Aporosa tetragona Tagane & V. S. Dang (Phyllanthaceae), a new species from Mt. Hon Ba, Vietnam

Abstract A new species, Aporosa tetragona Tagane & V. S. Dang, sp. nov., is described and illustrated from Mt. Hon Ba located in the Khanh Hoa Province, South Vietnam. This species is characterized by tetragonal pistillate flowers and fruits, which are clearly distinguishable from the other previously known species of the genus. The morphology and phylogeny based on rbcL and matK of this species indicated that the new species belongs to section Appendiculatae Pax & K. Hoffm.


Introduction
Th e genus Aporosa Blume (1825) (Phyllanthaceae) comprises 82 species of small to medium sized trees distributed in various environments in South and Southeast Asia. Th ey can be found in both primary and secondary forests, from lowland rain to dry deciduous, and as well as montane forest (up to 2200 m altitude in New Guinea) (Gagnepain 1927, Hô 2003, Schot 2004, Schot and van Welzen 2005. Th e genus is characterized by a dioecy, indumentum of simple hairs, petioles pulvinate at both base and apex, two glands on adaxial base of lamina (occasional), often with scattered disk-like glands on lower surface of lamina, axillary infl orescences, absence of petals, styles and disks, tiny staminate fl owers with a minute or absent pistillode, and dehiscent regmata with persistent stigmas, sometimes beaked and/or stiped (Pax andHoff mann 1922, Schot 2004).

Morphological observations
Th e new species was recognized by detailed comparisons with morphologically similar species through literature review, dry specimens from the herbaria ANDA, BK, BKF, BO, HN, KYO, SING, TNS, VNM, and digitized plant specimens available on the web (e.g. JSTOR Global Plants (https://plants.jstor.org/)).

DNA barcoding
Total DNA was extracted from silica-gel dried leaves collected in the fi eld. DNA extraction was performed by a modifi ed CTAB protocol (Doyle and Doyle 1987), as described in detail in Toyama et al. (2015). Amplifi cation and sequencing of the two DNA barcodes regions, rbcL and matK, were performed according to published protocols (Kress et al. 2009, Dunning andSavolainen 2010).

Phylogenetic analysis
In total, 22 accessions representing 14 species of Aporosa were included in phylogenetic analyses using DNA barcoding regions of rbcL (362 bp) and matK (392 bp) ( Table 1). In addition to the new species, Aporosa tetragona Tagane & V. S. Dang, four species, A. aurea Hook. f., A. microstachya (Tul.) Müll. Arg., A. penangensis (Ridl.) Airy Shaw and A. tetrapleura Hance, were newly sequenced in the present study. Th e remaining sequences were obtained from GenBank. Phyllanthus bokorensis Tagane was used as an outgroup. Th e sequence alignment was performed by ClustalW with default parameter implemented in MEGA v 6.06 (Tamura et al. 2013).
Th e Neighbor-joining methods (Saitou and Nei 1987) with Maximum Composite Likelihood distance matrix (Tamura et al. 2004) implemented in MEGA v 6.06 was used to construct the phylogenic trees. Confi dence values for individual branches were determined by bootstrap analysis with 10,000 repeated samplings of the data.

Results and discussion
Th e new species belongs to the section Appendiculatae as the leaf lamina has basal adaxial glands (Fig. 2C), disc-like glands unevenly scattered within the arches of the marginal veins throughout the abaxial surface of the lamina (Fig. 2B), papillate stigmas (Fig. 2D), and pubescent septae and column in the ovary (Fig. 2E) (Schot 2004), but is distinguished from previously known species by its tetragonal ovary of the pistillate fl ower and the fruit.
Th e Neighbor-joining tree based on rbcL and matK supports the separation of each morphologically defi ned section and the monophyly of sect. Appendiculatae, sect. Benthamianae and sect. Sundanenses with 98 %, 76 % and 85 % bootstrap probability, respectively (Fig. 3). Th e new species was placed in sect. Appendiculatae and clearly separated from other species of this section with a sister relationship to the clade including A. fi cifolia, A. odctandra var. octandra, A. planchoniana and A. villosa.
Th us, the species is morphologically distinct from known taxa and the phylogeny supports the separation from related species. Here, we defi ne the new species Aporosa tetragona Tagane & V. S. Dang. Diagnosis. Aporosa tetragona is distinct from all other Aporosa species by having a tetragonal ovary and fruit. Th e leaves are similar to Aporosa acuminata Th waites, but diff ering in not only fruit shape but also wider leaves (vs. 2-4 cm wide), larger pistillate fl owers (vs. 2-3.5 mm long), and glabrous fruits (vs. sparsely puberulous).
Distribution and habitat. Th is species is currently known only from Hon Ba Nature Reserve, Khanh Hoa Province, South Vietnam. Th e small populations were found at the edge of humid broad-leaved evergreen forest close to a stream, altitude 200-400 m.
Etymology. Th e specifi c epithet tetragona refl ects the quadrangular shape of the ovaries in the pistillate fl owers and fruits.
GenBank accession No. Toyama et al. V1976: LC050338 (rbcL), LC050339 (matK). Conservation status. Th e species is known only from the type locality in Mt. Hon Ba at 200-400 m altitude. It is suggested that Aporosa tetragona should be placed under the IUCN category 'Critically Endangered' (CR) (IUCN 2012) because of its limited distribution with an area of occupancy estimated to be less than 10 km 2 (criterion B2 a) and a small number of individuals estimated to be less than 250. Recent botanical inventories carried out in this narrow area along stream discovered several new species, including Dillenia tetrapetala Joongku Lee, T. B. Tran & R. K. Choudhary (Choudhary et al. 2012), Goniothalamus fl agellistylus Tagane & V. S. Dang (Tagane et  al. 2015) and Vanilla atropogon Schuit., Aver. & Rybková (Schuiteman et al. 2012), all of which are rare and endemic to Mt. Hon Ba. Th erefore further collection eff orts around this area are necessary to accurately understand the fl ora there and to update the conservation status of the species.