Generic affiliations of Canthium species placed under Pyrostria group B sensu Bridson (Vanguerieae, Rubiaceae) inferred from morphology and molecular data

Pyrostria sensu lato (s.l.) is regarded as one of the polyphyletic group within Vanguerieae formerly comprising of Pyrostria sensu stricto (s.s.), Pyrostria group A and Pyrostria group B delineated by the number of locules and geographical occurrence. Recent molecular phylogenetic studies within the genus have narrowed its circumscription that resulted in the merging of Pyrostria group A and Pyrostria s.s. Although some species of Pyrostria group B were already transferred to Pyrostria s.s. and Psydrax based on morphology, other representatives of the group remain unsettled. Bayesian and parsimony analysis of the combined ITS (nrDNA) and trnL-F (cpDNA) datasets showed a well-supported clade of the whole Vanguerieae containing four Philippine endemic representatives of Pyrostria group B. The placement of Canthium oligophlebium, Canthium obovatifolium and Canthium ramosii within Pyrostria s.s. (PP = 0.99; BS = 85%) is robustly supported likewise the affiliation of C. gynochthodes with Psydrax (PP = 0.94; BS = 85%). Morphological features shared by our species with Pyrostria s.s. and Psydrax further supports our molecular data. Our study supports the earlier hypothesis that Canthium oligophlebium, C. obovatifolium and C. ramosii should be placed under Pyrostria s.s. except for C. gynochthodes that grouped with Psydrax. Four new combinations are proposed in this study. The generic affiliations of other species of Pyrostria group B should be reinvestigated towards a more natural classfication in Vanguerieae.


Background
The tribe Vanguerieae has long been regarded as monophyletic since it is easily delineated from the rest of Rubiaceae by the presence of a unique type of pollen presenter above the style (Verdcourt 1987;Bridson 1992). Recent molecular phylogenetic studies of the tribe (Lantz et al. 2002;Bremer 2004, 2005) support previous classifications of Bridson (1985Bridson ( , 1986Bridson ( , 1992 in reinstating Keetia E. Phillips and Psydrax Gaertn. and raising Canthium subg. Afrocanthium Bridson to a genus level. Meanwhile, Vangueria Juss. was recircumscribed and is distinguished from its close relatives by having inflorescences borne from where leaves have fallen, small bracteoles in secondary branch and large fruit with 2 to 5 locules. The genus Canthium was regarded as highly polyphyletic and redelimited to include species with supraaxillary spines. Although major evolutionary lineages and new generic limits have been established within Vanguerieae utilizing * Correspondence: arriolaaxel@yahoo.com 3 The Graduate School, University of Santo Tomas, España, Manila 1015, Philippines morphology and molecular data, there are still understudied species. When Bridson (1987) reinstated the genus Pyrostria Comm. ex A. Juss., she lumped all Pyrostria species and representatives of Canthium with pair of persistent connate bracts (bracteate species) under Pyrostria s.l. and suggested informal groups (Pyrostria s.s., Pyrostria group A and Pyrostria group B) based on the number of locules and geographical occurrence. Both Pyrostria s.s. (pluri-locular ovary) and Pyrostria group A (bilocular ovary) have unisexual flowers and well represented in Madagascar, the latter extends from Africa to Arabia. Meanwhile, Pyrostria group B shares characters with Pyrostria group A in having unisexual flowers, bilocular ovaries, broad corolla tube, and 4-5 corolla lobes but the former radiates as far as SE Asia. The polyphyly of Pyrostria s.l. was initially addressed by Lantz and Bremer (2004) in recovering a clade composed mainly of dioecious species but failed to discussed further this group due to the poor internal support. Razafimandimbison et al. (2009) (Ruhsam et al. 2008).
There are still left unresolved endemic Philippine species placed under Pyrostria group B probably associated with Pyrostria such as C. brunneum (Merr.) Merr., C. ellipticum (Merr.) Merr., C. gynochthodes Baill., C. megacarpum (Merr.) Merr., C. obovatifolium (Merr.) Merr., C. oligophlebium (Merr.) Merr., C. ramosii (Merr.) Merr., and C. subcapitatum (Merr.) Merr. Available herbarium materials of these species are scarce and lack reproductive parts for confirmation. In this study, four species of Canthium informally placed under Pyrostria group B: C. gynochthodes, C. obovatifolium, C. oligophlebium, and C. ramosii were collected and challenged their phylogenetic positions within Vanguerieae utilizing molecular sequence data. Furthermore, type specimens were meticulously examined to confirm our molecular results. The present study is a good contribution in understanding a more robust phylogenetic evolutionary trends and lineages within the tribe.

Taxon sampling
This study is based on the examination of herbarium sheets from various herbaria as well as field observation. Canthium gynochthodes, C. obovatifolium, C. oligophlebium and C. ramosii were collected based on their type protologues. Collected samples (herbarium specimens and preserved reproductive structures in 70% ethanol) were deposited at the USTH for accessioning. Leaf samples were dried in silica-gel for DNA extraction (Chase and Hills 1991).

Molecular methods
Genomic DNA was extracted from silica gel-dried leaf samples using the DNeasy Plant Minikit (Qiagen, Germany). The entire ITS region (including the 5.8S gene) was amplified and sequenced using the primer pair P17F/26-82R and P16F/P25R (Popp and Oxelman 2001). Meanwhile, primer pair c/f were used for both amplification and sequencing of the trnL-F region (Taberlet et al. 1991). DNA amplification was carried out following the work of Alejandro et al. (2005Alejandro et al. ( , 2011. Amplified DNA was purified using the QIA-quick Purification Kit (Qiagen, Germany). Purified DNA was sent to MACROGEN Inc., Seoul, South Korea for sequencing.

Phylogenetic analysis
The ITS and trnL-F sequences were assembled and edited using the Codon Code Aligner version 3.0.1. Novel sequences of the four Philippine Canthium from each of the markers used were incorporated with several related sequences from the work of Lantz and Bremer (2004) taken from the GenBank (Table 1). Ixora coccinea L. and Mussaenda erythrophylla Schumach. & Thonn., considered as closely related to Vanguerieae were used as the outgroups. Sequences were aligned manually using Se-Al v.1.0al (Rambaut 1996).
Bayesian inference (BI) was used to estimate phylogenetic positions of the Philippine endemic Canthium species. The analysis was carried out using the MrBayes v.3.1.2p software (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003;Altekar et al. 2004). Model selection for the best-performing evolutionary models were determined under three model selection criteria: a) Akaike Information Criterion (AIC) (Akaike 1974), b) AICc (seconder order criterion of AIC, necessary for smaller samples) and c) the Bayesian Information Criterion (BIC) (Schwartz 1978). The selected models were HKY and GTR + G for the ITS and trnL-F, respectively. 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. For combined analyses, model selection as well as the settings is similar with that of the single-marker analysis, however there Table 1 Nucleotide sequence database accession numbers of taxa used in this study

Taxon
GenBank/EMBL Accession Number Canthium glaucum Hiern ssp. glaucum AJ617752 AJ620124 Canthium gynochthodes 1 HG937666 HG937663 Canthium inerme (L.f.) Kuntze AJ315120 AJ620125 were a total of three million running generations. Clades with posterior probability (PP) exceeding 0.95 were regarded as strongly supported. Parsimony analysis was conducted using PAUP ver-sion4.0b (Swofford 2000). Heuristic search was carried out to determine the most parsimonious trees utilizing a treebisection reconnection (TBR) branch swapping using 10,000 random addition sequences, with MULTREES option on. Consistency index (Kluge and Farris 1969) and retention index (Farris 1989) were calculated to determine if the data is far from being homoplasious. Bootstrapping was determined using 10,000 replicates, MULTREES option off, TBR branch swapping, and five random addition sequences. Clades receiving greater than 90% were considered strongly supported. Table 2 shows the matrix characteristics of the separate and combined ITS and trnL-F data sets. The aligned matrix of the 43 taxa of the ITS region includes a total of 691 positions, 190 base pairs (bp) of which are phylogenetically informative. The 43 sequences of trnL-F, have a total of 1,002 positions, 43 bp of which are informative. The combined ITS/trnL-F of the 43 taxa with 1,693 characters generated a total of 233 bp informative characters.

Phylogenetic analysis
The tree topologies of the separate ITS (PP = 1.00; BS = 100%) and trnL-F (PP = 0.89; BS = 90%) analyses (trees not shown) revealed a monophyletic Vanguerieae. Both trees resolved the phylogenetic positions of C. obovatifolium, C. oligophlebium and C. ramosii in Pyrostria clade with high support in ITS (PP = 0.96; BS = 89%) and trnL-F (PP = 1.00; BS = 85%). However, both separate analyses failed to resolve the placement of C. gynochthodes within the tribe and polytomies were observed for members of Canthium s.s and Psydrax.

Generic affiliations of species under Pyrostria group B
The results presented above clearly shows the polyphyly of Canthium as earlier observed by Lantz et al. (2002), Bremer (2004, 2005) and Razafimandimbison et al. (2009). The four Canthium (C. gynochthodes, C. obovatifolium, C. oligophlebium and C. ramosii) should be excluded from Canthium s.s. since these species are spineless.
The phylogenetic position of C. obovatifolium, C. oligophlebium and C. ramosii within the Pyrostria clade was already anticipated due to the presence of a persistent, basally paired, connate to acuminate bracts as observed in our recent collections and available herbarium sheets. The synapomorphic characters of Pyrostria such as dioecious sexuality and fleshy corolla with trichomes in the throat (Lantz and Bremer 2004) were also observed in our sampled Canthium species. The placement of these three Canthium species in Pyrostria s.l. was already suggested by Bridson (1987) but she was unsure of the placement in  the genus due to their geographical occurrence falling outside the known range, i.e. at that time Pyrostria was considered to be a predominately Afro-Madagascan genus. Although Pyrostria is mostly represented in Africa, Ruhsam et al. (2008) mentioned that the presence of SE Asian Pyrostria could probably be a disjunct part of their African relatives. There is a possibility that species under this genus may have undergone long range dispersal from Africa to Asia as in the case of Mussaenda L. (Alejandro et al. 2005).
Meanwhile, the phylogenetic placement of C. gynochthodes within Psydrax does not support the earlier suggestion of Bridson (1985) Alejandro, 11057 (PNH, USTH)] and revealed that bracts exist in younger inflorescences but totally absent in older ones. The presence of bracts on young inflorescences of C. gynochthodes will not affect its close relatedness with Psydrax. According to Bridson (1987) bracts may be present in some representatives of Psydrax, however, it is distinctive from the paired connate bracts of Pyrostria which are rare in Vanguerieae. The presence of bracts is not a cardinal character to delimit Psydrax. For instance, Bridson (1985) mentioned of the occurrence of bracts in the Indian Psydrax umbellata (Whit.) Bridson and unnamed Malayan species. Furthermore, examination of C. gynochthodes revealed that it posseses other diagnostic features of Psydrax such as coriaceous leaf blades, keeled stipules with truncate to triangular stipular base and falcate stipular apex, reflexed anthers, long style always exceeding the corolla tube, longer than wide stigmatic knob, cartilaginous seed and a very shallow to nearly inconspicuous apical crest without a lid-like area in the pyrene (Bridson 1985;Cheek and Sonke 2004). Additionally, the occurrence of a unique insertion of 40 bp in the trnL-F region of Psydrax that is non-alignable with other species of Vanguerieae (Lantz and Bremer 2004) exists in C. gynochthodes.
The close relatedness of species placed under Pyrostria group B with Pyrostria s.s. and Psydrax are supported by morphology and molecular data. Therefore, it is necessary to recollect the remaining species of Pyrostria group B to determine their correct generic affiliations within the tribe.
Distribution:-Luzon Island: Rizal; Mindanao Island: Davao Habitat:-In secondary forest; 500-900 m altitude. Phenology:-Flowering from March to December; Fruiting from Septmber to February Taxonomic notes: The smaller and fewer nerved leaves of P. oligophlebia approaches P. gynochthodes. However, P. oligophlebia differs from the latter by having persistent pair of bracts, many-flowered inflorescences and a longer petioles, peduncles and pedicel.
Distribution:-Luzon Island: Quezon Habitat:-In secondary forest; 200-350 m altitude. Phenology:-Fruiting August to December Taxonomic notes: Pyrostria oligophlebia and P. ramosii closely resemble each other due to their oblong shape leaf with acuminate apex. However, the latter have longer peduncles and few (2-4) flowered umbellate inflorescences as compared to the numerous (7-12) flowered inflorescence of the former.
Distribution:-Philippines, Taiwan Habitat:-In secondary forest; 500-900 m altitude. Phenology:-Flowering from March to June; Fruiting from June to December Taxonomic notes: Psydrax gynochthodes is comparable with P. obovatifolia, P. oligophlebia and P. ramosii. However it is delineated from the three species due to the absence of persistent acuminate-connate bracts. For the above reason, we do not agree with the observation of Bridson (1987) that P. gyncochthodes is closely associated with P. villarii Vidal and that the two species should be synonymize. Moreover, P. gynochthodes can be distinguised from the Philippine P. amplifolia by its smaller, thicker and darker glossy green leaves, umbellate to cymose inflorescences, longer peduncles and smaller fruits.

Conclusion
The generic affiliations of four species previously hypothesized under Pyrostria group B have been resolved based on morphology and molecular sequence data. We formally proposed three novel combinations in Pyrostria and a Psydrax. Other species of Pyrostria group B should be reinvestigated towards a more natural classfication in Vanguerieae. Furthermore, large number of Pyrostria and bracteate species temporarily placed under Canthium s.l should be sampled to fully understand the evolutionary dispersal of the genus.