Evaluation of species limits of Hortonia by DNA barcoding

* Corresponding author (sanathr@pdn.ac.lk) Abstract: A study was carried out to determine the species limits of the endemic genus Hortonia by DNA barcoding with a view to establishing conclusive molecular evidence regarding the speciation of Hortonia in Sri Lanka. Total DNA was extracted from all three species and the purity of the extracted DNA was confirmed. Internal transcribed spacer (ITS) region and the trnH-psbA region were first amplified by the polymerase chain reaction (PCR) with specific primers and ligated to a pBlueScript vector followed by plasmid purification. The purified plasmids containing the DNA of interest were subjected to sequencing. Sequence homology of ITS and trnH-psbA from all three species were compared using MacVector software. Between H. ovalifolia and H. floribunda, the ITS region showed a 2.37 % sequence divergence and the trnH-psbA region showed a 1.5 % sequence divergence. Between H. ovalifolia and H. angustifolia, the ITS region and trnH-psbA region showed a 3.36 % and 1.89 % sequence divergences, respectively. The percentage sequence divergence between ITS and trnH-psbA regions of H. floribunda and H. angustifolia were 3.36 % and 2.65 %, respectively. The high sequence divergence values clearly indicate that the genus Hortonia has three different species. Considering the percentage sequence divergence values, H. ovalifolia and H. floribunda are more closely related to each other than to H. angustifolia.


INTRODUCTION
Despite its relatively small size, Sri Lanka possesses a high level of biodiversity because of its topographic and climatic heterogeneity as well as its coastal influence.A noteworthy feature of Sri Lanka's biodiversity is the remarkable high proportion of endemic species among its flora and fauna.In the island's flora, the highest species diversity is recorded among the flowering plants.The endemic plant species diversity comprises 927 or 28 % of the flowering plants, of which 60 % are found in the lowland Wet Zone (Gunatilleke et al., 2008).
Sri Lanka is currently experiencing a tremendous increase in novel descriptions of its endemic diversity.Hortonia is a genus endemic to Sri Lanka, belonging to the family Monimiaceae Juss., which had possible origins in Gondwanaland about 100 −120 million years ago (Jayasekara, 1997).Hortonia is considered as a surviving genus of the ancestral plants from which the Monimiaceae alliance has evolved.Plants of this family are trees or shrubs and rarely climbers, and it comprises about 39 genera, where 440 species are widely spread in the tropical and subtropical regions of America (Leitao et al., 2000).This family occurs in Sri Lanka, Oceania, Polynesia, Australia, Malaysia, Madagascar and South America (Leitao et al., 2000).It is very rare in Africa and not reported from India.
Hortonia has had several taxonomic treatments: Wight (1853) considered three species (H.angustifolia, H. ovalifolia, H. acuminata); Hooker and Thomson (1855) described only one species, namely, H. floribunda while keeping angustifolia and acuminata as varieties; Thwaites (1864) too considered H. floribunda and placed angustifolia, ovalifolia and acuminata as varieties; a subsequent study described H. floribunda with ovalifolia as a variety and H. angustifolia with angustifolia and acuminata as varieties (Trimen, 1895).Later, Hortonia was considered as one species during a study on the phylogenetic affinities of the Monimiaceae based on cpDNA gene and spacer sequences.These data were the basis of Renner's assertion that H. ovalifolia and H. angustifolia were identical with H. floribunda (Renner, 1998).
The most recent revision of the family, however, recognizes three species of Hortonia; H. ovalifolia, H. floribunda and H. angustifolia (Dassanayake, 1996).Leaf morphology has been used as the main character to key the three species.According to this revision, leaves elliptic, rounded at base and apex belong to H. ovalifolia, leaves lanceolate to narrowly ovate and major veins arched belong to H. floribunda and leaves narrowly lanceolate to narrowly elliptic with major veins parallel to margin belong to H. angustifolia.In terms of the conservation status, H. ovalifolia is endangered while H. angustifolia is critically endangered (IUCN, 2007), and according to our studies, H. floribunda also has become increasingly difficult to locate (unpublished data).A study was undertaken to investigate the phytochemistry of all three species, based on the near identity of TLC patterns and the mosquitolarvicidal activity of the leaf extracts of the three species.Previously, the authors have reported the isolation of several new butenolides with mosquitolarvicidal activity from the leaves of all three species (Ratnayake et al, 2001(Ratnayake et al, , 2008a(Ratnayake et al, , 2008b)).It was concluded from these studies that the three species were phytochemically very similar.In another attempt Yakandawala and Rubasinghe (2003) re-evaluated the species limits of Hortonia using a large number of morphological characters and concluded the presence of three species, H. ovalifolia, H. floribunda and H. angustifolia.With a view to establishing conclusive molecular evidence regarding the speciation of Hortonia in Sri Lanka, the work described herein aimed to differentiate the species using DNA barcoding.
The surge in the application of molecular biology information to answer systematic and evolutionary questions has resulted in significant contributions to both plant and animal systematics.Among the different approaches used in molecular systematics, DNA sequencing has become one of the most widely used, particularly for species identification and discrimination among closely related species.DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, the DNA barcode.

METHODS AND MATERIALS
Leaf samples of H. angustifolia, H. floribunda and H. ovalifolia were collected from Kanneliya, Hakgala Botanic Gardens and the Adams Peak area in September 2009.The plants were identified at the National Herbarium of Royal Botanic Gardens, Peradeniya where voucher specimens are kept and the leaf samples were kept at -20 o C until use.Total DNA was extracted using a recommended procedure (Keb et al., 2002).Purity of the extracted DNA was confirmed by spectrophotometry.DNA from all three species were subjected to polymerase chain reaction (PCR) using high fidelity KOD plus DNA polymerase (Toyobo, Osaka, Japan) and universal primers for the internal transcribed spacer (ITS) region and the trnH-psbA region.The primer sequences were as follows: ITS5af (5'-CCTTATCATTTAG AGGAAGGAG-3') (Stanford et al., 2000), ITS4r (5'-TCCTCCGCTTATTGATATGC-3') (White et al., 1990): trnHf (5'-CGCGCATGGTGGATTCACAATCC-3') and psbA3f (5'-GTTATGCATGAACGTAATGCTC-3') (Sang et al., 1997).The PCR conditions for both ITS region and trnH-psbA regions were 3 min at 94 o C for heating, followed by 30 cycles of 30 s at 94 o C for denaturing, 30 s at 49 o C for annealing and 1 min at 68 o C for extension.
Amplified ITS and trnH-psbA regions were purified using a GeneClean kit and phosphorylated using T 4 polynucleotide kinase.Phosphorylated PCR products were ligated to a pBlueScript vector previously digested with EcoR V.The ligated vector was transformed to E.coli strain JM109 and the cells were grown at 37 o C in Luria Bertani (LB) medium containing ampicillin for 12−15 h.Cells were harvested by centrifugation, followed by plasmid purification and the purified plasmids containing the DNA of interest were subjected to sequencing using a 3100 sequencer (Applied Biosystems).Sequence homology of ITS and trnH-psbA from all three species were compared using MacVector software.

RESULTS
The sequence lengths of ITS and trnH-psbA regions of all three species were 802 and 527 base pairs, respectively.Between H. ovalifolia and H. floribunda, the ITS region showed a sequence difference in 19 base pairs accounting for 2.37 % sequence divergence and the trnH-psbA region showed a sequence difference in 8 base pairs accounting for 1.5 % sequence divergence.Between H. ovalifolia and H. angustifolia, the ITS region showed a sequence divergence in 27 base pairs accounting for 3.36 % sequence divergence and the trnH-psbA region showed a sequence difference in 10 base pairs accounting for 1.89 % sequence divergence.Moreover, the percentage sequence divergence between ITS and trnH-psbA regions of H. floribunda and H. angustifolia were 3.36 % and 2.65 %, respectively (Figures 1, 2 and Table 1).There was no divergence at all in both pairs of loci when sequences from different individuals of the same species were compared.

DISCUSSION
Methods to identify species using short orthologous DNA sequences have been initiated to facilitate biodiversity studies.For plant molecular systematic investigations at the species level, the ITS region of nuclear ribosomal cistron (18S-

Figure 1 :
Figure 1: Comparison of nucleotide sequences of ITS region.Nucleotide sequence alignment was performed using Mac Vector software.

Table 1 :
5.8S-26S) is the most commonly sequenced locus(Alvarez & Wendel, 2003).With the exception of ferns, the ITS region has shown broad utility across photosynthetic eukaryotes and fungi.In plant phylogenetic studies the ITS region is widely employed for species level discrimination.A recent study proposed that the trnH-psbA intergenic spacer plastid region that has good priming sites, and a shorter length with interspecific variation would be suitable as a universal barcode for land plants (Kress et al., 2005).Both ITS and trnH-psbA regions therefore have the potential to discriminate among closely related plant species.Percentage sequence divergence between the three species of the genus Hortonia Intraspecific variation in both these DNA barcodes is known to be extremely low, compared with interspecific variation.Notwithstanding phytochemical similarities, the high sequence divergence values clearly indicate that the genus Hortonia has three different species because a 0.5 % sequence divergence is sufficient to discriminate among plant species(Kress et al., 2005).Considering the percentage sequence divergence values, H. ovalifolia and H. floribunda are more closely related to each other than to H. angustifolia.This corroborates with the recent work on re-evaluation of the species limits of Hortonia by empirical methods (Yakandawela & Rubasinghe, 2003).
Figure 2: Comparison of nucleotide sequences of trnH-psbA region.Nucleotide sequence alignment was performed using Mac Vector software