing tolerance QTL contribute to genetic trade‐offs across environments for both survival and overall fitness. A major regulator of freezing tolerance, CBF2, is implicated as a candidate gene for one of the trade‐off freezing tolerance QTL. Our study provides some of the first evidence of a trait and gene that mediate a fitness trade‐off in nature.     

Survival and growth patterns of white spruce (Picea glauca [Moench] Voss) rangewide provenances and their implications for climate change adaptation

Intraspecific assisted migration (ISAM) through seed transfer during artificial forest regeneration has been suggested as an adaptation strategy to enhance forest resilience and productivity under future climate. In this study, we assessed the risks and benefits of ISAM in white spruce based on long‐term and multilocation, rangewide provenance test data. Our results indicate that the adaptive capacity and growth potential of white spruce varied considerably among 245 range‐wide provenances sampled across North America; however, the results revealed that local populations could be outperformed by nonlocal ones. Provenances originating from south‐central Ontario and southwestern Québec, Canada, close to the southern edge of the species' natural distribution, demonstrated superior growth in more northerly environments compared with local populations and performed much better than populations from western Canada and Alaska, United States. During the 19–28 years between planting and measurement, the southern provenances have not been more susceptible to freezing damage compared with local populations, indicating they have the potential to be used now for the reforestation of more northerly planting sites; based on changing temperature, these seed sources potentially could maintain or increase white spruce productivity at or above historical levels at northern sites. A universal response function (URF), which uses climatic variables to predict provenance performance across field trials, indicated a relatively weak relationship between provenance performance and the climate at provenance origin. Consequently, the URF from this study did not provide information useful to ISAM. The ecological and economic importance of conserving white spruce genetic resources in south‐central Ontario and southwestern Québec for use in ISAM is discussed.     

Nucleotide diversity at two phytochrome loci along a latitudinal cline in Pinus sylvestris

Forest tree species provide many examples of well-studied adaptive differentiation, where the search for the underlying genes might be possible. In earlier studies and in our common conditions in a greenhouse, northern populations set bud earlier than southern ones. A difference in latitude of origin of one degree corresponded to a change of 1.4 days in number of days to terminal bud set of seedlings. Earlier physiological and ecological genetics work in conifers and other plants have suggested that such variation could be governed by phytochromes. Nucleotide variation was examined at two phytochrome loci (PHYP and PHYO, homologues of the Arabidopsis thaliana PHYB and PHYA, respectively) in three populations: northern Finland, southern Finland and northern Spain. In our samples of 12-15 sequences (2980 and 1156 base pairs at the two loci) we found very low nonsynonymous variation; pi was 0.0003 and 0.0002 at PHYP and PHYO loci, respectively. There was no functional differentiation between populations at the photosensory domains of either locus. The overall silent variation was also low, only 0.0024 for the PHYP locus. The low estimates of silent variation are consistent with the estimated low synonymous substitution rates between Pinus sylvestris and Picea abies at the PHYO locus. Despite the low level of nucleotide variation, haplotypic diversity was relatively high (0.42 and 0.41 for fragments of 1156 nucleotides) at the two loci.     

Environmental and genetic effects on flowering differences between northern and southern populations of Arabidopsis lyrata (Brassicaceae)

Arabidopsis lyrata (Brassicaceae) is a close outcrossing relative of A. thaliana. We examine flowering time variation of northern and southern A. lyrata populations in controlled environmental conditions, in a common garden experiment with A. thaliana, and in the field. Southern populations of A. lyrata flowered earlier than northern ones in all environmental conditions. Individuals from southern populations were more likely to flower in short days (14 h light) than northern ones, and all populations had a higher probability of flowering and flowered more rapidly in long days (20 h). The interaction of population and day length significantly affected flowering probability, and flowering time in one of two comparisons. The common garden experiment demonstrated differences between populations in the response to seed cold treatment, but growth chamber experiments showed no vernalization effect after 4 wk of rosette cold treatment. In a field population in Norway, a high proportion of the plants flowered in each year of the study. The plants progressed to flowering more rapidly in the field and common garden than in the growth chamber. The genetic basis of these flowering time differences here can be further studied using A. thaliana genetic tools.     

Extremely low genetic variability and highly structured local populations of Arabidopsis thaliana at higher latitudes

The genetic diversity and population structure of Arabidopsis thaliana populations from Norway were studied and compared to a worldwide sample of A. thaliana to investigate the demographic history and elucidate possible colonization routes of populations at the northernmost species limit. We genotyped 282 individuals from 31 local populations using 149 single nucleotide polymorphism markers. A high level of population subdivision (F(ST) = 0.85 ± 0.007) was found indicating that A. thaliana is highly structured at the regional level. Significant relationships between genetic and geographical distances were found, suggesting an isolation by distance mode of evolution. Genetic diversity was much lower, and the level of linkage disequilibrium was higher in populations from the north (65-68°N) compared to populations from the south (59-62°N); this is consistent with a northward expansion pattern. A neighbour-joining tree showed that populations from northern Norway form a separate cluster, while the remaining populations are distributed over a few minor clusters. Minimal gene flow seems to have occurred between populations in different regions, especially between the geographically distant northern and southern populations. Our data suggest that northern populations represent a homogenous group that may have been established from a few founders during northward expansions, while populations in the central part of Norway constitute an admixed group established by founders of different origins, most probably as a result of human-mediated gene flow. Moreover, Norwegian populations appeared to be homogenous and isolated compared to a worldwide sample of A. thaliana, but they are still grouped with Swedish populations, which may indicate common colonization histories.     

Chloroplast DNA intraspecific phylogeography of plants from the Pacific Northwest of North America

Molecular studies of plants from the Pacific Northwest of North America suggest a recurrent pattern of genetic differentiation and geographic structuring. In each of five angiosperms and one fern species representing diverse life histories, cpDNA data indicate two clades of populations that are geographically structured. A northern group comprises populations from Alaska to central or southern Oregon, whereas populations from central Oregon southward to northern California form a southern group. In several of these species, a few populations having southern genotypes may have survived in glacial refugia further north in the Olympic Peninsula, Queen Charlotte Islands, and Prince of Wales Island. Allozyme data reveal a similar pattern of differentiation in several other plants from the Pacific Northwest. North-south partitioning of genotypes has also been reported for several animal species from this region. On a broader geographic scale, northsouth partitioning of genotypes has also been observed in other plants from western North America having a variety of geographic distributions. Some species also display a reduction of genetic variability in the northern portion of their range compared to the south. The data suggest strongly that past glaciation profoundly influenced the genetic architecture of the flora and fauna of the Pacific Northwest. Two alternative hypotheses are advanced to explain the geographic structuring of genotypes. First, past glaciation may have created discontinuities in the geographic distributions of plant species, with populations surviving in several well-isolated northern and southern refugia. Following glaciation, migration of genetically differentiated, once-isolated populations resulted in the formation of a continuous geographic distribution with a major genetic discontinuity. Alternatively, plants survived and subsequently migrated northward from a southern refugium, and a genotype became fixed in one or a few populations at the leading edge of recolonization. Subsequent long-distance dispersal from this leading edge resulted in a relatively uniform northern genotype that differs from the southern genotype(s). Whatever the underlying mechanism, Pleistocence glaciation may have molded the intraspecific genetic architecture of both plants and animals from the Pacific Northwest in a geographically similar manner. Future studies should seek to obtain a comprehensive phylogeography for regions that includes a diversity of both plants and animals.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Quantifying latitudinal clines to light responses in natural populations of Arabidopsis thaliana (Brassicaceae)

Evidence of adaptation in Arabidopsis thaliana (Brassicaceae) phenotypic traits has rarely been shown. We demonstrate latitudinal clines in two A. thaliana traits: hypocotyl responses to red and far-red light. Natural populations of A. thaliana were sampled along a latitudinal gradient from southern to northern Norway. Seeds from maternal families within each population were subjected to 1 wk of constant red, far-red, blue, white, and dark treatments. Hypocotyl lengths were measured for each maternal family within each population. Significant variability within and among populations in hypocotyl responses for the various treatments was found. There was a significant latitudinal cline in hypocotyl responses for red and far-red treatments, with northern populations being more de-etiolated than southern populations. These results suggest that northern populations are more responsive to red and far-red light than southern populations. Thus, differentiation of seedling traits in natural populations of A. thaliana seems in part to be mediated by the phytochrome pathway. There was no correlation between hypocotyl responses and flowering time for any treatment. This suggests that flowering time variability and variability in hypocotyl responses may not be governed by genes shared between the pathways, such as those involved in photoreception or the circadian clock.     

Local adaptation primes cold‐edge populations for range expansion but not warming‐induced range shifts

According to theory, edge populations may be poised to expand species’ ranges if they are locally adapted to extreme conditions, or ill‐suited to colonise beyond‐range habitat if their offspring are genetically and competitively inferior. We tested these contrasting predictions by transplanting low‐, mid‐, and high‐elevation (edge) populations of an annual plant throughout and above its elevational distribution. Seed from poor‐quality edge habitat (one of two transects) had inferior emergence, but edge seeds also had adaptive phenology (both transects). High‐elevation plants flowered earlier, required less heat accumulation to mature seed, and so achieved higher lifetime fitness at and above the range edge. Experimental warming improved fitness above the range, but eliminated the advantage of local cold‐edge populations, supporting recent models in which cold‐adapted edge populations do not facilitate warming‐induced range shifts. The highest above‐range fitness was achieved by a ‘super edge phenotype’ from a neighbouring mountain, suggesting key adaptations exist regionally even if absent from local edge populations.     

Latitudinal divergence in a widespread amphibian: Contrasting patterns of neutral and adaptive genomic variation

Stochastic effects from demographic processes and selection are expected to shape the distribution of genetic variation in spatially heterogeneous environments. As the amount of genetic variation is central for long‐term persistence of populations, understanding how these processes affect variation over large‐scale geographical gradients is pivotal. We investigated the distribution of neutral and putatively adaptive genetic variation, and reconstructed demographic history in the moor frog (Rana arvalis) using 136 individuals from 15 populations along a 1,700‐km latitudinal gradient from northern Germany to northern Sweden. Using double digest restriction‐site associated DNA sequencing we obtained 27,590 single nucleotide polymorphisms (SNPs), and identified differentiation outliers and SNPs associated with growing season length. The populations grouped into a southern and a northern cluster, representing two phylogeographical lineages from different post‐glacial colonization routes. Hybrid index estimation and demographic model selection showed strong support for a southern and northern lineage and evidence of gene flow between regions located on each side of a contact zone. However, patterns of past gene flow over the contact zone differed between neutral and putatively adaptive SNPs. While neutral nucleotide diversity was higher along the southern than the northern part of the gradient, nucleotide diversity in differentiation outliers showed the opposite pattern, suggesting differences in the relative strength of selection and drift along the gradient. Variation associated with growing season length decreased with latitude along the southern part of the gradient, but not along the northern part where variation was lower, suggesting stronger climate‐mediated selection in the north. Outlier SNPs included loci involved in immunity and developmental processes.     

Ecotype differentiation in the face of gene flow within the diving beetle Agabus bipustulatus (Linnaeus, 1767) in northern Scandinavia

The repeated occurrence of habitat-specific polyphyletic evolved ecotypes throughout the ranges of widely distributed species implies that multiple, independent and parallel selection events have taken place. Ecological transitions across altitudinal gradients over short geographical distances are often associated with variation in habitat-related fitness, these patterns suggest the action of strong selective forces. Genetic markers will therefore contribute differently to differences between ecotypes in local hybrid zones. Here we have studied the adaptive divergence between ecotypes of the water beetle Agabus bipustulatus along several parallel altitudinal gradients in northern Scandinavia. This water beetle is well known for its remarkable morphological variation associated with mountain regions throughout the western Palaearctic. Two morphological ecotypes are recognised: a montane type with reduced flight muscles and a lowland type with fully developed muscles. Using a multilocus survey of allozyme variation and a morphological analysis with landmark-based morphometrics, across thirty-three populations and seven altitudinal gradients, we studied the local adaptive process of gene flow and selection in detail. Populations were sampled at three different elevations: below, at and above the tree line. The results indicate that the levels of divergence observed between ecotypes in morphology and allele frequencies at α-Glycerophosphate dehydrogenase relative to those shown by neutral molecular markers reflects local diversifying selection in situ. Four main lines of evidence are shown here: (1) A repeated morphological pattern of differentiation is observed across all altitudinal transects, with high reclassification probabilities. (2) Allele and genotype frequencies at the α-Gpdh locus are strongly correlated with altitude, in sharp contrast to the presumable neutral markers. (3) Genetic differentiation is two to three times higher among populations across the tree line than among populations at or below. (4) Genetic differentiation between ecotypes within independent mountain areas is reflected by different sets of allozymes.     

Adaptation to temperate climates

Only model organisms live in a world of endless summer. Fitness at temperate latitudes reflects the ability of organisms in nature to exploit the favorable season, to mitigate the effects of the unfavorable season, and to make the timely switch from one life style to the other. Herein, we define fitness as Ry, the year-long cohort replacement rate across all four seasons, of the mosquito, Wyeomyia smithii, reared in its natural microhabitat in processor-controlled environment rooms. First, we exposed cohorts of W. smithii, from southern, midlatitude, and northern populations (30-50 degrees N) to southern and northern thermal years during which we factored out evolved differences in photoperiodic response. We found clear evidence of evolved differences in heat and cold tolerance among populations. Relative cold tolerance of northern populations became apparent when populations were stressed to the brink of extinction; relative heat tolerance of southern populations became apparent when the adverse effects of heat could accumulate over several generations. Second, we exposed southern, midlatitude, and northern populations to natural, midlatitude day lengths in a thermally benign midlatitude thermal year. We found that evolved differences in photoperiodic response (1) prevented the timely entry of southern populations into diapause resulting in a 74% decline in fitness, and (2) forced northern populations to endure a warm-season diapause resulting in an 88% decline in fitness. We argue that reciprocal transplants across latitudes in nature always confound the effects of the thermal and photic environment on fitness. Yet, to our knowledge, no one has previously held the thermal year constant while varying the photic year. This distinction is crucial in evaluating the potential impact of climate change. Because global warming in the Northern Hemisphere is proceeding faster at northern than at southern latitudes and because this change represents an amelioration of the thermal environment and a concomitant increase in the duration of the growing season, we conclude that there should be more rapid evolution of photoperiodic response than of thermal tolerance as a consequence of global warming among northern, temperate ectotherms.     

POTENTIAL FITNESS TRADE‐OFFS FOR THERMAL TOLERANCE IN THE INTERTIDAL COPEPOD TIGRIOPUS CALIFORNICUS

Thermal adaptation to spatially varying environmental conditions occurs in a wide range of species, but what is less clear is the nature of fitness trade‐offs associated with this temperature adaptation. Here, populations of the intertidal copepod Tigriopus californicus are examined at both local and latitudinal scales to determine whether these populations have evolved differences in their survival under high temperature stress. A clear pattern of increasing high temperature stress tolerance is seen with decreasing latitude, consistent with temperature adaptation. Additionally, there is also evidence for significant variation in thermal tolerance on a smaller scale. The competitive fitness of pairs of northern and southern copepod populations were also examined under a series of lower, more moderate temperatures. These fitness assays show that the southern populations that have the best survival under extreme high temperatures have lowered competitive fitness at the lower temperatures tested, whereas the fitness of the southern populations exceeded that of the northern populations at the highest temperatures tested. Combined, these results suggest that there may be evolutionary trade‐offs between performance at high and stressful temperatures and fitness at moderate temperatures in this species.     

Genetic Variation in Wild and Hatchery Stocks of Suminoe Oyster (Crassostrea ariakensis) Assessed by PCR-RFLP and Microsatellite Markers

Genetic variation in wild Asian populations and U.S. hatchery stocks of Crassostrea ariakensis was examined using polymerase chain reactions with restriction fragment length polymorphism (PCR-RFLP) analysis of both the mitochondrial COI gene and the nuclear internal transcribed spacer (ITS) 1 region and using 3 microsatellite markers. Hierarchical analysis of molecular variance and pairwise comparisons revealed significant differentiation (P < 0.05) between samples from the northern region, represented by collections from China and Japan, and 2 of 3 samples from southern China. PCR-RFLP patterns were identified that were diagnostic for the northern (N-type) and southern (S-type) groups. Microsatellite marker profiles were used to assign each oyster to one of the two northern or two southern populations. Results for more than 97% of the oysters were consistent with the PCR-RFLP patterns observed for each individual in that oysters with N-type patterns were assigned to one of the northern populations and those with S-type patterns to one of the southern populations. At one site of the Beihai (B) region in southern China a mix of individuals with either the N-type or S-type PCR-RFLP genotypes was found. No heterozygotes at the nuclear ITS-1 locus were found in the sample, possibly indicating reproductive isolation in sympatry. Microsatellite assignment test results of the B individuals were also consistent with identifications as either the N-type or S-type based on PCR-RFLP patterns. The parental population for one hatchery stock was this B sample, which initially was composed of almost equal numbers of northern and southern genetic types. After hatchery spawns, however, more than 97% of the progeny fell into the northern genetic group by PCR-RFLP and microsatellite assignment test analyses, indicating that the individuals with the southern genotype contributed little to the spawn, owing to gametic incompatibility, differential larval survival, or a difference in timing of sexual maturity. Overall, results suggested that oysters collected as C. ariakensis in this study, and likely in other studies as well, include two different sympatric species with some degree of reproductive isolation.