Genet. Res., Camb. (2008), 90, pp. 111–118. f 2008 Cambridge University Press 111 doi:10.1017/S001667230700897X Printed in the United Kingdom ‘Junk’ DNA and phenotypic evolution in Silene section Siphonomorpha THOMAS R. MEAGHER1* AND DENISE E. COSTICH2 1 Centre for Evolution Genes and Genomics, School of Biology, University of St Andrews, St Andrews, Fife KY16 9TH, UK 2 USDA-ARS, Cornell University, 175 Biotechnology Bldg, Ithaca, NY 14853-2703, USA (Received 12 September 2007 and in revised form 24 October 2007) Summary One of the long-standing mysteries in genomic evolution is the observation that much of the genome is composed of repetitive DNA, resulting in inter- and intraspecific variation in nuclear DNA content. Our discovery of a negative correlation between nuclear DNA content and flower size in Silene latifolia has been supported by our subsequent investigation of changes in DNA content as a correlated response to selection on flower size. Moreover, we have observed a similar trend across a range of related dioecious species in Silene sect. Elisanthe. Given the presence of sex chromosomes in dioecious Silene species, and the tendency of sex chromosomes to accumulate repetitive DNA, it seems plausible that dioecious species undergo genomic evolution in ways that differ from what one might expect in hermaphroditic species. Specifically, we query whether the observed relationship between nuclear DNA content and flower size observed in dioecious Silene is a peculiarity of sex chromosome evolution. In the present study we investigated nuclear DNA content and flower size variation in hermaphroditic species of Silene sect. Siphonomorpha, as close relatives of the dioecious species studied previously. Although the nuclear DNA contents of these species were lower than those for species in sect. Elisanthe, there was still significant intra- as well as interspecific variation in nuclear DNA content. Flower size variation was found among species of sect. Siphonomorpha for petal claw and petal limb lengths, but not for calyx diameter. This last trait varies extensively in sect. Elisanthe, in part due to sex-specific selection. A negative correlation with nuclear DNA content was found across populations for petal limb length, but not for other floral dimensions. We conclude that impacts of nuclear DNA content on phenotypic evolution do manifest themselves in hermaphroditic species, so that the effects observed in sect. Elisanthe, and particularly in S. latifolia, while perhaps amplified by the genomic impacts of sex chromosomes, are not limited to dioecious taxa. 1. Introduction floral evolution leading to sex differences in flower size and other secondary sex characteristics (Meagher, The evolution of gender polymorphisms in plant 1999). Given that dioecy is based on very specific populations is characterized by complexity on many genomic effects, such as sex-limited gene expression levels (Charlesworth, 2006). For example, gender ex- and in the extreme the evolution of sex chromosome pression is evolutionarily linked to many aspects heteromorphism, it is possible that floral evolution in of floral evolution, including variation in flower size dioecious species is driven by genomic processes that and overall plant life-history. In species that exhibit are specific to dioecy. On the other hand, genomic gender dimorphism, such as dioecy, sex-specific effects underlying floral evolution, and in particular drivers of floral evolution are relatively easy to study flower size evolution, might be independent of gender in isolation. Indeed, quantitative genetic models have expression. In the present paper, we explore whether been applied to good effect to understand sex-specific genomic effects that influence flower size in dioecious species are also manifested in closely related her- * Corresponding author. e-mail: [email protected] maphroditic species. T. R. Meagher and Denise E. Costich 112 One of the long-standing mysteries in genomic evolution is the observation that much of the genome is composed of repetitive DNA in the form of trans- posable elements and other forms of repetitive se- quences (Flavell et al., 1983; Heslop-Harrison, 2000; Kidwell & Lisch, 2001; Meagher & Vassiliadis, 2005). 17 Such repetitive DNA has typically been regarded as 12 superfluous to the function of the genome in generat- 3 15 13 ing phenotypes, and the introduction of repetitive 14 sequences has often been interpreted as a deleterious 4 5 effect (Charlesworth et al., 1994). More recently, our continuing investigation of the relationship between 16 variation in repetitive DNA, manifested as variation in nuclear DNA content and flower size, suggests that 11 repetitive DNA may play an effective role in adaptive 6 7 9 evolution (Meagher et al., 2005). 8 10 The discovery of a negative correlation between nuclear DNA content and flower size in Silene lati- Fig. 1. Map of Spain showing locations of the sampled folia (Meagher & Costich, 1994) has been supported populations of Silene sect. Siphonomorpha (circles, by investigation of changes in DNA content as a corre- S. andryalifolia; diamonds, S. coutinhoi; squares, lated response to selection on flower size (Meagher & S. fernandezii; triangles, S. mellifera; inverted triangles, Costich, 1996), and the effect has been observed across S. nutans). Precise coordinates for each locality are given in Table 1. a range of related species in Silene sect. Elisanthe (Meagher & Costich, 2004). This work has intriguing implications for the relationship between DNA con- non-combining region of Y chromosomes could pro- tent variation and phenotypic evolution. We attribute vide a reservoir that could then elevate probabilities of the observed relationship to indirect impacts of re- transposable element establishment to other parts of petitive DNA on patterns of gene expression. For the genome, such as the autosomes, through lateral example, tandem repetitive DNA is known to influ- transmission. Thus, we query whether the observed ence local protein-DNA interactions; and dispersed relationship between nuclear DNA content and flower repetitive DNA, such as transposable elements, is size observed in dioecious Silene is a peculiarity of sex known to affect gene expression through insertion chromosome evolution. More specifically, would a into regulatory regions of genes. On the basis of our negative correlation between flower size and nuclear observations, we have proposed that repetitive DNA DNA content appear in the absence of dioecy? underlying DNA content variation is a major driver The present study investigated nuclear DNA in quantitative phenotypic variation (Meagher & content and flower size variation in Silene sect. Costich, 1996; Meagher & Vassiliadis, 2005). Siphonomorpha, a group comprised of hermaphrodite A consistent feature of species of Silene sect. species. We addressed the following questions: What Elisanthe is that they are all dioecious, and there is a is the extent of nuclear DNA content and flower size well-developed model of sex chromosome evolution variation among sect. Siphonomorpha species? Does within this group (Ainsworth, 1999; Charlesworth, the negative correlation between nuclear DNA con- 2002; Westergaard, 1958). Indeed, S. latifolia was one tent and flower size previously observed in dioecious of the first known examples of the XX (female) and species of Silene hold up for hermaphroditic species? XY (male) mode of sex determination (Blackburn, What insights into genome evolution are suggested by 1923, and Winge, 1923, cited in Lengerova et al., the pattern of distribution of nuclear DNA content 2003). This species has also been an important object across species of sect. Siphonomorpha (hermaphro- of study in the development of evolutionary models of ditic) in contrast to sect. Elisanthe (dioecious)? Y chromosome evolution, in which the Y chromo- some is predicted to accumulate non-coding repetitive 2. Materials and methods DNA sequences (Nicolas et al., 2005). Given the presence of sex chromosomes in dioecious This study focuses on five species in Silene sect. Silene species, and the tendency of sex chromosomes Siphonomorpha: S. andryalifolia, S. coutinhoi, S. fer- to accumulate repetitive DNA, it seems plausible that nandezii, S. mellifera and S. nutans. These species dioecious species undergo genomic evolution in ways are native to the Iberian peninsula, though S. nutans that differ from what one might expect in her- is more widespread (Talavera, 1990). In May–June maphroditic species. For example, one might specu- of 1998, populations of each species were located in late that accumulation of transposable elements in the Spain (Fig. 1, Table 1); floral measurements (calyx ‘Junk’ DNA and phenotypic evolution 113 Table 1. Collection localities for Silene sect. Siphonomorpha populations sampled Silene sp. Site name Map no. Longitude Latitude Nfield Ngh S. andryalifolia Pen˜ on de Alga´mitas, Sevilla 6 x5.18 37.01 25 30 S. andryalifolia Ermita de Pruna, Sevilla 7 x4.32 37.46 25 19 S. andryalifolia El Torcal de Antequera, Malaga 10 x4.55 36.95 25 29 S. andryalifolia Zuheros, Cordoba 11 x4.31 37.54 25 32 S. coutinhoi Ermita de San Frutos, Segovia 12 x3.87 41.33 25 25 S. coutinhoi Herva´s, Ca´ceres 14 x5.87 40.27 25 29 S. coutinhoi La Orbada, Salamanca 17 x5.47 41.13 25 22 S. fernandezii Penas Blancas, Malaga 8 x5.18 36.49 15 33 S. mellifera Rio Tajun˜ a, Madrid 3 x2.70 40.59 25 21 S. mellifera Belmonte de Tajo, Madrid 4 x3.34 40.13 25 25 S. mellifera El Torcal de Antequera, Malaga 9 x4.51 36.98 25 29 S. mellifera Puerto del Barrancazo, Albacete 16 x2.42 38.58 25 8 S. nutans Puerto de Berzocana, Cacares 5 x5.45 39.42 12 18 S. nutans Silla de Felipe II, Madrid 13 x4.15 40.57 25 15 S. nutans Herva´s, Ca´ceres 15 x5.87 40.27 25 16 Map reference numbers for each population as well as latitude and longitude (in degrees) were those used in Fig. 1. Sample sizes for field-based floral measurements (Nfield) and greenhouse-based DNA content assays (Ngh) are also shown.