SPACER ( ITS ) SEQUENCES OF NUCLEAR RIBOSOMAL DNA INDICATE MONOPHYLY OF THE GENUS PHYTOLACCA L . ( PHYTOLACCACEAE )

Relationships within the family Phytolaccaceae sensu lato were examined based on internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA). The study revealed Phytolacca L. as taxonomically the most difficult genus in the family with completely unknown phylogeny. Molecular evidence was used from nrDNA ITS sequences of about 90% of the species for maximum parsimony analyses, and the molecular phylogenetic analyses defined a monophyletic Phytolacca. This first molecular phylogenetic study of Phytolacca concludes that the relationships among the species within the genus do not show harmony with the generic classification based on morphology. These results set the stage for a more detailed phylogenetic analysis of Phytolacca. Introduction The angiosperm family Phytolaccaceae sensu lato comprises a weedy, and polyphyletic genera (APGIII, 2009) of largely tropical and subtropical plants that have been placed, almost without exception, in Centrospermae under either the order Chenopodiales or Caryophyllales (Nowicke, 1969). The genus Phytolacca L. (family Phytolaccaceae) is commonly known as ‘pokeweeds’ comprises about 20 species (Nowicke, 1969) of perennial herbs, shrubs and trees, nearly cosmopolitan, mostly native to South America, with a few species in Africa and Asia (Shu, 2003). The genus Phytolacca possess alternate, simple leaves, pointed at the end, with entire or crinkled margins; the leaves can be either deciduous or evergreen; the stems are green, pink or red; the flowers are greenish-white to pink, produced in long racemes at the ends of the stems; they develop into globose berries 4−12 mm in diameter, green at first but dark purple to black after ripening (Nowicke, 1969). The generic name is derived from the Greek word phyton, meaning plant, and the Latin word lacca, meaning a red dye (Umberto, 2000). Phytolaccatoxin and phytolaccigenin, which are poisonous, are present in many species of the genus Phytolacca. The active principles for analgesic, anti-inflammatory, bactericidal, fungicidal, mitogenic and molluscicide action have been reported from several species of Phytolacca (Hernández et al., 2013). The active principles have also been found in methanolic extracts of fruit of P. tetramera Hauman, which is a source of saponins with fungicidal action (Escalante et al., 2002; Santecchia et al., 2002). The African soapberry plant, P. dodecandra L’Her., locally called endod, produces a range of triterpenoid Corresponding author. Email: alimohammad@ksu.edu.sa International Biological Material Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, South Korea. Email: joongku@kribb.re.kr Present address: Division of Plant Management, National Institute of Ecology, Choongnam, Secheon-gun, Maseo-myeon, Geumgang-ro, 1210, 325-813, South Korea


Introduction
The angiosperm family Phytolaccaceae sensu lato comprises a weedy, and polyphyletic genera (APGIII, 2009) of largely tropical and subtropical plants that have been placed, almost without exception, in Centrospermae under either the order Chenopodiales or Caryophyllales (Nowicke, 1969).The genus Phytolacca L. (family Phytolaccaceae) is commonly known as 'pokeweeds' comprises about 20 species (Nowicke, 1969) of perennial herbs, shrubs and trees, nearly cosmopolitan, mostly native to South America, with a few species in Africa and Asia (Shu, 2003).The genus Phytolacca possess alternate, simple leaves, pointed at the end, with entire or crinkled margins; the leaves can be either deciduous or evergreen; the stems are green, pink or red; the flowers are greenish-white to pink, produced in long racemes at the ends of the stems; they develop into globose berries 4−12 mm in diameter, green at first but dark purple to black after ripening (Nowicke, 1969).
The generic name is derived from the Greek word phyton, meaning plant, and the Latin word lacca, meaning a red dye (Umberto, 2000).Phytolaccatoxin and phytolaccigenin, which are poisonous, are present in many species of the genus Phytolacca.The active principles for analgesic, anti-inflammatory, bactericidal, fungicidal, mitogenic and molluscicide action have been reported from several species of Phytolacca (Hernández et al., 2013).The active principles have also been found in methanolic extracts of fruit of P. tetramera Hauman, which is a source of saponins with fungicidal action (Escalante et al., 2002;Santecchia et al., 2002).The African soapberry plant, P. dodecandra L'Her., locally called endod, produces a range of triterpenoid saponins possessing very potent and useful biological properties, including antifungal, antiprotozoan, spermicidal and insecticidal activities (Lemma et al., 1979).Because of its fastgrowing nature, P. dioica L. is frequently planted as a shade tree in the tropics.Nowicke (1969) reported the use of berries and the young sprouts, and leaves of some species of Phytolacca as an adulterant of red wine and poke salad, respectively.
The generic composition and phylogeny of Phytolaccaceae have long been controversial.The phylogenetic studies have substantially added new results to our knowledge of phylogeny of the family Phytolaccaceae (Brown and Varadarajan, 1985;Downie et al., 1997;Cuenoud et al., 2002;Lee et al., 2013).Nowicke (1969) referred Phytolacca as the most difficult genus in the family Phytolaccaceae sensu lato, and classified under three subgenera and six sections (Table 1).However, comprehensive information on phylogeny of the genus Phytolacca is lacking.

Molecular methods
Total genomic DNA was extracted by use of the DNeasy Plant Mini Kit from Qiagen (Valencia, CA, USA).The nrDNA ITS regions were amplified using the primers ITS1 and ITS4 (White et al., 1990).The DNA amplification for 35 cycles was carried out through PCR.Initial denaturation was carried out at 94°C for 5 min, followed by denaturation at 94°C for 1 min, annealing at 48°C for 1 min, extension at 72°C for 1 min, and the final extension at 72°C for 5 min.The PCR products were purified using SolGent PCR Purification kit-Ultra (SolGent, Daejeon, South Korea).For sequencing, the Big Dye Terminator chemistry (ABI) and an ABI 3100 Avant capillary sequencer were used.All sequences were BLAST-searched in GenBank.

Sequence alignments and phylogenetic analyses
Sequences were edited using the ABI Sequence Navigator (Perkin-Elmer/Applied Biosystems, USA).Sequence alignment was performed using Clustal X version 1.81 (Thompson et al., 1997), and subsequently adjusted manually using BioEdit (Hall, 1999).Information on sequence alignment can be made available from the corresponding author.Data were exported as a nexus file and subsequently analyzed using Maximum Parsimony (MP) in PAUP* 4.0b10 (Swofford, 2002).The MP analysis was performed with the following settings: heuristic search algorithms with tree bisection reconnecting (TBR) branch swapping, MULPARS in effect, all characters equally weighted, gap treated as missing characters, zero-length branches collapsed, random addition sequence set to 1000 replicates, and branch swapping limited to 10,000,000 rearrangements per replicate.When maximum parsimony trees were saved, a strict consensus tree was constructed.Bootstrap analysis was performed using 1000 replicates, with the random addition sequence set to 10, and branch swapping limited to 10,000,000 rearrangements per replicate.

Sequence characteristics
The combined length of the entire ITS region (ITS1, 5.8S and ITS2) from taxa analyzed in the present study ranged from 609−631 nucleotides (nt).The length of the ITS1 region and GC contents ranged from 220−232 nt and 56%−63%, the 5.8S gene was 166 nt long, the length of the ITS2 region and the GC content ranged from 221−235 nt and 55%−63%, respectively.Data matrix has a total number of 654 nt characters of which 423 nt characters were constant, 88 nt characters were variable but parsimony-uninformative, and 143 nt characters were parsimony-informative.

Phylogenetic analyses
The parsimony analysis of the entire ITS region resulted a total number of four maximally parsimonious trees (MPTs) with a total length of 252 steps, a consistency index (CI) of 0.7110, a homoplasy index (HI) of 0.2890, rescaled consistency index (RC) of 0.5361 and a retention index (RI) of 0.7540 (Fig. 1).
The generic composition of Phytolacca has long been controversial principally due to common occurrence of intraspecific variability and hybridization (Fassett and Sauer, 1950;Sauer, 1951).Walter (1909) placed 26 species of Phytolacca into three subgenera based on the degree of connation of the carpels: free, connate at the base with the apices free, or completely united carpels.The subgenus Pircunia (Moq.)H. Walter Nowicke (1969) did not consider the names assigned to hybrid origin.Nowicke (1969) recognized a total of 20 species in the genus Phytolacca and classified them into resulting from the analysis of nrDNA ITS sequences are strongly supported as a monophyletic group (100% BS).However, the relationships among the species within the genus do not show harmony with the previous generic classification based on morphology.In the present analysis, a total number of 143 out of 654 (21%) sites of sequence data set were phylogenetically informative, so further sampling of additional taxon and addition of more regions are needed for the robust phylogeny of the genus Phytolacca.We herein based on the present analysis hypothesize that the intraspecific classification of Phytolacca should be recircumscribed into subgenus Phytolacca (P.acinosa, P. americana, P. bogotensis, P. brachystachys, P. dodecandra, P. heterotepala, P. icosandra, P. meziana, P. octandra, P. purpurascens, P. rivinoides, P. rugosa, P. sanguinea and P. thyrsiflora), subgenus Pircuniopsis (P.dioica, P. tetramera and P. weberbaueri), and P. heptandra should be treated under an independent subgenus.This treatment, as a hypothesis, however, needs testing and further data would help to clarify their true intraspecific affinities.

Fig. 1 .
Fig. 1.The bootstrap strict consensus of four maximally parsimonious trees of Phytolacca L. species based on the ITS sequence with gaps being treated as missing data (252 steps, CI= 0.71, HI= 0.28, RC= 0.53 and RI= 0.75).Bootstrap values greater than 50% in 1000 replicates are shown above lines.