Two new species and a new species record of Aglaia (Meliaceae) from Indonesia

Abstract Two new species of Aglaia from Indonesia are described, Aglaia monocaula restricted to West Papua, and Aglaia nyaruensis occurring on Borneo (Kalimantan, Brunei, Sabah and Sarawak). A phylogenetic analysis using nuclear ITS and ETS, and plastid rps15-ycf1 sequence data indicates that the two new species of Aglaia are also genetically distinct. Aglaia monocaula belongs to sectionAmoora, while A. nyaruensis is included in section Aglaia. A dichotomous key, drawings and three-locus DNA barcodes are provided as aids for the identification of the two new species of Aglaia. In addition, the geographic range of Aglaia mackiana (section Amoora) is expanded from a single previously known site in Papua New Guinea to West Papua, Indonesia.


Introduction
The classification of the family Meliaceae continues to be refined (Muellner et al. 2003, b, Muellner and Mabberley 2008, Pennington and Muellner 2010, Köcke et al. 2015, Clarkson et al. 2016, Gama et al. 2020 and new taxa are still being discovered and described (e.g., Pannell 2004Pannell , 2019. Aglaia Lour. is the largest genus of the family, and, with at least 120 arborescent species, presents more taxonomic problems in species delimitation than any other genus of the family (Pannell 1992, 1995, 1998a, b, 2004, Muellner et al. 2005, 2008a. Aglaia forms an important component of the moist tropical forest in the Indomalesian region. The distribution range comprises the tropics of southeastern Asia from Sri Lanka and India to Australia (Queensland, Northern Territory, and Western Australia) and eastwards to the island of Samoa in Polynesia and north to the Mariana (Saipan, Roti, and Guam) and Caroline Islands (Palau and Ponape) in Micronesia (Pannell 1992). A monograph of the entire genus throughout its range has been published (Pannell 1992), of the Malesian species, including New Guinea, in Flora Malesiana (Pannell 1995), and of the Bornean species in the Tree Flora of Sabah and Sarawak (Pannell 2007).
From the 1990s, the genus received increasing scientific focus due to its bioactivity potential (Muellner et al. 2005, and references therein), including inhibiting activity against Ebola-, corona-, Zika-, Chikungunya-and hepatitis E-viruses (Müller et al. 2018(Müller et al. , 2020. A recent research call in the field of "Biodiversity and Health", funded by the German Federal Ministry of Education and Research, has led to a surge of interest in the taxonomic investigation of plant groups of potential interest for the future development of new antiinfective compounds. A taxonomic survey of Aglaia in Indonesia in the course of this research program has led to the discovery of two new species. We here describe these two new species from Indonesia. In addition, we report a new record of Aglaia from Indonesia, previously known only from Papua New Guinea.

Morphology
The two new species are described based on field observations and examination of herbarium specimens at BO, FHO, K, L, and A, using morphological characters that distinguish them from all other species in the genus Aglaia. Descriptions were written from herbarium specimens. Measurements were made with a tape-measure and calipers. The structure of the indumentum and its distribution was observed and described under a dissecting microscope at magnifications of more than 20×. Flowers were rehydrated by boiling in tap water. They were placed on a glass slide covered with 1 mm graph paper for scale and dissected, measured and described under a dissecting microscope. Additional information on locality, habitat, ecology, plant form, bark and wood characters and fruits was collected in the field and taken from herbarium labels. Conservation threat assessment followed IUCN Categories and Criteria (IUCN 2012).

DNA extraction, amplification, and sequencing
Total genomic DNA was extracted for representative samples of each species of Aglaia described herein (Table 1) using a Macherey-Nagel NucleoSpin Plant II kit. The protocol was modified by adding 40 ul β-mercaptoethanol and 2% polyvinylpyrrolidone (PVP). ITS was amplified either as a whole using the primer combination 17SE_m/26SE_m (Grudinski et al. 2014) or, if this failed, adding two internal primers (F1_ITS/R1_ITS, Muellner et al. 2005) to amplify the first and second part of the ITS region separately. ETS was amplified using the primers 18S_ETS (Baldwin and Markos 1998) and a newly designed primer, 18S_MEL [5'-GTG TGA GTG ATT GGA T-3'; this study]. The plastid region rps15-ycf1 was amplified using the primer pair rps15-IGSR/ycf1-IGSR (Prince 2015).
For all amplifications, we used the Phusion High-Fidelity DNA Polymerase (New England BioLabs, Ipswich, MA, United States) according to the manufacturer's protocol. Annealing temperature for ITS (whole region or in two parts) and ETS was 51.5 °C, and for rps15-ycf1 51 °C. PCR products were cleaned using the NucleoSpin Extract II Kit (Macherey-Nagel, Düren, Germany). Sequencing reactions and analyses were run by LGC Genomics (Berlin, Germany).
All sequences were assembled and edited using Geneious (v7.06, Kearse et al. 2012). Consensus sequences were aligned using MUSCLE (v.3.8.31 Edgar 2004) as implemented in Geneious, and all alignments were thoroughly checked and further refined manually. For ITS, sequences were explored for the presence of several structural motifs, allowing for the detection of pseudogenes: the conserved angiosperm motif GGCRY-(4 to 7n)-GYGY-CAAGGAA (Liu and Schardl 1994); the conserved (C1-C6) and variable (V1-V6) domains determined for plant ITS2 sequences (Hershkovitz and Zimmer 1996); and the conserved angiosperm motif 5´-GAATTGCAGAATCC-3´ within the 5.8S rRNA gene (Jobes and Thien 1997). Secondary structure predictions were confirmed by hemi-compensatory base changes and full compensatory base changes that preserved the predicted folding pattern. Sequences for the new species were deposited in GenBank (http://www.ncbi.nlm.nih. gov/; Table 1). Voucher information, geographic origin, and GenBank accession numbers for all samples included in this study are provided in Suppl. material 1: Table S1.

Phylogenetic analyses
Newly generated sequences of ITS were combined with the data from Muellner et al. (2008b) and an improved and reduced version of the data matrix used in Grudinski et Tree, 3-10 m high, unbranched, with a terminal tuft of spirally inserted leaves; bole 4 cm in diameter; latex white. Twigs greyish-brown with large orange-brown pustules, densely covered with orange-brown and dark brown compact stellate hairs at the apex, glabrescent on older wood.
Leaves 47-70 cm long, 28-32 cm wide; petiole 11-30 cm long; the petiole, rachis and petiolules with few to numerous hairs like those on the twigs, glabrescent. Leaflets 15, the laterals opposite or subopposite, coriaceous, lamina 7-16 cm long 2-5.5 cm wide, elliptical, slightly up-curved at the margins, cuneate at the slightly asymmetrical base, tapering to an acuminate apex, the acumen obtuse and 10-12 mm long; lateral veins 5-14, ascending and curved upwards near the margin, not anastomosing, lateral veins and reticulation subprominent; midrib prominent below with sparse stellate scales, absent from lower leaflet surface, upper and lower leaflet surfaces minutely rugulose; petiolules 10-15 mm on lateral leaflets, slender, 20-40 mm long on terminal leaflet, all with a dark blackish-brown, slightly swollen, region at the base of the petiolules.
Inflorescences not seen. Infructescence 11 cm long, 7 cm wide; peduncle 6 cm long, the peduncle and fruit stalks with few to numerous hairs like those on the twigs, glabrescent. Fruits 2.8 cm long, 1.8 cm wide, ovoid, pericarp bright scarlet or pinkish-red, inner pericarp white, dehiscent with three locules each containing 1 seed; seed white where attached to the central axis of the fruit by a large hilum, aril orange.
Distribution. Known only from the area around Ayawasi village in West Papua.
Ecology. Primary open forest on limestone ridge to 600 m, with an abundant growth of moss. Fruits eaten by kuskus.
Use. Wood used for house beams. Vernacular. sapa sai (K.Yumte) Etymology. The specific epithet of Aglaia monocaula refers to the unbranched habit of this small tree.
Conservation. This species is known from only two fruiting specimens collected near Ayawasi village and is therefore assessed to be Data Deficient (provisional). Further collecting and monitoring is necessary to allow more conclusive estimations about the rareness and vulnerability of the species. However, the collections seen were made 24 and 25 years ago, so the likelihood of obtaining further material from this species is not great.
Additional specimen. Indonesia. West Papua: top ridge of limestone hills south of Ayawasi village, fr., 1 May 1996, Polak 1221 (FHO) Notes. This new species is represented by two fruiting specimens of monocaul trees that have leaves with a long petiolule on the terminal leaflet.
Section Aglaia 2. Aglaia nyaruensis C.M. Pannell, sp. nov. urn:lsid:ipni.org:names:77210864-1 Fig. 2 Diagnosis. Aglaia nyaruensis resembles A. foveolata, from which it differs in its smooth leaflet lower surface, with the lateral veins and reticulation not prominent. These characters, combined with numerous pits on the leaflet upper and lower surfaces, make this species unique in the genus.
Tree, 10-22 m high, bole 8 m, diameter 20 cm, outer bark smooth, greyish or greyish-brown, shallowly fissured and lenticellate, inner bark pink, brownish-green or brown and fibrous, sapwood pale yellow, heartwood white; latex white or absent. Young twigs densely covered with reddish-brown stellate hairs and scales.
Leaves 25-31(-60) cm long, 14-21(-34) cm wide; petiole 7-9 cm long, the petiole, rhachis and petiolules densely covered with reddish-brown stellate hairs. Leaflets 11-15, the laterals subopposite, lamina 5.5-11.0 cm long 2.5-4.0 cm wide, narrowly oblong or elliptical, pale brownish-green when dry, rounded to cordate at the asymmetrical base, acuminate at apex with the acute acumen narrow and to 15 mm long; lateral veins 10-15, ascending and curved upwards near the margin, anastomosing some distance from the margin and with further reticulation between this and the margin of the leaflet, with shorter lateral veins in between. Midrib prominent below, lateral veins and reticulation not raised and barely visible in dried leaflets, the midrib on the upper surface of leaflet with numerous pale brown stellate hairs and scales, the midrib on the lower surface densely covered with reddish brown stellate hairs and scales, numerous on the lower leaflet surface when young, glabrescent, becoming sparse on the mature lamina near the midrib and absent from the rest of both surfaces of the lamina, with numerous pits on both surfaces; petiolules to 3 mm long on lateral leaflets to 10 mm long on terminal leaflet.
Male inflorescence 20 cm long, 10 cm wide; peduncle 8-9 cm long, the peduncle, rachis and first branches densely covered with reddish-brown stellate hairs and scales; higher orders of inflorescence branches with numerous reddish-brown stellate hairs and scales. Male flower 2 mm long, 2 mm wide; pedicel 1 mm long, the calyx and pedicel densely covered with reddish-brown stellate hairs and scales; calyx cup-shaped, deeply divided into five rounded lobes, petals 5, 1.75 mm long, 1 mm wide, yellow, obovate; staminal tube 1.5 mm long, 1.5 mm wide, obovoid with a wide mouth 1.5 mm across, anthers 6, 0.75 mm long, 0.25 mm wide, inserted half way down the tube inside and protruding through the aperture; ovary 0.5 mm long, 0.5 mm wide, ovoid, densely covered with brown stellate hairs and scales on the outside, with two locules each containing one vestigial ovule. Female flowers not seen.
Distribution. One record each from Kalimantan, Brunei, Sabah and Sarawak. Ecology. Peat swamp forest, swampy forest on white sand, on ultrabasic soil or on yellow-brown sandy soil over Tertiary clays, with deep litter and abundant humus and living roots. Altitude to 400 m. Vernacular name. Jalongan sasak (Bejang b. Sitam). Etymology. The specific epithet of Aglaia nyaruensis refers to the type locality, Nyaru Menteng in Kalimantan.
Conservation (provisional). This species is known from one locality each in Kalimantan, Brunei, Sabah and Sarawak and is therefore considered to be Vulnerable.

New record for West Papua, Indonesia
Aglaia mackiana Pannell, Kew Bull. 52(3): 715. 1997. Fig. 3 Remark. Previously known only from the type locality in Papua New Guinea, this tall tree species in section Amoora, has the largest fruits recorded for the genus Aglaia. Collections from West Papua are of immature fruits and flower buds.
Distribution. Indonesia, two records from West Papua. In Papua New Guinea, known only from the type locality in Chimbu Province.
Ecology. Primary lowland forest on the coastal plain and to 450 m altitude; canopy 25-45 m high; associate species include Celtis, Sterculia, Pometia, Ficus, Oncospermum, and sundry Rubiaceae. Canopy tree to 45 m tall, branching above; bole c. 1 m diameter, buttressed below; bark tan, smooth, somewhat round flaky; fruits 12-16 cm diameter, light brown, lactiferous, 3-lobed. In Papua New Guinea, the fruit either dehisces on the tree and the seeds fall to the ground or the whole fruit falls from the tree and dehisces on impact with the ground. The seeds are swallowed whole by the Dwarf Cassowary and defaecated at up to 1000 m from the parent trees (Mack 1995a, b;Pannell 1997). A fruit bat (probably Dobsonia moluccensis) carries seeds shorter distances, reportedly less than 100 m, away from the parent tree. Germination is semi-hypogeal, within a few days of deposition of seeds; the two large cotyledons persist at ground level for up to two years after germination. Vernacular. 'sapa peka' (Wanda Ave 4394) Etymology. Named after Andrew Mack, who discovered this species in the course of his field work on the Dwarf Cassowary.
Conservation. This species is known from only three localities, two in Papua and one in Papua New Guinea. It is therefore assessed to be Data Deficient (provisional). Further collecting and monitoring is necessary to allow more conclusive estimations about the rareness and vulnerability of the species. However, the collections seen were made 24, 25, 27, and 28 years ago, so the likelihood of obtaining further material from this species is not great.

New species inserted into a truncated version of the existing key to the species of Aglaia in Malesia
To accommodate the three species the following couplets (in bold, labelled (i) and (ii), can be inserted into the existing key to Malesian Aglaia by Pannell (1995, pp. 201-212)] 1a Leaf always (10)

Markers and trees
The final lengths of our alignments were 1006 bp (ITS), 515 bp (ETS), and 600 bp (rps15-ycf1). The results of our phylogenetic analyses of the combined nuclear data were largely congruent with the infrageneric relationships of Grudinski et al. (2014). Individual analysis of the plastid data, however, resulted in a largely unresolved tree (tree not shown). Furthermore, the combination of all markers led to an overall decrease of resolution and support as compared to the nuclear dataset (partly due to the high degree of missing data in the rps15-ycf1 data). Given that we did not find any strongly supported disagreements between the plastid and nuclear data, we here present only the results of the analyses of the combined nuclear (ITS and ETS) dataset (Fig. 4). Both new species were found to be phylogenetically well supported. Aglaia monocaula was found to be closely related to other members of section Amoora (A. meridionalis, A. australiensis) from Australia. All samples of A. nyaruensis formed a strongly supported clade (pp = 0.99), with an accession of A. elliptica from Kalimantan as sister species. Both species belong to section Aglaia and have an indumentum of stellate hairs and scales.

DNA barcodes
Three-locus DNA barcodes (Table 1) are provided as aids for the identification of the two new species of Aglaia. On purpose, we did not use the 2-locus combination of rbcL and matK as originally recommended by the CBOL Plant Working Group (Hollingsworth et al. 2009a) or other previously recommended chloroplast markers, as previous phylogenetic and barcoding studies provided evidence for their insufficient taxonomic resolution at species level in the Meliaceae (e.g., Muellner et al. 2003, Muellner et al. 2011. ITS was proposed by Kress et al. (2005) as potential barcoding region, and has repeatedly been suggested as additional marker in case resolution with plastid markers was not sufficient in the group under investigation Hollingsworth et al. 2009b; also compare the review by Shneyer 2009; and many others since then). found that parts of the plastid gene ycf1 were very variable across flowering plants, indicating that this marker might be a useful barcode region. Finally, tests across several Meliaceae genera indicated that ETS might be another useful, i.e. informative, marker.