Rinorea niccolifera (Violaceae), a new, nickel-hyperaccumulating species from Luzon Island, Philippines

Abstract A new, nickel-hyperaccumulating species of Rinorea (Violaceae), Rinorea niccolifera Fernando, from Luzon Island, Philippines, is described and illustrated. This species is most similar to the widespread Rinorea bengalensis by its fasciculate inflorescences and smooth subglobose fruits with 3 seeds, but it differs by its glabrous ovary with shorter style (5 mm long), the summit of the staminal tube sinuate to entire and the outer surface smooth, generally smaller leaves (3–8 cm long × 2–3 cm wide), and smaller fruits (0.6–0.8 cm diameter). Rinorea niccolifera accumulates to >18,000 µg g-1 of nickel in its leaf tissues and is thus regarded as a Ni hyperaccumulator.


Introduction
Rinorea Aublet (Violaceae) is a pantropical genus of forest shrubs and trees. It is the second most species-rich genus in the family after Viola L., with an estimated total of 225-275 species throughout the tropics (Wahlert and Ballard 2012). In the Malesian region, only 11 species are recognized in the genus, with four species attributed to the Philippines (Jacobs and Moore 1971). However, the very broad circumscriptions of the widespread Rinorea bengalensis (Wall.) Kuntze and Rinorea javanica (Blume) Kuntze in the taxonomic revision of Jacobs and Moore (1971) needs a closer re-examination. A few new taxa from Borneo have subsequently been added for the region (e.g. Forman and Ahmad 1996, Jarvie and Stevens 1998, Stevens 2000. In Rinorea, at least three species are known to hyperaccumulate the heavy metal nickel. Rinorea bengalensis (Wall.) Kuntze was the fi rst nickel hyperaccumulator species of Rinorea discovered with up to 17,500 μg g -1 (dry weight) based on herbarium specimens from throughout Southeast Asia, including the Philippines . A subsequent analysis of herbarium material of 70 other species of Rinorea from Central and South America, Africa, and Asia also revealed another species, Rinorea javanica (Blume) Kuntze, as a nickel hyperaccumulator with up to 2,170 μg g -1 in its leaf tissues . More recently, Proctor et al. (1994) reported another, yet unnamed, nickel-hyperaccumulating species of Rinorea from Mt Piapi on Karakelong Island, northeast of Sulawesi in Indonesia with up to 1,830 μg g -1 foliar Ni.
Th e ability to absorb certain metals and metalloids (chemical elements with properties in between those of metals and non-metals, also referred to as semi-metals) from the soil and to accumulate them in shoot tissues in exceptionally high and normally toxic concentrations without any evidence of physiological stress is rather rare among plants (Baker and Brooks 1989;Reeves and Baker 2000;Kramer 2010). Metal hyperaccumulation has recently been suggested to have had multiple origins within the angiosperms (Cappa and Pilon-Smits 2013). Th e more than 500 plant taxa thus far recorded as metal hyperaccumulators represent only a very small portion of all known angiosperms (Reeves and Baker 2000, Kramer 2010, van der Ent et al. 2012. Th e largest number of species, approximately 450, distributed in a wide range of angiosperm families, hyperaccumulate the metal nickel and generally occur on serpentine or ultramafi c soils (van der Ent et al. 2012, Pollard et al. 2014.
Apart from their unusual and interesting ecology and physiology, hyperaccumulator plants have received considerable attention owing to the possibility of exploiting their accumulation traits for practical applications, especially in the development of so-called environmentally green technologies, e.g. phytoextraction, phytoremediation of heavy metal in contaminated soils, or phytomining to recover commercially valuable metals in plant shoots from mineralized sites (Chaney et al. 1997, Brooks and Robinson 1998, McGrath and Zhao 2003, Reeves 2003, Pilon-Smits 2005, Rascio and Navari-Izzo 2011.
In the Philippines, much of the forest fl ora on ultramafi c or serpentine soils (Fernando et al. 2008) remain underexplored. Field surveys in a number of sites in the archipelago have revealed some new species (e.g. Hoff mann et al. 2003, Fernando andRodda 2013), including several species that are able to accumulate heavy metals in their above-ground tissues (Baker et al. 1992, Hoff mann et al. 2003, Gotera et al. 2014. In this paper, we describe a new species of Rinorea discovered in remnant forest on ultramafi c soils that is also a nickel-hyperaccumulator. Th is species is, thus far, known only from small populations in the northern section of Zambales Province on Luzon Island in the Philippines. Th is area is part of the Zambales Ophiolite Complex (Rossman et al. 1989, Zhou et al. 2000, Yumul 2004 which is host to several metallic mineral deposits (e.g. chromium, nickel) (Osberger et al. 1988, Bacuta et al. 1990, Yumul et al. 2003.

Materials and methods
Th e morphology of the species presented here was based on fi eld, vegetative, and reproductive characters. Field characters were recorded on site. Vegetative characters were observed and measured from press-dried specimens and seedlings and reproductive characters from fresh specimens and from material preserved in 70% ethanol. Detailed morphological measurements were made using digital calipers and a calibrated eye piece under a dissecting microscope. Herbarium specimens were also consulted and compared at CAHUP, LBC, PNH, and PUH, including additional material, e.g. images of type specimens of Southeast Asian and Philippine Rinorea available online at BISH, K, L, MO, NY, and US. All photographs, except where indicated, were taken in the fi eld in the natural habitat of the species. Conservation threat assessment follows IUCN Categories and Criteria (IUCN 2012).
Field semi-quantitative screening for nickel accumulation in this species was performed on site on leaf samples, thoroughly washed in distilled water, crushed in a mortar and pestle, and tested on fi lter paper previously impregnated with 1% of the nickelspecifi c colorimetric reagent, dimethylglyoxime, dissolved in 95% ethanol (Baker et al. 1992;Reeves et al. 1996Reeves et al. , 1999. Formation of pink or magenta color indicated exceptionally high (above 1,000 μg g -1 ) concentration of Ni in the dry plant matter. Tissue samples of roots, stems and leaves, and of soil from the rhizosphere ( c. 30-100 cm) of each plant sampled were also collected. Th ese were subsequently subjected to laboratory elemental analyses for nickel (Ni) and two other heavy metals, copper (Cu) and cobalt (Co). Th e plant samples were thoroughly washed in distilled water and then oven-dried at 60 °C. Each sample was later weighed into borosilicate test tubes and ashed in a muffl e furnace for 4-5 hours, with the fi nal temperature of 500 °C being maintained for the last 2 hours. Th e ash was then taken up in 5 ml of warm 2 M HCl and the digest fi nally made up to an appropriate volume (5-20 ml) then analyzed for Ni, Cu, and Co content using atomic absorption spectrophotometer. Th e soil samples were digested with aqua regia (3:1 concentrated hydrochloric acid: nitric acid), then diluted appropriately for metal analyses of Ni, Cu, and Co using an atomic absorption spectrophotometer. Details of this method follow in general that described by Reeves et al. (1996).
Distribution. Endemic in the Philippines. Luzon Island, Zambales Province, Municipalities of Sta. Cruz and Candelaria.
Habitat and ecology. Th is species grows in forests on ultramafi c soils, usually along gullies or sloping areas with large boulders or rocks at elevations of 320-825 m. In its type locality, Rinorea niccolifera was observed growing with Syzygium longissimum Etymology. Th e specifi c epithet niccolifera refers to the ability of this species to hyperaccumulate the heavy metal nickel in its stem and leaf tissues (from niccolum -Neo Latin for nickel, and; fer -to yield, to contain). Conservation status. Following the IUCN Categories and Criteria (IUCN 2012), we regard this species as Endangered (EN B2ab(ii,iii,iv)). Its habitat is severely fragmented and is so far recorded only from three adjacent localities. Its current known area of occupancy is estimated to be less than 500 km 2 , and a continuing decline is observed, inferred or projected in its (a) extent of occurrence; (b) area of occupancy; and (c) area, extent and/or quality of habitat. Much of the habitat of this new species is subject to open pit mining.
Key to the species of Rinorea in the Philippines

Metal hyperaccumulation in Rinorea niccolifera
Field screening for Ni accumulation in Rinorea niccolifera using the colorimetric reagent, dimethylglyoxime (Baker et al. 1992, Reeves et al. 1996, 1999 indicated high levels in the leaves (Figure 3). Subsequent chemical analyses of the plant tissues in the laboratory revealed foliar nickel concentrations varying from 7,168 to 18,388 μg g -1 on dry weight basis (Table 1). Th e data shown in Table 1 is based on six sets of plant tissue samples of Rinorea niccolifera collected from two sites. Th e range of foliar Ni concentration on dry weight basis is similar to that reported for Rinorea bengalensis with 15,400-17,500 μg g -1 (Brooks andWither 1977, Reeves 2003, Jopony and Tongkul 2011) on ultramafi c soils. It is, however, higher when compared with Rinorea javanica, 2,170 μg g -1  or Rinorea sp., 1,830 μg g -1 (Proctor et al. 1994). As this species surpasses the 10,000 μg g -1 Ni accumulation level in the leaves, it is regarded as a 'hypernickelophore' following the Ni accumulation category of Jaff ré and Schmid (1974) and Boyd and Jaff ré (2009). Th e cobalt (Co) accumulation in R. niccolifera (21.35-51.46 μg g -1 ) ( Table 1) was low but is within the range recorded by  for R. bengalensis (0.5-545 μg g -1 ) and R. javanica (3-670 μg g -1 ). All these fi gures are above the normal concentrations (0.03-2 μg g -1 ) of cobalt in plants, which according to Reeves (2006) rarely exceeds 20 μg g -1 . Copper (Cu) accumulation (Table 1) was also within normal range of concentrations (5-25 μg g -1 ) for plants (Reeves 2006).