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

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. ([2005], [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

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 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 version4.0b (Swofford [2000]). Heuristic search was carried out to determine the most parsimonious trees utilizing a tree-bisection 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.

Sequence characteristics

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.

Bayesian and parsimony analyses of the combined ITS/trnL-F data sets (Figure 1) shows a robustly supported Vanguerieae (PP = 1.00; BS = 99%) (Figure 1). The majority rule consensus tree of the combined ITS/trnL-F data sets (Figure 1) supports the monophyly of the included genera and recovered tree topologies similar with Lantz and Bremer ([2004]). For instance, the monophyly of Canthium s.s. is supported (PP = 0.96; BS = 67%) and is closely related to the large flowered group (PP = 1.00; BS = 85%); Keetia (PP = 1.00; BS = 64%) and Afrocanthium (PP = 1.00; BS = 89%) as sister taxa is likewise supported (PP = 0.98; BS = 72%); and the monophyly of Psydrax (PP = 0.94; BS = 85%) and Pyrostria s.s. (PP = 0.99; BS = 85%) were also sustained. The combined data analysis agrees with single marker analyses on the close relatedness of C. obovatifolium, C. oligophlebium and C. ramosii with Pyrostria (PP = 0.99; BS = 85%). Meanwhile, C. gynochthodes is finally resolved within Psydrax (PP = 0.94; BS = 85%).

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]), Lantz and 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]) in placing the species under Pyrostria s.l.. Bridson may have assigned this SE Asian species under Pyrostria group B due to the presence of bracts which resembles that of Pyrostria although Baillon ([1879]) did not mention the occurrence of this character. However, we examined herbarium sheets of C. gynochthodes [Gaerlan, F.J.M 0542753 (L, PNH); Romero, E.M. 0542751 (L, PNH); Soejarto, D.D. 0219674 (L, PNH); Arriola and Alejandro, 12442 (PNH, USTH); Arriola and 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.

Taxonomic treatment

We present here novel combinations of four species that were included in our study (Figure 2).

Pyrostria obovatifolia (Merr.) Wong, Magdaleno & Alejandro, comb. nov. Basionym: Canthium obovatifolium (Merr.) Merr., Philipp. J. Sci. 35 (1928) 8. Plectronia obovatifolia Merr., Philipp. J. Sci. C12 (1917) 167. Philippines, Luzon, Tayabas Prov. Mount Dalindingan, Sept. 1916, Ramos and Edano 26526 (holotype: PNH destroyed; lectotype: designated here K!; isolectotypes: US, HUH) (Figure 2A).

Shrub to small tree less than 3 m high; branches terete to a more or less quadrangular and glabrous. Leaves obovate, 3.5-7.5 × 1.0-3.0 cm, glabrous on both sides; apex rounded; base acute to acuminate; visible lateral nerves 3 to 4 on each side of the midrib; petiole 2.5-8.0 mm, glabrous. Stipules triangular to broadly ovate, 5.0-6.0 × 1.0 mm, glabrous on both sides. Female inflorescences axillary on 3.0-5.5 mm long peduncles, 6-flowered; peduncular bracts present, 3.0-5.5 mm long, triangular to broadly triangular, glabrous on both sides, enclosing the young inflorescence; pedicels erect, 3.0-4.0 mm long at flowering. Female flowers: calyx limb glabrous; tube 1.2- 2.5 mm long; lobes acuminate, 0.2 × 0.4 mm. Corolla 5-merous, white, glabrous outside; tube tubular, 0.8-1.2 mm long, hairs present at the throat; lobes broadly triangular, 2.0-2.5 × 1.0-1.2 mm, recurved. Stamens exerted, attached to corolla tube; anthers narrowly ovate to ovate, 0.3 mm long, exserted. Style including stigmatic knob 3.0-3.9 mm long; stigmatic knob 1 mm long, with a shallow cleft above, style not recessed into the stigmatic head; disk glabrous. Ovary 2-locular. Male flower unknown. Fruits ovoid 8.5-10.5 mm, glabrous.

Distribution:-Luzon Island: Ilocos Norte, Quezon

Habitat:-In secondary forest; 200-350 m altitude.

Phenology:- Flowering from March to June; Fruiting May to December

Taxonomic notes: This species approaches P. subsessifolia by its elliptic to ovate leaf shape but differs by its less conspicuous lateral nerves, shorter bracts and non- keeled fruits.

Pyrostria oligophlebia (Merr.) Pacia, Quiogue & Alejandro, comb. nov. Basionym: Canthium oligophlebium (Merr.) Merr., Philipp. J. Sci. 35 (1928) 8. Plectronia oligophlebia Merr., Philipp. J. Sci. 17 (1921) 442. Philippines, Luzon, Rizal Prov. Mount Susong Dalaga, Aug. 1917, Ramos and Edano 29342 (holotype, PNH destroyed; lectotype: designated here US; isolectotype: HUH, K !) (Figure 2B)

Shrub to small tree, less than 5.0 m high; branches quadrangular to more or less terete and glabrous. Leaves broadly lanceolate to oblong, 3.0-5.5 × 1.0-2.0 cm, glabrous on both sides; apex acute; base acute; visible lateral nerves 2 to 4 on each side of the midrib; petiole 0.7-1.0 mm, glabrous. Stipules broadly triangular to ovate, 1.0-2.0 × 1.0 mm, glabrous on both sides. Female inflorescences axillary on a glabrous peduncle less than 2 mm long, 7-12 flowered; peduncular bracts present, 2.5-3.0 mm long, triangular to broadly triangular, glabrous on both sides, enclosing the young inflorescence; pedicels erect, 4.0-5.0 mm long at flowering, persistent. Female flowers: calyx limb glabrous 2-2.7 mm long; lobes shortly toothed, 0.1 × 0.3 mm. Corolla 4-merous, white, glabrous outside; tube tubular, 0.8-1.2 mm long, hairs present at the throat; lobes broadly ovate, 2.0-2.5 × 1.0-1.2 mm. Stamens attached to corolla tube adjacent to the throat; anthers ovate, 0.3 mm long, exserted. Style including stigmatic knob 1.0-2.5 mm long; stigmatic knob 1 mm long, with a shallow cleft above; disk glabrous. Ovary 2-locular. Male flower unknown. Fruits ovoid 6.0-6.5 mm, glabrous with distinct indentation when dry.

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.

Pyrostria ramosii (Merr.) Arriola, Paraguison & Alejandro, comb. nov.

Basionym: Canthium ramosii (Merr.) Merr., Philipp. J. Sci. 35 (1928) 9. Plectronia ramosii Merr., Philipp. J. Sci. 7 (1921) 443. Philippines, Luzon, Tayabas Prov., Mount Umiray, June 1917, Ramos and Edano 28973 (holotype, PNH destroyed; lectotype: designated here NY!; isolectotype: K!) (Figure 2C)

Tree less than 9 m high; branches terete, glabrous. Leaves broadly lanceolate to oblong, 2.5-9.5 × 1.5-3.9 cm, glabrous on both sides; apex triangular to acuminate; base attenuate; visible lateral nerves 3 to 4 on each side of the midrib; petiole 1.5-2.0 mm, glabrous. Stipules triangular, 3.5-4.0 × 1.5-2.0 mm, glabrous on both sides. Inflorescences 4 flowered umbels; pedicels erect, 6.5-7.0 mm, persistent glabrous, peduncular bracts present, 0.5-1.5 mm long, triangular, glabrous on both sides. Infructiscence glabrous; stalk glabrous. Fruits ovoid to didymous 6.5-8.5 mm, 2 -celled, glabrous.

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.

Psydrax gynochthodes (Baill.) Arriola, Yayen & Alejandro, comb. nov.

Basionym: Plectronia gynochthodes (Baill.) Merr., Enum. Philipp. Fl. Pl. 3 (1923) 536. Canthium gynochthodes Baill., Adansonia 12 (1879) 199. Philippines, Luzon, Batangas Prov., 1917, Cuming 1848 (holotype, K!) (Figure 2D)

Tree, less than 7 m tall; branches flattened to subterete, glabrous. Stipules glabrous on both sides, triangular to ovate, 2-4.5 mm, keel prominent on the abaxial side. Petioles 1.0-5.0 × 0.3-0.5 mm, glabrous; leaf blades leathery, elliptic to elliptic-oblong or obovate, 3-8.5 × 1.5-5 cm, base acute to broadly acute, apex obtuse, glabrous throughout, glossy; lateral nerves 3-4 on each side of the midrib, domatia present. Inflorescences axillary, 10-12(-several) flowered, glabrous; peduncle 0.8-2.5 mm, glabrous; pedicel 4.0-10.0 mm, puberulent. Calyx tube infundibuliform, 2-3 × 2.0-2.5 mm, glabrous; lobes triangular 1.5-2.0 mm. Corolla tube infundibuliform, 1.0-1.5 mm, glabrous outside, with ring of white hairs inside; lobes 4, triangular, glabrous outside and inside. Anthers 4, reflexed. Style 2-6 mm, exceeding corolla tube, stigmatic-knob longer than wide, 0.3 × 0.2 mm, bifid. Ovary bilocular, 1-ovule per locule. Fruits ovoid to didymous, distinctly broader than long, 7.5-8.0 × 9-10 mm, green when young, glabrous, calyx limb persistent; Seeds 2, obliquely ovoid, ventrally flattened, 2-5.5 × 3 mm, apical crest very shallow to nearly inconspicuous, pyrene cartilaginous.

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.