Pollen morphology of Philippine species of Phyllanthus ( Phyllanthaceae , Euphorbiaceae s . l . )

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INTRODUCTION
Phyllanthus is the largest genus of the Phyllanthaceae (Eu phorbiaceae s.l.). It contains about 830 species (Govaert et al. 2000). On the basis of a large molecular analysis of Phyllanthus and its relatives, Kathriarachchi et al. (2006) recommended the inclusion of Breynia, Glochidion, Reverchonia and Sauropus to form a large monophyletic genus Phyllanthus s.l. This genus would then comprise c. 1 269 species (Govaert et al. 2000). They found that the subgenera Conami, Emblica, Eriococcus, Gomphidium (sensu Schmid 1991) and Xylophylla (except for P. sellowianus) are monophyletic, whereas the subgenera Isocladus, Kirganelia and Phyllanthus appeared to be not monophyletic. Most clades obtained in their analyses were supported by palynological characters.
In the study of the West Indian species of Phyllanthus, Webster (1956) discovered the extraordinary diversity of pollen morphology within the species using light microscope (LM). Punt (1967) showed two series of Phyllanthus pollen types and proposed their evolutionary trends. The precise usage of pollen characters in taxonomy for the small pollen size, usually around 20 -30 μm diam is difficult, therefore the aid of scanning electronic microscope (SEM) in the observation of the exine ornamentation is necessary. At least 30 pollen types were reported in 220 species of Phyllanthus studied using LM and/or SEM (Erdtman 1952, Webster 1956, 1986, Köhler 1965, Punt 1967, 1980, 1987, Punt & Rentrop 1973, Bor 1979, Meewis & Punt 1983, Long & Yu 1984, Rossignol et al. 1987, Lobreau-Callen et al. 1988, Chen & Wu 1997, Webster & Carpenter 2002, Wei et al. 2002, Sagun & Van der Ham 2003, Santiago et al. 2004).
In the Philippines, more than 30 species of Phyllanthus have been reported previously and can be classified into five subgenera and eleven sections (Elmer 1908, 1910, Robinson 1909, Merrill 1912, 1914, 1920, 1923, Quisumbing & Merrill 1928, Airy Shaw 1983, Hoffmann et al. 2003. The minute unisexual flowers and the variable leaf morphology caused the classification of some species to remain in doubt. In order to provide new evidence for the species classification, we undertook the present pollen morphological study of Philippine Phyllanthus species.

MATERIAL AND METHODS
Pollen samples were collected from specimens deposited in the following herbaria: L, NHU, PUH (Table 1).
Per sample, anthers of 1-3 flowers were placed in 1.5 ml centrifuge tubes, immersed in acetic acid for 30 min until soft, crushed by a glass bar to release the pollen grains, and then acetolyzed (Erdtman 1952). The acetolyzed pollen was then dehydrated in a series of 70 %, 90 % and 100 % ethanol and pipeted onto a filter membrane to be air dried. Under a thin layer of gold coating, the pollen grains were examined and photographed using a Hitachi S-800 Scanning Electron Microscope. Pollen size was based on measuring 2-4 pollen grains per sample. The terminology used in this paper follows Huang (1972) and Punt et al. (2007).

RESULTS
Eleven pollen types were recognized in the Philippine Phyl lanthus species studied ( Note -The number of lumina per areola varies: P. lanceifo lius and P. samarensis: ± 30, P. virgatus: ± 60, and Phyllanthus aff. samarensis: ± 70. Phyllanthus aff. samarensis differs from the other species of the P. virgatus type by its colpus margins consisting of two parallel muri rather than four.
Note -The P. maritimus type is similar to the P. casticum type, but differs by the presence of granules in the lumina.

Pollen types and infrageneric classification
Five of the 11 pollen types in Philippine Phyllanthus each occur in a single section (  Punt 1967Punt , 1987. Transferring P. lanceifolius to sect. Macraea should therefore be considered. Pollen morphologically, subg. Gomphidium, including three pollen types, is most heterogeneous in Philippine Phyllanthus. The P. maritimus type (sect. Adenoglochidion) is 3-syncolporate. The P. glochidioides type and P. securinegoides type (both sect. Gomphidium) have 4-colporate pollen. They differ by their exine ornamentation and the morphology of the colpus margins. Merrill (1914) found P. securinegoides hard to classify. He noticed that P. securinegoides did not fit in sect. Gomphidium, because the scarce styles are united, spreading and prominently cleft. Hence, he suggested that a new section should be described. Pollen morphology confirms Merrill's opinion.
Phyllanthus sp. A has not been assigned to a section and subgenus yet. Its pollen (Pilate type), is similar to the pilate pollen of P. attennutus (subg. Conami, sect. Hylaeanthus; Webster & Carpenter 2002), but has a 4-colporate apertural system.

Pollen types and phylogenetic relationships
Subgenera Isocladus, Kirganelia and Phyllanthus are not monophyletic, based on ITS and matK DNA sequence analyses (Kathriarachchi et al. 2006). According to these data, sect.
Paraphyllanthus is more primitive than sect. Macraea (subg. Isocladus), sect. Anisonema more primitive than sect. Cicca (subg. Kirganelia) and P. amarus more primitive than sect. Uri naria (subg. Phyllanthus). Punt (1967) considered an increased number of colpi as a derived feature. Thus, the P. erythrotrichus type of sect. Paraphyllanthus would be more primitive than the P. virgatus type of sect. Macraea, and the P. amarus type of sect. Phyllanthus is more primitive than the P. urinaria type of sect. Urinaria. In these cases the pollen morphological trend proposed by Punt is concordant with the DNA sequence data. According to Punt (1967), the colporate P. acidus type of sect.
Cicca is more primitive than the syncolporate P. casticum type of sect. Anisonema. This conflicts with the DNA sequence data. However, comparing the ornamentation in detail, the P. casticum type of sect. Anisonema has a primitive reticulate ornamentation and the P. acidus type of sect. Cicca has an advanced rugulate-reticulate ornamentation. This result tells us that the ornamentation trend is concordant with the DNA sequence data of Kathriarachchi et al. (2006).