Plant Syst Evol (2013) 299:1419–1431 DOI 10.1007/s00606-013-0804-z ORIGINAL ARTICLE The evolution of Dianthus polylepis complex (Caryophyllaceae) inferred from morphological and nuclear DNA sequence data: one or two species? Mohammad Farsi • Maryam Behroozian • Jamil Vaezi • Mohammad Reza Joharchi • Farshid Memariani Received: 3 December 2012 / Accepted: 22 March 2013 / Published online: 6 April 2013 Ó Springer-Verlag Wien 2013 Abstract Dianthus polylepis complex consists of two Keywords Caryophyllaceae Á Dianthus polylepis already known endemic species, Dianthus polylepis and complex Á Morphology Á Molecular phylogeny Á Iran D. binaludensis, in Khorassan-Kopetdagh floristic prov- ince. The taxonomic position of these species has long been debated. The aim of the present study is to shed light Introduction on the evolutionary relationships of the members of the complex using morphological and molecular data. In One of the fundamental problems in plant taxonomy con- morphological study, firstly, 56 vegetative and floral cerns with the plant identification, especially for distin- characters were measured on 33 specimens of the both guishing closely related or recently evolved species species. Multivariate analyses were performed on 25 (out (Rieseberg et al. 2006; Fazekas et al. 2009; Yan et al. of 56) significantly discriminating morphological traits. In 2011). molecular study, we sequenced alleles obtained from a Dianthus L. (Caryophyllaceae) with over 300 species region between 2nd and 6th exons of the gene coding for worldwide is characterized by its extensive morphological the enzyme dihydroflavonol 4-reductase copy1 (DFR1). variability at both inter- and intraspecific levels (Erhardt Morphological results show that most of a priori identified 1990, 1991; Friedman et al. 2001; Bloch et al. 2006). accessions were not grouped in a posteriori classification. It Identification of some of the species in the genus is prob- is difficult to discriminate D. polylepis from D. binalud- lematic due to their morphological plasticity (Sultan 1987; ensis in morphological continuum among the accessions. West-Eberhard 1989; Crespi et al. 2004). In reality, this Results obtained from the molecular data indicated no variability seems to be due to variations occurred in sexual monophyly for the members of the D. polylepis complex. organs resulting from cross pollination (Collin and Shykoff Consistency between the morphological and molecular 2003). results shows that D. polylepis and D. binaludensis were Linder (2008) compiled several examples of rapid not morphologically and molecularly well differentiated. radiations in plants. This process appears to be common in Therefore, we propose a new combination as D. polylepis plants (Kadereit et al. 2004; Linder 2008; Bittkau and subsp. binaludensis. Comes 2009). Many phylogenetic studies revealed that the evolutionary radiations have been occurred in Dianthus (Balao et al. 2010; Valente et al. 2010). A consequence of radiations, however, is that the rates of morphological diversity could be raised making species delimitation dif- M. Farsi Á M. Behroozian Á M. R. Joharchi Á F. Memariani Research Centre for Plant Sciences, ficult (Linder 2008; Balao et al. 2010). On the other hand, Ferdowsi University of Mashhad, Mashhad, Iran polyploidy, hybridization, and genome duplication, com- mon evolutionary forces in plants, act as potential drivers & J. Vaezi ( ) of plant radiations (Ramsey and Schemske 1998; Otto Department of Biology, Faculty of Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran 2007; Paun et al. 2009; Soltis and Soltis 2009; Wood et al. e-mail: [email protected] 2009). These processes are among phenomena occurring in 123 1420 J. Vaezi et al. the complexes and complicate the study of taxonomy and ranges and also on southern and southeastern extensions of evolution in certain plant groups. However, in addition to Khorassan-Kopetdagh ranges, while distribution range of morphological studies, including data from ecology D. binaludensis is locally restricted to Binalud Mountains (Mandakova and Munzbergova 2006), cytology (Balao (Fig. 1). Jafari and Behroozian (2010) reported the same et al. 2009; Al-Saghir 2010), and molecular methods ploidy level (2n = 2x = 30) for the both species. (Oxelman et al. 1997; Linder et al. 2006; Pillon et al. 2007) The problematic taxonomic position of D. polylepis could assist in resolving taxonomic problems of species compared with D. binaludensis (hereafter the D. polylepis complexes. complex) is still uncertain. Our main objective is to unravel The internal transcribed spacer (ITS) region of the the evolutionary history of this complex. To address this nuclear ribosomal DNA is highly employed in phyloge- goal, we used morphological data and sequence data from netic publications (Baldwin et al. 1995; Volkov et al. the gene coding for the enzyme dihydroflavonol 4-reduc- 2007). The markers like rDNA nrITS and chloroplast DNA tase (DFR). The enzyme dihydroflavonol 4-reductase evolve slowly compared to speciation in recently radiated functions most obviously in the reduction of three taxa and thus, may not provide enough information to dihydroflavonols (dihydrokaempferol, dihydroquercetin, resolve relationships (Baldwin et al. 1998; Sang 2002; and dihydromyricetin) to leucoanthocyanidins (Martens Small et al. 2004; Whittall et al. 2006). Lu et al. (2002) et al. 2003). This gene is presented as a small, tandemly showed that analyses of ITS sequences provided low res- arrayed three-gene family (DFR-A, DFR-B, and DFR-C; olution among Dianthus species distributed in China. This Inagaki et al. 1999) in the genus Ipomoea or with two homogeneity of the accessions seems to be due to the functional copies (DFR1 and DFR2; Xie et al. 2004)in radiation phenomenon where there has not been possibly Medicago. However, we used the dihydroflavonol enough time to differentiate the ribotypes in this recently 4-reductase copy1 (DFR1) copy in the present phylogenetic evolved taxon. study. Low-copy nuclear genes may provide more information to resolve relationships and therefore, could overcome the problem of low resolution within recent radiations Materials and methods (reviewed in Sang 2002; Small et al. 2004). Thus, numer- ous investigations have attempted to evaluate the use of Plant materials low-copy nuclear genes to reconstruct phylogenies and to estimate their evolutionary impact (Fan et al. 2004; Syring Morphological analyses were carried out with 33 herbar- et al. 2005; Janssens et al. 2007; Tu et al. 2008; Duarte ium [Ferdowsi University of Mashhad Herbarium et al. 2010; Naumann et al. 2011). (FUMH)] and field-collected Dianthus specimens includ- Our current study is focused on Dianthus polylepis Bien. ing 18 and 15 individuals of D. polylepis and D. binalud- ex Boiss. as a species complex in Khorassan-Kopetdagh ensis, respectively. Of these, 3 and 10 specimens floristic province, northeast of Iran and southern Turk- (D. binaludensis and D. polylepis, respectively) were menistan. This species has a large range of variation in included in the molecular analysis (Table 1). Accessions morphological traits. For this reason, taxonomists did not were chosen to represent the extensive morphological reach the same conclusion to identify specimens of the diversity that can be found particularly in distribution range species in different geographical areas. Rechinger (1983) of D. polylepis. One individual of Dianthus crinitus subsp. described D. binaludensis Rech.f. as a new species from tetralepis was included as outgroup in the molecular study Binalud Mountains. He considered D. binaludensis and D. (Table 1). The specimens of each species included in this polylepis to be distinct, independent species (in ‘‘Flora work are classified a priori using Flora Iranica (Rechinger Iranica’’, Rechinger 1988), while Assadi (1985) declared 1988). For the molecular analysis, the leaf materials of these two species as synonyms and nominated it as D. collected specimens were dried in silica gel and stored at polylepis. In Flora Iranica (Rechinger 1988) D. polylepis is room temperature. Voucher specimens of the samples distinguished from D. binaludensis by bract numbers, collected were deposited in FUMH. length of calyx, branches of stem, length of fimbria, width of calyx, and length of calyx dents. However, these traits Morphological character analysis have never been assessed in a phylogenetic context, and thus whether they can be used as discriminating variables Fifty-six morphological characters (including quantitative to define the monophyly of D. polylepis and D. binalud- and qualitative) were measured and/or scored (Table 2). ensis remained ambiguous. Dianthus polylepis is widely All quantitative measures were made using a ruler with the distributed in northeast of Iran and partly in southern precision of 1 mm. All qualitative traits were numerically Turkmenistan, on Hezar-Masjed and Kopetdagh mountain codified as binary or multi-status criteria (from 0 to 9, 123 The evolution of Dianthus polylepis complex 1421 Fig. 1 Geographical distribution of Dianthus polylepis (black circles) and D. binaludensis (white circles)in Khorassan-Kopetdagh mountain ranges. The localities are based on herbarium records (FUMH) and distribution data in the ‘‘Flora Iranica’’ (Rechinger 1988). The recorded specimen by Rechinger (1988) from Almeh in Golestan National Park (the question mark) has not been confirmed by Akhani (1998) depending on the states of each trait). Univariate analyses L. (GenBank Accession No. AF267172),