Recurrent parent similarity index Six characters (upper petal reflexing, lateral petal reflexing, pistil length, stamen length, lateral petal width and nectar volume) for which QTLs have been mapped 6,7 were measured on two flowers from each plant. There was a significant difference between the multivariate flower phenotypes of wild-type and ‘mutant’ NILs in both the M. lewisii (multiple analysis of variance, MANOVA, F ¼ 18.18, Wilks’ l ¼ 0.32, P , 0.0001) and M. cardinalis (MANOVA, F ¼ 11.00, Wilks’ l ¼ 0.56, P , 0.0001) genetic backgrounds (PROC GLM, SAS Institute). Least-squares means for each trait within each NIL genotypic class were normalized to the difference between trait means of the two parental species 7 , setting the recurrent parent trait value at 100% and the nonrecurrent parent at 0%. Lower recurrent parent similarity (RPS) values are evidence of linkage drag, whereas values larger than 100% represent measurement error or heterosis. In the M. lewisii genetic background, the wild-type plants had a mean RPS index across all traits of 91% (range 66–108%), whereas their ‘mutant’ sibs had a value of 80% (range 51–103%). In the M. cardinalis genetic background, the wild-type plants had a mean RPS index of 95% (range 49–129%), whereas their ‘mutant’ sibs had a value of 80% (range 46–155%). Although ‘mutant’ NILs show more linkage drag than the wild type, we judge the difference to be small. Nectar volume, which is known from our F 2 experiments to have a marked effect on hummingbird visitation 8 , has RPS index values that are very close to one another in the NILs: 105% and 103% in the M. lewisii background, and 46% and 53% in the M. cardinalis background. This suggests that differences in nectar production between pairs of NILs did not affect pollinator visitation patterns. Pollinator visitation For each of two field experiments conducted to measure pollinator visitation, 50 pink or dark pink (YUP/___) and 50 yellow-orange or red (yup/yup) plants were drawn at random from five BC 4 S 1 (M. lewisii) or BC 4 (M. cardinalis) NIL families. Assessments of pollinator visitation were performed at Mather (California, USA), the site where much of the previous work on these two species of Mimulus has been done 5 . Pollinator observations were carried out from dawn to evening, with a 1–2 h break at midday when pollinators were least active. Dates of observation were 18–30 August 1999 for M. cardinalis NILs, and 18–27 July 2000 for M. lewisii NILs. These dates correspond closely to the peak flowering times of natural populations of the two Mimulus species. We chose to do the experiments in different years so that pollinators were faced with a binary choice of flower phenotypes, as would be the case for a newly arisen mutation. Plants were placed at random on a 1m £ 1 m grid to produce the experimental arrays (a black bear visit reduced the total sample size in the M. lewisii NIL array from N ¼ 100 to N ¼ 99). A pollinator visit was counted if it appeared that the pollinator probed the flower and contacted the anthers or stigma. Bumblebees and hummingbirds were the only pollinators observed. We observed 1,090 bumblebee visits to the M. lewisii NILs, 180 bumblebee visits to the M. cardinalis NILs, 201 hummingbird visits to the M. lewisii NILs, and 3,738 hummingbird visits to the M. cardinalis NILs. The number of flowers on each plant was recorded daily, along with the number of hours spent observing. Visitation rates were calculated by dividing the total number of pollinator visits across all days by the aggregate number of hours in which visits could have occurred to each flower (flower-hours). For the M. lewisii NILs, both bumblebee and hummingbird pollinator observations were carried out simultaneously, with 47,159 flower-hours for the wild-type NILs and 138,648 flower-hours for the ‘mutants’. For the M. cardinalis NILs, separate pollinator observation periods were required to keep track of the large number of hummingbird visits. During the bumblebee observation periods, there were 16,291 flower-hours for the ‘mutant’ NILs and 13,556 flower-hours for the wild-type. During the hummingbird observation periods, there were 11,505 flower-hours for the ‘mutant’ NILs and 9,520 flower-hours for the wild type. Received 15 July; accepted 3 October 2003; doi:10.1038/nature02106. 1. Orr, H. A. & Coyne, J. A. The genetics of adaptation: a reassessment. Am. Nat. 140, 725–742 (1992). 2. Gillham, N. W. Evolution by jumps: Francis Galton and William Bateson and the mechanism of evolutionary change. Genetics 159, 1383–1392 (2001). 3. Fisher, R. A. 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Grant, V. Historical development of ornithophily in the western North American flora. Proc. Natl Acad. Sci. USA 91, 10407–10411 (1994). Acknowledgements We thank A. Angert, K. Kay, andD. Grosenbacher for field observations of pollinators, P. Beardsley and S. Stefanovic for field assistance, and B. Watson for genotyping. We are grateful to F. Nicholson and the Carnegie Institution of Washington for allowing us to use the Mather field station. Y. Sam provided helpful comments on the manuscript. This work was supported by an award from the National Science Foundation. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to H.D.B. (toby@u.washington.edu). .............................................................. Light-induced hormone conversion of T 4 to T 3 regulates photoperiodic response of gonads in birds Takashi Yoshimura 1 , Shinobu Yasuo 1 , Miwa Watanabe 1 , Masayuki Iigo 3 , Takashi Yamamura 1 , Kanjun Hirunagi 2 & Shizufumi Ebihara 1 1 Division of Biomodeling, Graduate School of Bioagricultural Sciences, Nagoya University, and 2 The Nagoya University Museum, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan 3 Department of Applied Biological Chemistry, Faculty of Agriculture, Utsunomiya University, Mine-Machi, Utsunomiya, Tochigi 321-8505, Japan ............................................................................................................................................................................. Reproduction of many temperate zone birds is under photoperi- odic control. The Japanese quail is an excellent model for study- ing the mechanism of photoperiodic time measurement because of its distinct and marked response to changing photoperiods. Studies on this animal have suggested that the mediobasal hypothalamus (MBH) is an important centre controlling photo- periodic time measurement 1–8 . Here we report that expression in the MBH of the gene encoding type 2 iodothyronine deiodinase (Dio2), which catalyses the intracellular deiodination of thyrox- ine (T 4 ) prohormone to the active 3,5,3 0 -triiodothyronine (T 3 ), is induced by light in Japanese quail. Intracerebroventricular administration of T 3 mimics the photoperiodic response, whereas the Dio2 inhibitor iopanoic acid prevents gonadal growth. These findings demonstrate that light-induced Dio2 expression in the MBH may be involved in the photoperiodic response of gonads in Japanese quail. The molecular mechanism of photoperiodic or seasonal time measurement is not well understood in any organism studied so far. In birds, the MBH—which includes the nucleus hypothalamicus posterior medialis (NHPM), the infundibular nucleus and the median eminence—is an important centre controlling photoperi- odic time measurement (Supplementary Figs 1 and 2). For example, introduction of a lesion to the nucleus hypothalamicus posterior medialis and/or the infundibular nucleus resulted in loss of photo- periodic response of the gonads 1–3 even though the gonadotrophin- releasing hormone (GnRH) system of the lesioned animal had been left intact 4 . Electrical stimulation of this area increases luteinizing hormone secretion 5 and induces testicular growth 6 . Furthermore, c-Fos expression has been reported in these structures as a result of photostimulation for one long day (20/4 h light/dark cycle) 7,8 and deep-brain photoreceptors are thought to be localized in the infundibular nucleus 9 . Recently, we have also observed the expression of circadian clock genes in the MBH, and proposed that the clock in the MBH may function as the ‘photoperiodic clock’ 10 . These observations indicate that all of the essential machin- ery for photoperiodic time measurement is localized in the MBH. Single light pulses within the photo-inducible phase increase letters to nature NATURE | VOL 426 | 13 NOVEMBER 2003 | www.nature.com/nature 178 © 2003 Nature Publishing Group