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Early stages of ecological speciation can create populations with an ecology and reproduction timing distinct from those of related populations. Landscape genetic models incorporating environmental heterogeneity and population-specific reproductive traits enable the processes of population genetic differentiation to be inferred. We investigated genome-wide genetic variation in ecotypic populations of Solidago virgaurea sensu lato, a herbaceous plant inhabiting a wide range of habitats (woodlands, serpentine barrens and alpine grasslands) and displaying remarkable variation in flowering time. Simultaneous evaluation of environmental factors revealed an overwhelming effect of soil type differences on neutral genetic differentiation, compared with elevational differences. This result probably reflects the abrupt environmental changes generated by geological boundaries, whereas mountain slopes exhibit clinal changes, facilitating gene exchange between neighbouring populations. Temporal isolation was positively associated with genetic differentiation, with some early-flowering serpentine populations having allele frequencies distinct from adjacent nonserpentine populations. Overall, this study highlights the importance of ecological processes and of evolution of flowering time to promote genetic differentiation of S. virgaurea populations in a complex landscape.

The often patchy distribution of serpentine geology can lead to abrupt changes in soil and microclimates. Thus, serpentine areas provide an ideal natural setting to understand how divergent selection drives the process of local adaptation in edaphically specialized plants. When the serpentine ecotype is surrounded by a related nonserpentine ecotype, a balance of natural selection and potential gene flow should maintain the different ecotypes over very short distances. We aimed to reveal the mechanisms allowing soil ecotypes of a goldenrod species to co‐occur sympatrically in Japan. We performed field surveys to characterize microenvironments and flowering phenology of each ecotype, common garden and reciprocal transplant experiments and artificial crossing, and population genetic analysis to investigate the levels of genetic differentiation between ecotypes. Growth chamber experiments show that serpentine plants showed lower specific leaf area (SLA) and greater resource allocation to their root systems than did their nonserpentine counterparts, a potential adaptation to drier and less fertile soil condition in serpentine habitats. Reciprocal transplant studies demonstrated a clear pattern of local adaptation in the plant growth rate. Importantly, serpentine populations completed flowering by midsummer versus late summer in nonserpentine plants. This pattern is consistent with the hypothesis that early flowering ensures reproductive success, before the microclimatic conditions becomes severe in open habitats. Although prezygotic isolation was a strong barrier to gene flow, genetic differentiation was very low, indicating a recent origin for the serpentine ecotypes and/or gene flow at low frequencies. Synthesis. The findings indicate that the early flowering times of serpentine ecotypes, which would have been selected for by microclimates in serpentine areas, can play roles in local adaptation, but also population isolation via a by‐product of diverged reproductive timings. This study contributes to general understanding of the initial stages of plant ecological speciation under potential gene flow in very small geographic scales.

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The aldo-keto reductase (AKR) superfamily is a rapidly growing group of NAD(P)(H)-dependent oxidoreductases that metabolize carbohydrates, steroids, prostaglandins, and other endogenous aldehydes and ketones, as well as xenobiotic compounds. 1) Currently there are more than a hundred known members of this superfamily classified into 14 families.2) The largest family, AKR1, is subdivided into five subfamilies: AKR1A) mammalian aldehyde reductases; AKR1B) mammalian aldose reductases; AKR1C) hydroxysteroid dehydrogenases (HSDs) and prostaglandin F synthases; AKR1D) D 4 -3-ketosteroid-5b-reductases; and AKR1E) mouse keto-reductase. The subfamilies are defined by a Ͼ60% identity in amino acid sequence among subfamily members, but the AKR1C subfamily includes some members that have not been studied as to their enzymatic properties or functions. One such member is AKR1C19 that was found by mouse genomic analysis and cDNA isolation from the liver and gastrointestinal tract. 3) Although AKR1C19 is postulated to be a 3(20)a-HSD-like enzyme based on its highest sequence identity (72%) with human 3(20)a-HSD (AKR1C1) in the AKR1C subfamily, 2) a mouse counterpart of AKR1C1 is shown to be an NADP(H)-dependent 3a/3b/20a-HSD (AKR1C18), 4,5) which shares a lower sequence identity (65%) with AKR1C19. To determine the functional relationship of AKR1C19 with AKR1C18, we examined the enzymatic properties of the recombinant AKR1C19. MATERIALS AND METHODSChemicals Prostaglandins were obtained from Cayman Chemicals (Ann Arbor, MI, U.S.A.), steroids were from Sigma Chemicals and Steraloids (Newport, RI, U.S.A.), and resins for column chromatography were from Amersham Biosciences (Piscataway, NJ, U.S.A.). trans-Benzene dihydrodiol was synthesized by the method of Platt and Oesch. 6) a-and b-3-Hydroxyhexobarbitals (3HBs) were gifts from Dr. R. Takenoshita (University of Fukuoka, Japan). All other chemicals were of the highest grade that could be obtained commercially.cDNA Isolation The cDNA for AKR1C19 was amplified by reverse transcription (RT)-PCR from the total RNA of an 8-week-old male ICR mouse liver. The preparation of the total RNA and RT were carried out as described previously. 7) PCR was performed with Pfu DNA polymerase (Stratagene). The sense primer (5Ј-ATGAGTTCCAAACAGCAA) and antisense primer (5Ј-ACTAAAATTCATCAGAAAAG) correspond to positions 1-18 and 954-973, respectively, of the sequence of a transcript of the AKR1C19 gene. The PCR products of 973 base pairs were ligated into pCR T7/CT-TOPO vectors (Invitrogen), and the expression constructs were transfected into Escherichia coli BL21 (DE3) pLysS according to the protocol described by the manufacturer. The inserts of the cloned cDNAs were sequenced using a CEQ2000XL DNA sequencer (Beckman Coulter) to confirm that the deduced amino acid sequences of the cDNAs are identical to that of AKR1C19 reported by Vergnes et al. 3) Expression and Purification of Recombinant Protein The E. coli cells were cultured in a LB medium containing ampicillin (50 mg/ml) at 37°C until the absorban...