c Indian Academy of Sciences RESEARCH ARTICLE Phylogeny and biogeography of Alyssum (Brassicaceae) based on nuclear ribosomal ITS DNA sequences YAN LI 1,2,3, YAN KONG1,3, ZHE ZHANG1,3, YANQIANG YIN1,3,BINLIU1∗, GUANGHUI LV2 and XIYONG WANG1 1Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, People’s Republic of China 2College of Resource and Environment Science, Xinjiang University, Urumqi 830046, People’s Republic of China 3University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China Abstract The genus Alyssum consists of about 195 species native to Europe, Asia and northern Africa. All species were assigned to six sections. Previous molecular phylogeny studies indicate that Alyssum is polyphyletic. However, the divergence time and dispersal of the genus are not well studied. In this study, the phylogenetic relationships within the genus Alyssum were studied with nrDNA ITS sequences obtained from five sections. The divergence time was estimated by fossil calibration and the biogeography was examined by spread analysis. The phylogeny indicated two main lineages: lineage 1 includes the section of Alyssum, Gamosepalum and Psilonema; lineage 2 includes the section of Odontarrhena, Meniocus and Clypeola.The phylogenetic relationship was not congruent with the previous sectional classifications. The age of Alyssum was dated to the upper Miocene. Molecular data suggested the diversification of Alyssum in Mediterranean areas and wide-ranging distribution such as North Africa, eastward into Central Asia and immigration into North America. Climatic aridification and arid/semiarid areas established in the Pliocene/Pleistocene could have provided favourable conditions for the migration and diversification of Alyssum. [Li Y., Kong Y., Zhang Z., Yin Y., Liu B., Lv G. and Wang X. 2014 Phylogeny and biogeography of Alyssum (Brassicaceae) based on nuclear ribosomal ITS DNA sequences. J. Genet. 93, 313–323] Introduction united with Alyssum (Warwick et al. 2008). Moreover, A. klimesii is most closely related to Ptilotrichum canescens ∼ The genus Alyssum consists of 195 species (Warwick et al. (Al-Shehbaz 2002), and it should belong to the tribe 2006) that are native to Europe, Asia and Northern Africa Arabideae (Al-Shehbaz et al. 2006). However, A. klimesii (Alshehbaz 1987). Species richness and diversity are con- formed the sister group with Crucihimalaya with a super fined to the Mediterranean and Turkey, and only a few network analysis of several genes (Koch and Matschinger species are distributed in North Africa, Central Asia, Siberia 2007). Warwick et al.(2008) suggested that A. klimesii and North America (Dudley 1964a, b). The genus comprises should be placed in Camelineae. German and Al-Shehbaz annual and perennial herbaceous plants and (rarely) small (2010) classified A. klimesii in the tribe Crucihimalayeae. shrubs, with oblong–oval leaves and yellow or white flowers The species of Alyssum are divided into six sections: (pink and purple in a few species), stellate, stalked or ses- Meniocus (Desv.) Hook, Psilonema (C. A. Meyer) Hook, sile trichomes, and dehiscent silicle fruit (Ball and Dudley Alyssum, Gamosepalum (Hausskn.) Dudley, Tetradenia 1964). (Spach) Dudley, and Odontarrhena (C.A.Meyer)Koch. Recent phylogenetic analyses indicate that Alyssum is Infrageneric phylogeny studies of section Odontarrhena polyphyletic (Al-Shehbaz et al. 2006; Warwick et al. 2008). have been conducted (Mengoni et al. 2003; Cecchi et al. Morphological evidence and internal transcribed spacer 2010), which demonstrate the monophyletic character of (ITS) sequencing data demonstrate that Clypeola should be section Odontarrhena. However, these investigations were mostly concentrated on the European taxa and focussed on the evolutionary dynamics of nickel hyperaccumulation. The inclusion of taxa from Asia and North America was ignored ∗ For correspondence. E-mail: [email protected]. in these studies, even though the information about these Keywords. Brassicaceae; phylogeny; ITS; divergence time; biogeography; Alyssum. Journal of Genetics, Vol. 93, No. 2, August 2014 313 YanLietal. groups is important for the biogeography and global dispersal 45 s annealing at 52◦C, and 1 min elongation at 72◦C; and of the genus Alyssum. a final elongation of 10 min at 72◦C. PCR products were The divergence time and migration of Alyssum are checked for length and concentrations on a 1.2% agarose gel still unclear. As indicated by Cecchi et al.(2010), the in TAE buffer. The gel was stained with nontoxic SYBR section Odontarrhena had a younger divergence time in Green. Before sequencing, the PCR products were purified Europe, with a mean ITS genetic distance of ∼2%, but using Sigma GenElute PCR Clean-up Kit (Sigma-Aldrich, there is no further investigation for the divergence time St Louis, USA) following the manufacturer’s protocol. of this genus (Cecchi et al. 2010). Section Odontarrhena Sequencing was performed on the automated ABI 3730 DNA is characterized by the prevalence of species that are dis- Analyzer, and the original amplification primers were used tributed on serpentine soils. They accumulate high concen- as double-strand sequencing primers. tration of nickel (>1000 μg/g dry mass), hitherto known as nickel hyperaccumulators. Previous study suggests that section Odontarrhena spread from the origin centre of Phylogenetic analysis Turkey based on the highest number of nickel hyperaccu- The ITS sequencing data were aligned using Clustal W mulators (Brooks and Radford 1978). However, the phylo- (Thompson et al. 1994) as implemented in MEGA 5.0 genetic relationships depicted by Mengoni et al.(2003) con- (Tamura et al. 2011). The maximum parsimony (MP) tree flicted with Brooks’ hypothesis. Mengoni argued that the was constructed using MEGA 5.0 with the tree-bisection- present area of distribution of the species might reflect histor- reconnection (TBR) search method. Gaps were treated as ical phenomena such as spreading immediately for the more missing data. Bootstrap values were calculated for analy- cold-sensitive species after the Ice Age. sis sampling trees (BEAST) 1000 replicates. The Bayesian In the present study, nuclear ITS of ribosomal DNA phylogenetic tree was constructed using the Bayesian evo- (include ITS1, 5.8S RNA and ITS2) with a larger collection lutionary software package BEAST ver. 1.7.4 (Drummond of Alyssum from five of six sections and broader geographic et al. 2012). Two independent runs of 45 million genera- distribution areas were used to (i) examine the phylogenetic tions were undertaken by sampling every 1000th generation. status of Alyssum and compare it with the traditional sec- Each run was checked using Tracer ver. 1.5 (Drummond tional classification; (ii) determine the infrageneric relation- and Rambaut 2007) for having reached a sufficient effective ship; and (iii) draw some conclusions of the Alyssum crown sample size (ESS over 100, as suggested by the authors) and stem age. in relevant statistics. The two runs (log-files and trees-files) were then combined with LogCombiner ver. 1.7.4. The maxi- mum clade credibility (MCC) tree was produced from 81,002 Materials and methods post-burn-in trees using TreeAnnotator ver. 1.7.4. Finally, Taxon sampling FigTree ver. 1.5 (http://tree.bio.ed.ac.uk/) was used to display and print the molecular phylogenetic tree. A total of 90 ITS sequences from 63 species were obtained The best fitting nucleotide substitution models were cho- for phylogenetic analysis. A total of 78 accessions from 53 sen using the program MrModeltest ver. 2.3 (Nylander Alyssum species, five Crucihimalaya taxa and four Clype- 2004). The model selected by Bayesian information criterion ola taxa were included in the sequences. ITS region for five (BIC) in the program was chosen for the BEAST analysis accessions representing three species of Alyssum from China and for the MP analysis, the criterion applied was the Akaike was sequenced. Aethionema grandiflorum was selected as information criterion (AIC) following Koch et al.(2010). the outgroup following studies on Brassicaceae (Galloway et al. 1998;Kochet al. 2001; Mathews and McBreen 2008; Couvreur et al. 2010; Franzke et al. 2011). Detailed sequence Divergence time estimates information, geographic origin and GenBank accession num- Based on the markers and calibration points used divergence- bers were provided in table 1. time estimates in Brassicaceae have been discussed widely. In the present study, divergence time was estimated by BEAST using estimated Brassicaceae age, macrofossil DNA extraction, PCR amplification and sequencing calibration and mutation rate of ITS. The primary BEAST Total DNA was extracted by the CTAB protocol (Doyle estimation was based on the age of Brassicaceae which was 1987). PCR amplifications of ITS region was performed in a dated 54.3 Mya (95% highest probability density (HPD) volume of 50 μL, containing 0.2 mM dNTPs, 0.5 μM of each interval: 45.2–64.2) by Beilstein et al.(2010). The secondary primer, 0.5 μL DMSO, 1.5 mM MgCl2 and2UofTaq poly- BEAST estimation used the macrofossils of Clypeola from merase (Takara Bio, Shanghai, China). The ITS region was upper Miocene dated 5–11 Mya (Schulz 1936).Therateof amplified using the primers ITS5 and ITS4 designed by mutation of ITS sequences for calculation was adopted in White et al.(1990). The amplifications were conducted under this research. The mutation rates for ITS region were vari- the following conditions: 5 min initial denaturation at 95◦C; able among plants and the value was between 1.72 × 10−9 35 cycles of amplification with 30 s denaturation at 95◦C, and 1.71 × 10−8 mutations per site per year in herbaceous 314 Journal of Genetics, Vol. 93, No. 2, August 2014 ITS for the phylogenic relationship of the genus Alyssum Table 1. List of studied taxa including voucher geographical origin and GenBank accession numbers. Genus Species Location Section Accession number Alyssum A. alpestre Italy Odontarrhena AY237957.1 A. alyssoides New Zealand Psilonema AF401114.1 A. alyssoides Wyoming, USA Psilonema EF514595.1 A.