Molecular Evolution of Phytochromes in Cardamine nipponica (Brassicaceae) Suggests the Involvement of PHYE in Local Adaptation

Given that plants are sessile organisms, traits involved in adapting to local environments and/or in monitoring the surrounding environment, such as having photoreceptors, are significant targets of natural selection in plant evolution. To assess the intraspecific adaptive evolution of photoreceptors, we investigated sequence variations in four phytochrome genes (PHYA–C and PHYE) of Cardamine nipponica (Brassicaceae), an endemic Japanese alpine plant. The genealogies of haplotypes and genetic differentiations showed inconsistent patterns of evolution across phytochromes, suggesting that evolutionary forces were distinct in phytochromes of C. nipponica. An overall low level of nucleotide diversity in phytochrome genes suggests that the evolution of phytochromes is constrained by purifying selection within C. nipponica, which is consistent with previous findings on phytochromes. However, PHYE alone exhibited a non-neutral pattern of polymorphisms (Tajima''s D = 1.91, P < 0.05) and an accumulation of nonsynonymous substitutions between central and northern Japan. In particular, the PHY domain, which plays an important role in stabilizing the active form (Pfr) of phytochromes, harbored a specific nonsynonymous fixation between regions. Thus, our finding indicates that local adaptation is involved in the evolution of PHYE in C. nipponica and is the first to suggest the involvement of PHYE in local adaptation.GIVEN that plants are sessile organisms, adapting to the surrounding environment and monitoring environmental changes such as temperature, aridity, and day length are important not only for survival, but also for reproductive success. Thus, natural selection has likely played a significant role in selecting for such traits in the evolution of plants. In particular, various developmental responses are influenced by the light environment (known as photomorphogenesis); for example, germination, de-etiolation, shade avoidance, and flowering are regulated by light signals (Whitelam and Devlin 1997; Whitelam et al. 1998; Smith 2000; Mathews 2006). Thus, plants have obtained sophisticated systems, including photoreceptors, to monitor light signals such as intensity, direction, quality, and duration. In particular, phytochromes, which sense red and far-red light, are among the most studied photoreceptors and play a major role in developmental pathways as well as in evolutionary history.Phytochromes have two photoreversible conformations: an inactive red-light-absorbing form (Pr) and an active far-red-light-absorbing form (Pfr). Red light converts Pr to Pfr, while far-red light converts Pfr back to Pr. At least three phytochromes are widely known in angiosperms (PHYA–C; Mathews et al. 1995), and five have been identified in Arabidopsis thaliana (PHYA–E; Sharrock and Quail 1989; Clack et al. 1994). According to phylogenetic analyses, these gene families are clustered into two major groups, PHYA/C and PHYB/D/E (Alba et al. 2000), and the duplication of these two major clusters occurred prior to angiosperm radiation (Mathews et al. 1995). Further duplication resulted in PHYA and PHYC and PHYB/D and PHYE, although some groups, such as monocots and poplars, lack PHYE (Mathews and Sharrock 1996), and PHYD was duplicated from PHYB specifically in the Brassicaceae (Mathews and McBreen 2008). The evolutionary consequences following gene duplications have recently been reported. Positive selection was involved in the functional divergence following duplications in PHYA and PHYC and PHYB/D and PHYE, whereas purifying selection suppressed the divergence in each group (Yang and Nielsen 2002). Moreover, adaptive evolution was involved in the evolution of PHYA in early angiosperms (Mathews et al. 2003).Because light signals differ among habitats, such as forest understory, canopy, and open meadow, as well as among populations at high and low latitudes and altitudes, the functions of phytochromes should be both a target for natural selection and adapted to the local light environment. This hypothesis was recently supported by studies of A. thaliana, which showed that amino acid substitutions in PHYA, PHYB, and PHYC may be responsible for intraspecific phenotypic differences among accessions (Maloof et al. 2001; Balasubramanian et al. 2006; Filiault et al. 2008). In addition to the conclusions of the Arabidopsis studies, the importance of PHYB2 for local adaptation was also suggested in studies of Populus tremula (Ingvarsson et al. 2006, 2008). Thus, determining polymorphisms in phytochrome genes and their geographic distribution could demonstrate the importance of phytochromes for local adaptation.A previous phylogeographic study on Cardamine nipponica Franch. et Savat. (Brassicaceae), an endemic Japanese alpine plant, found a higher level of fixation of nonsynonymous substitutions among populations in a partial sequence of PHYE (Ikeda et al. 2008b), indicating the involvement of PHYE in local adaptation. In the Japanese archipelago, alpine flora are distributed on high mountaintops from central to northern Japan. Nearly half of the Japanese alpine flora species are also found in Arctic regions [such as Dryas octopetala (Rosaceae), Loiseleuria procumbens (Ericaceae), and Diapensia lapponica (Diapensiaceae)], indicating that Arctic plants significantly contributed to the Japanese alpine flora. According to recent phylogeographic investigations, most alpine plants show strong genetic differentiation between populations from central and northern Japan, suggesting a history of vicariance in the Japanese archipelago (Fujii et al. 1997, 1999; Senni et al. 2005; Fujii and Senni 2006; Ikeda et al. 2006, 2008a,b; Ikeda and Setoguchi 2007, 2009). Most importantly, the genetic structure of 10 nuclear genes in C. nipponica revealed that haplotypes of most loci were closely related in each region and diverged from those in the other region. Moreover, the isolation with migration model (Nielsen and Wakeley 2001; Hey and Nielsen 2004, 2007) demonstrated no migration between the two regions after the regions were split, suggesting that the divergence in functional genes occurred following the vicariance between regions (H. Ikeda, N. Fujii and H. Setoguchi, unpublished results). In a partial sequence of PHYE (∼700 of the 3700 bp), three nonsynonymous substitutions were fixed between central and northern Japan, whereas nine other loci showed few nonsynonymous substitutions between the two regions. Thus, local adaptation in PHYE may have occurred following the vicariance between central and northern Japan, while genetic drift following the vicariance happened to fix the nonsynonymous substitutions. Further investigations that evaluate evolutionary patterns along the functional domains of phytochromes, including determining the entire sequences of PHYE, may demonstrate the involvement of PHYE in local adaptation.In this study, to test the hypothesis that PHYE has been involved in local adaptation between central and northern Japan, we determined the entire sequences of PHYE from the entire distribution range of C. nipponica and examined polymorphisms and their geographic structures. Furthermore, to assess whether the signature of local adaptation was exclusively detected in PHYE or whether other phytochromes were also involved, we determined the entire sequences of PHYA–C and examined their polymorphisms.