Research Article - (2014) Volume 4, Issue 3
1Department of Biology, Collage of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran
2Department of Biology, Garmsar Branch, Islamic Azad University, Garmsar, Iran
Linaria Miller With more than 150 taxa is the largest genus of the tribe Antirrhineae. This study was carried out on the species of Linaria that growing in Iran. Some of these species are native to Iran. Internal transcribed spacer (ITS) sequences were obtained for 37 samples representing 6 sections of Linaria recognized by recent taxonomic treatments from Iran. In addition, we used 82 previously ITS sequences from GenBank to test the monophyly of Linaria. Phylogenetic analysis were conducted using Bayesian inference and maximum likelihood. The results indicate that Linaria species from Iran constituted a monophyletic group within the Antirrhineae. data analysis indicates that the classification of species according winged or wingless seeds so not true, so seed morphology especially seed wing that have been considered as useful for the taxonomy of Linaria species, appears to be a homoplasious character in Linaria. Bayesian inference and maximum parsimony analyses confirmed Two sections of Linaria including Macrocentrum and Versicolores from Iran are monophyletic, while monophyly of sections Linaria, Diffusae, Supinae and Speciosae are unsupported by this results. To determine the evolution of Linaria use of morphological characteristics coupled with molecular data will be most effective.
Iran, Linaria, phylogeny, ITS.
Linaria Miller With more than 150 taxa is the largest genus of the tribe Antirrhineae [28]. distributed in the Northern hemisphere, its species living in Europe, Asia and North Africa [19]. Linaria was identified as a taxonomic being as soon as the time of pre-Linnaean botanists [22,29]. At first Linnaeus (1753) identified Linaria species inner genus Antirrhinum [20]. Miller (1754) first logical description for Linaria supplied [21]. Linaria species are annual or perennial herbs, with heteromorphic shoots, sessile leaves and racemose inflorescences [26]. for the taxonomy of Linaria species, seed morphology was applicable. Small seeds of Linaria were encircled by capsules, they were winged seeds or wingless seeds. The wingless seeded species by Viano [32,33], and the winged seeded species by Valde´s (1970) established [30]. The winged seeds species designated Discoideae and the wingless seeds species introduced Oblongae by Boissier (1879) [3]. Viano’s theory called wingless and winged seeds formed distinct natural sister lineages in Linaria species [32,33]. Sutton(1988) recognized seven sections mainly based on seed morphology within Linaria widely accepted today [28]. Species of sections Linaria, Supinae and Pelisserianae have discoid and usually winged seeds. While species of sections Versicolores, Speciosae, Diffusae and Macrocentrum have nondiscoid and wingless seeds. In Iran, 35 species of linaria belonging to the sections Linaria (20 spp.), Speciosae (6 spp.), Supinae (4 spp.), Macrocentrum (2 spp.), Diffusae (2 spp.) and Versicolores (1 spp.) are present [3,23,28,7,8,9,10,11,12,13,14,15,16,17]. Linaria species that distribution in different regions of Iran, showed in Table 1 [7]. The aims in this study were to test the naturalness of the seven sections of Sutton’s (1988) classification that six sections of them are present in Iran and to test the hypothesis of a basal divergence between two lineages of Linaria, one with winged seeds and the other with wingless seeds [28].
Table 1: Distribution of Linuria species in Iran
Geographical abbreviations: C, enter Iran; S, southern Iran; SE, south-eastern Iran; N, northern Iran; NE, north-eastern Iran; NW, northwestern
Iran; E, eastern Iran; W, western Iran [7].
Samples were collected in the field and dried in silica gel or obtained from herbaria in Iran (TARI, IAUH), [18]. Phylogenetic reconstructions were performed in 37 samples of Linaria presented in six sections from Iran. Table 2 lists all taxa used in this study and summarizes sources, voucher specimen data and GenBank accession numbers. Total DNA was extracted using the DNeasy Plant Mini kit (Qiagen, Germany). We amplified the ITS region (ITS1- 5.8S-ITS2) of the nuclear ribosomal DNA using primer combinations AB101 and AB102 primers: a forward primer AB101annealing, 5'-ACG AAT TCA TGG TCC GGT GAA GTG TTC G-3', and a reverse primer (AB102) annealing, 5'-TAG AAT TCC CCG GTT CGC TCG CCG TTA C-3'. The PCR protocol for ITS region included: 25 cycles of 1 min denaturation (94°C), 1 min annealing (54°C), and 2 min, 30 s elongation (72° C), with two additional seconds elongation per cycle [5].
Table 2: List of taxa investigated in our analysis and herbaria where the vouchers are deposited with Genbank accession numbers
TARI= herbarium of Research Institute of Forests and Rangelands, IAUH= Islamic Azad University Avicennia herbarium
Phylogenetic Analysis
Phylogenetic reconstructions were performed in 37 sampels of Linaria in six sections from Iran. In this study We used the ITS sequence of 34 species of Linaria, 37 species of Antirrhinum, 4 species of Galvezia, 2 species of Maurandya and 2 species of Kickxia from GenBank. List of non-Iranian taxa used in our analysis with GenBank accession numbers showed in Table 3. We also used the ITS sequence of Lafuentea rotundifolia, from GenBank as the outgroup [1]. Matrices were analyzed with PAUP*4.0b10, with the following options: heuristic search with 1,000 random-addition-sequence replicates; tree bisection-reconnection (TBR) branch swapping; “collapse zero length branches;” saving all most parsimonious trees. Character state changes were treated as equally weighted. Nonoverlapping parsimony informative indels were coded as binary characters and added to the end of the data matrix. Relative clade support was estimated using 1,000 bootstrap, replicates in PAUP* via “full heuristic” searches and simple taxon addition. The consistency index (CI) and retention index (RI) were used to assess the amount of homoplasy present in the data. The best-fitting substitution model (GTR+I+G) was determined under the Akaike Information Criterion (AIC) in Model selected [24]. BI was performed in MrBayes ver. 3.1.2 [25]. A 50% majority-rule consensus tree with Bayesian posterior probabilities (PPs) of clades was calculated after removing the first 10% generations as burn in.
Table 3: List of non-Iranian taxa used in our analysis with GenBank accession number
The data set of the ITS region included 611 characters, 224 of them potentially parsimony informative. Strict consensus phylogeny trees, with 1014 steps was included consistency index (CI)=0.4872, retention index (RI)=0.8971 and homoplasy index (HI)= 1.83. Within the Antirrhineae four major clades were identified which have been given the name of one typical genus: the Antirrhinum clade (2 genera; PP = 0.99; BS = 60%), the Maurandya clade (2 genera; PP = 0.99; BS = 90%), the Kickxia clade (2 genera; PP = 1; BS = 100%), and all sampled species of Linaria were formed Linaria clade (PP = 0.99; BS = 100%). A little supported relationships between major clades were existed. Linaria clade included five major clades (A–E). Clade A included two species (L. chalepensis and L. armenica from Iran) of sect. Macrocentrum (PP = 1; BS = 100%). Clade B included one Iranian species (L. iranica) and two other species of sect. Versicolores (PP = 0.99; BS = 100%). Pelisserianae constituted clade C (PP = 0.69; BS = 71%). L. triornithophora is sister to clade B. This is not recognized in Iran. All sampled species of sect. Supinae (L. arvensis, L. simplex, L. kavirensis and L. micrantha from Iran) and three species of sect. Diffusae were formed Clade D (PP = 0.99; BS = 79%). Clade E (PP = 0.98; BS = 60%) was formed by all sampled species of sect. Linaria (L. vulgaris, L. odora, L. khorasanensis, L. striatella, L. farsensis, L. bamianica, L. michauxii, L. pyramidalis subsp. pyramidalis, L. pyramidalis subsp. kopetdaghensis, L. fastigiata, L. remotiflora, L. nurensis, L. kurdica subsp. kurdica, L. kurdica subsp. pycnophylla, L. lineolata, L. karajensis, L. elymatica, L. azerbaijanensis, L. khalkhalensis, L. shahroudensis and L. guilanensis from Iran), all sampled species of sect. Speciosae (L. mazandaranensis, L. genistifolia, L. golestanensis, L. dalmatica, L. grandiflora and L. orientalis from Iran), and six species of sect. Diffusae (L. bousherensis1, L. bousherensis2 and L. albifrons from Iran). A little supported relationships among main clades were existed. Best support was occurred for two sister-group relationship between clades D and E (PP = 0.98; BS = 100%). Relationship between two sister-group clades B and C was supported by BI (PP = 0.69; BS = 71%). On the other side, relationships among other clades remained indefinite in our results.
In this study we provide the first phylogenetic analysis about Linaria from Iran. At first the monophyly of Linaria on the eight sampled species exponent the seven sections of Sutton’s classification, proposed by Vargas et al. [31]. Also in recent phylogenetic study based on ITS sequences by Ferna´ndez et al. (2013), Monophyly of Linaria was supported [6]. This analysis include 37 sampels of Linaria from Iran. Phylogenetic relationships between Linaria species in this study are according to those obtained by Vargas et al. (2004) and Ferna´ndez et al. (2013) based on the ITS sequences [6,31]. In this ITS phylogeny study, our species of Linaria organized a intensely supported monophyletic group (figure 1). morphological characters that are not occured elsewhere in the Antirrhineae tribe and basic chromosome number (x=6) affirmed the monophyly of Linaria. [31,28]. According to our obtained data the genus Linaria was determined as a paraphyletic group (figure 1). This results as Ferna´ndez et al. (2013) finding, Viano’s theory, separation of natural sister lineages in Linaria species based on with wingless and winged seeds forms, were refused. [32,33]. Our analysis represent Sections Macrocentrum and Versicolores to be monophyletic (figure 1). Other sections including Linaria, Speciosae, Supinae and Diffusae do not constitute as monophyletic groups. Species of sect. Macrocentrum (L. chalepensis and L. armeniaca, clade A; figure 1) describe with some characters including the adaxial lobe of the calyx is shorter than abaxial lobes and lateral appendage present at the base of each stamen filament is small. Sutton (1980), based on these characters are specific between Linaria, separated them from sect. Versicolores. [27]. Section Versicolores is including just one species in Iran (L. iranica). Sutton (1988) was defined morphological character for sect. Versicolores that not found elsewhere in the Antirrhineae tribe [27]. This character was including separated style with distinct stigmatic region. The species L. iranica, that is endemic of Iran, has a bifid style. Champagnat (1961) With specific pattern of seedling progression is affirmed the naturalness of sect. Versicolores[4]. Four sections of Linaria including Speciosae, Diffusae, Supinea and Linaria, conversely sects. Macrocentrum, Pelisserianae and Versicolores, were not determined as monophyletic in this study. Relationships between species of sects. Speciosae and Linaria were showed in clade E. Previously Sutton (1988) was shown a identical morphological relationship among species of sections Linaria and Speciosae based on the perennial duration, erect stems, alike leaf, flower, and capsule morphology [28]. But there is a remarkable difference in seed form among species of both sections, also sect. Linaria have winged seeds and sect. Speciosae have wingless seeds. Three species of sect. Diffusae were also included in clade D (L. haelava, L. joppensis and L. warionis). This species formed a monophyletic group within clade D. Clade D also included species of sect. Supinae (L. arvensis, L. simplex, L. kavirensis and L. micrantha from Iran and other species of sect. Supinae). This results was supported the monophyly of species of sect. Supinae. The naturalness of sect. Supinae was affirmed union of both plastid and nuclear sequences in a merging-based analysis permitted the discovery of hybridization and imperfect lineage grouping [2]. Another species of sect. Diffusae, including L. bousherensis1, L. bousherensis2, L. albifrons_IR, L. albifrons, L. flava, and L. triphylla, constituted a monophyletic group within clade E. The main traits species of sect. Speciosae and sect. Diffusae including wingless seeds, entire style, normal adaxial lobe of calyx. Actually, separated species from both of sections is difficult. But chiefly morphological differences between species of sect. Diffusae and sect. Speciosae including annuals duration with ascending stems in sect. Diffusae and perennials duration with erect stems in sect. Speciosae. At first Valde´s (1970) was proposed that sect. Diffusae was apparently polyphyletic, But Sutton (1988) believed that it was constituted a heterogeneous group [28]. Because of low phylogenetic resolution within clade E, we required extra markers for expose the phylogenetic relationships.
Figure 1: Phylogenetic relationships of 37 sampels of Linaria from Iran, 34 species of Linaria, 37 species of Antirrhinum, 4 species of
Galvezia, 2 species of Maurandya and 2 species of Kickxia on the basis of the analysis of internal transcribed spacer sequences
Numbers above branches are Bayesian posterior probabilities. Numbers below branches are maximum likelihood percentage bootstrap values
The results indicate that Linaria species from Iran constituted a monophyletic group within the Antirrhineae. data analysis indicates that the classification of species according winged or wingless seeds so not true, so seed morphology especially seed wing that have been considered as useful for the taxonomy of Linaria species, appears to be a homoplasious character in Linaria. Two sections of Macrocentrum and Versicolores based on conformity special morphological traits and ITS sequences organized separated developmental lineages, therefor should be retained in classification of Linaria. But, naturalness of secttions Supinae, Linaria, Speciosae, and Diffusae were not supported. For carefully determined of Linaria location we need to more molecular markers and more Linaria sampling from all sections.
This article is extracted from my Phd thesis. We have finally thank from Islamic Azad University -Tehran Science and Research Branch for providing the facilities necessary to carry out the work.
