A New Record of Pyrostria from the Philippines Inferred from Molecular and Morphological Data Submitted to Rey G. Tantiado In Partial Fulfillment of the Requirement in BIO 218A: Systematic Biology Submitted by Louregine S. Navarra BS Biology (Premed Track) October 2014 Introduction Background Information of the study The Vanguerieae belongs to subfamily Ixoroideae of the Rubiaceae with 600 to 700 species of trees, shrubs, lianescent shrubs and geofructices that are distributed throughout tropical Africa, Madagascar, Asia and the Pacific. Members of the tribe are characterized by a combination of axillary inflorescences, valvate corolla, a pendulous ovule, and a swollen stylar knob. Among the genera of the tribe, only Canthium Lam. is represented in the Philippines. Canthium sensu lato (s.l.) is one of the most problematic groups of Vanguerieae since the presence of a 2-locular ovary is the only cardinal character used to delimit the genus. As a result, Canthium s.l. forms a heterogeneous group which contains elements that have more pronounced generic differences than many of the accepted genera in Vanguerieae. Purpose of the Study The study aims to determine the affiliation of Philippine endemic C. subsessilifolium (Merr.) Merr. in the context of the present-day generic concepts in the Vanguerieae. In addition, the study aims to provide a species description, botanical illustration and conservation status assessment of the species. Also, this study aims to revise the whole Philippine Vanguerieae. Assumptions In the course of taxonomic revision of Canthium s.s. only species from Africa and Madagascar received attention. Conversely, there are numerous Asian species of Canthium including Philippine representatives left unresolved. In the Philippines, there are 20 currently recognized species of Canthium whose delimitation is still based on the work of Merrill (1928). As most of the endemic Philippine Canthium are without spines, their generic affinities should be reexamined. The assumptions are justifiable. Such that, the authors’ assumptions are based on molecular data like the internal transcribed spacer (ITS) and the trnL-F from the nuclear and chloroplast genomes, respectively, to better understand the phylogenetic relationships of plant species. Procedure The study involved techniques like DNA amplification, Sequence alignment, Bayesian inference, Model selection, Parsimony analysis, Consistency index, Retention index and Bootstrapping. There are no errors in the procedure outlined by the author and I would like to add no further changes in the experimental design. Data The data are all accurate, clear and relevant as it used QIAquick Purification Kit in the DNA amplification. Sequences alignment for the ITS and trnL-F were assembled and edited using the CodonCode Aligner v3.0.1. Bayesian inference was carried out using the MrBayes v.3.1.2p software and Model selection was determined using Akaike Information Criterion (AIC), AICc (seconder order criterion of AIC, necessary for smaller samples) and the Bayesian Information Criterion (BIC). Also, in analyzing single marker, the best performing model was selected and one million generation was considered with a sample frequency of 1000 and four parallel chains. Parsimony analysis was conducted using PAUP version4.0b and the most parsimonious trees were determined using heuristic search, tree-bisection reconnection (TBR) branch swapping using 10,000 random addition sequences, with MULTREES option on. Bootstrapping was determined using 10,000 replicates, MULTREES option off, TBR branch swapping, and five random addition sequences. Clades receiving a bootstrap (BS) value greater than 90% were considered strongly supported. Results The consistency and retention index of both the separate and combined datasets ranged from 0.60 to 0.93, which confirmed that data are far from being homoplasious and thus reliable in reconstructing phylogenies. The majority rule consensus tree of the combined ITS-trnL-F showed a highly supported monophyletic Vanguerieae (PP=1.00, BS=100%). The molecular results of the study served to confirm the hypothesis on the close affinity of C. subsessilifolium with Pyrostria. Conclusions and Implications The data and the conclusion support each other. The study implies a new phylogenetic position of Canthium subsessilifolium within the Pyrostria clade. The consequence of the study is that there would be a need to revise the whole Philippine Vanguerieae and it would be very time consuming. This would also imply, that systematists will need to verify if other species do really belong to the same clade or subgroup. Recommendations From the accomplished study, it is recommended that the experimentation and observation of the study be done using other systematic concepts and principles on data analysis. Literature Cited Molecular phylogeny and taxonomic revision of the Philippine endemic Villaria Rolfe (Rubiaceae) by Alejandro, G.J.D., Meve, U., Mouly, A., Thiv, M., Liede-Schumann, S. 2011. New combinations and names in Peponidium and Pyrostria (Vanguerieae – Rubiaceae) by Razafimandimbison, S.G., Lantz, H., Bremer, B. 2007. Phylogeny inferred from morphology and DNA data: characterizing well-supported groups in Vanguerieae (Rubiaceae) by Lantz, H., Bremer, B. 2004. Polyphyly of Mussaenda inferred from ITS and trnT-F data and its implication for generic limits in Mussaendeae (Rubiaceae) by Alejandro, G.J.D., Razafimandimbison, S.G., Liede-Schumann, S. 2005. Studies in African Rubiaceae – Vanguerieae: a new circumscription of Pyrostria and a new subgenus, Canthium subgen. bullockia by Bridson, D.M. 1985. The Philippine Rubiaceae genera: updated synopsis in INTKEY databases of the DELTA System by Alejandro, G.J.D., Liede, S., 2003. The reinstatement of Psydrax (Rubiaceae, subfam. Cinchonoideae, tribe Vanguerieae) and a revision of the African species by Bridson, D.M. 1985. Two new combinations in Pyrostria (Rubiaceae-Vanguerieae) from Thailand by Utteridge, T.M.A., Davis, A.P. 2009.