Borneocola (Zingiberaceae), a new genus from Borneo

Abstract A new genus from Borneo, Borneocola Y.Y.Sam, is described here. The genus currently contains eight species previously classified as members of the Scaphochlamys Baker. The finding is based on the results of the morphological and molecular studies of Scaphochlamys throughout its geographical range and its closely allied sister groups, Distichochlamys M.F.Newman and Myxochlamys A.Takano & Nagam. Borneocola is nested within the tribe Zingibereae and its monophyly is strongly supported by both ITS and matK sequence data. The genus is characterised by several thin, translucent and marcescent floral bracts, absence of coloured streaks on the labellum and capitate stigma with two dorsal knobs. The genus is distributed in northwest Borneo and all species are very rare and highly endemic.


Introduction
Southeast Asia is the centre of diversity for the family Zingiberaceae. Here, new taxa are continuously being discovered and named, both at the generic and specifi c levels. Several of the recent discoveries were further supported by the phylogenetic analyses which give a better understanding of the evolutionary relationships within the family (Kress and Larsen 2001;Kress et al. 2010;Leong-Škorničková et al. 2011). During the revision of the genus Scaphochlamys throughout its entire geographical range by the fi rst author, some distinctive morphological traits were observed in several Bornean species, suggesting they might represent a separate group from the Peninsular Malaysian taxa. Th is hypothesis was confi rmed by the phylogenetic analyses which are presented here and the eight species previously included in the genus Scaphochlamys are recircumscribed in this paper as a new genus, Borneocola Y.Y.Sam.
Th e genus Scaphochlamys was described by Baker (1892) in the Flora of British India with Scaphochlamys malaccana Baker from Mt. Ophir (now known as Gunung Ledang), Peninsular Malaysia, chosen as the type species. Holttum (1950) carried out the fi rst comprehensive revision of the genus in which he recognised 19 species, all of which were recorded in the peninsula. When Smith (1987) reviewed the tribe Hedychieae in Borneo, she applied the generic delimitation defi ned by Holttum and recognised fi ve Scaphochlamys species in Borneo. Out of the fi ve, S. polyphylla and S. petiolata were formerly placed in the genus Haplochorema K.Schum. Sakai and Nagamasu (2006) discovered that H. gracilipes K.Schum. also have the characteristics of Scaphochlamys and eff ected the transfer. Recent years have seen a surge in the new species discovered from Borneo bringing the total number of Bornean Scaphochlamys to 14 (Poulsen and Searle 2005, Meekiong et al. 2011, Ooi and Wong 2014Meekiong 2015).
Distichochlamys M.F.Newman and Myxochlamys A. Takano & Nagam. are sister genera to Scaphochlamys with several unique characteristics clearly separating them from Scaphochlamys (Newman 1995, Searle and Hedderson 2000, Kress et al. 2002, Ngamriabsakul et al. 2004, Takano and Nagamasu 2007. However, the distinction, based on morphological characters, became ambiguous as several taxa described recently exhibit exceptions to the usual generic characters. For example, S. calcicola A.D.Poulsen & R.J.Searle, a species named in 2005 from Sarawak, has a distichous infl orescence, a distinguishing character for the genus Distichochlamys M.F.Newman. Larsen and Newman (2001) also reported another Scaphochlamys species with a distichous infl orescence from north Peninsular Malaysia. A current study on the morphology of Scaphochlamys also revealed that some species display the characteristics of Distichochlamys and Myxochlamys. To test the validity of the current generic concept of Scaphochlamys and closely related genera Distichochlamys and Myxochlamys, we have examined their relationship by utilising ITS and matK markers together with the analysis of the morphology across these genera.

Morphological study
Th e morphological study was based on living plants in the forest, cultivated plants in the nursery of the Forest Research Institute Malaysia and specimens in the herbaria of AAU, BKF, C, FI, E, K, KEP, KLU, PSU, SAN, SAR and SING. A total of 372 herbarium specimens were examined in this study which includes 29 Scaphochlamys species and four Borneocola species (the types of another four Borneocola species were not yet deposited in the herbaria).Th e morphological characters examined in the study were habit; position of the rhizome, thickness and colour; height of leafy stem, its base (whether swollen to form a bulbous base); distance between leafy stems; characters of bladeless sheath such as colour, indumentum, number and length; ligule length, indumentum and shape; petiole length, indumentum, whether channelled or rounded in cross section; number of leaves per leafy stem; lamina colour on both surfaces, size, shape, venation, texture, indumentum, apex and base; length of the infl orescence and infructescence, arrangement of the fl oral bracts on the rachis, characters of fl oral bracts and bracteoles (colour, indumentum, texture, shape); size, colour and shape of calyx, fl oral tube, corolla lobes, labellum, staminodes, stamen, ovary.

DNA extraction, amplification and sequencing
Fresh leaves from the cultivated plants or silica-dried materials from plants collected in the fi eld were used for genomic DNA extraction.
For the ITS, the genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, California, USA) following the manufacturer's protocol. Two primers, ITS 5P (5'-GGAAGGAGAAGTCGTAACAAGG-3') and ITS 8P (5'-CACGCTTCTCCAGACTACA3') (Moller and Cronk 1997) were used to amplify the ITS region during the polymerase chain reaction (PCR). Th e thermal cycle of PCR for the amplifi cation of the ITS sequences is initial denaturation at 94°C for 2 minutes, 40 cycles of denaturation at 94°C for 30 seconds, primers annealing at 48°C for 2 minutes, an extension at 72°C for 45 seconds and fi nal extension at 72°C for 7 minutes. Th e PCR products were then purifi ed using MinElute Gel Extraction Kit (Qiagen, Valencia, California, USA).

Sequence alignment and phylogenetic analysis
Raw sequence data were assembled and edited manually using BioEdit software ver. 7.2.5 (Hall 1999). DNA sequences were aligned with the CLUSTALW 1.83 software package, with default settings and multiple alignments (Th ompson et al. 1994). Alignments of the matK sequences of cpDNA and the ITS sequences of nrDNA were combined. Gaps were deleted.
A total of 100 individuals including 54 taxa of Scaphochlamys and allied species were used. Th e three Siphonochilus species were used as an outgroup (Kress et al. 2002). Materials, accession numbers for the sequences, vouchers and references to the literature are presented in Table 1 at the end of this paper. Th ree datasets which comprise ITS, matK and ITS+matK combined, each containing 82, 78, and 61 taxa, were constructed. Th ese three datasets were analysed using three methods: maximum parsimony, maximum likelihood and Bayesian analysis. A maximum parsimony (MP) analysis was performed with MEGA 6 (Tamura et al. 2013). Heuristic searches were conducted with RANDOM addition, SPR branch swapping and MULPARS options. Support for each branch was estimated with a bootstrap analysis, with 1000 replications (Felsenstein 1985), in a heuristic search with RANDOM addition and TBR branch swapping. Th e maximum likelihood (ML), based on the Tamura-Nei model (Tamura and Nei 1993), was also determined with MEGA 6 (Tamura et al. 2013). Neighbor-Join and BioNJ algorithms were applied to a matrix of pairwise distances estimated with the maximum composite likelihood approach; then, the topology that had the best log likelihood value was selected. Bootstrap analysis under the MP criterion was conducted with "fast" stepwise, addition searches, with 1000 replicates. In addition, a Bayesian analysis was carried out with MrBayes software ver. 3.1.2 (Huelsenbeck and Rohnquist 2001;Rohnquist and Huelsenbeck 2003). Th e best fi tting substitution model (the GTR+G model for nrDNA datasets, the GTR+G model for cpDNA datasets and the GTR+I+G model for cpDNA+nrDNA datasets) was selected for Bayesian analysis based on a series of hierarchical likelihood ratio tests, implemented in MrModeltest software ver. 2.3 (Nylander 2004). Th e analysis was performed with the selected model and two simultaneous runs of two million generations with four chains, sampling every 100 generations. Each analysis reached stationarity (i.e. when the average standard deviation of split frequencies between runs was ≤ 0.01) well before the end of the run. Burn-in trees were discarded and the remaining trees and their parameters were saved. A 50 % majority rule consensus tree was constructed. Th e results of the Bayesian analysis were reported as the posterior probability (PP; Huelsenbeck and Rohnquist 2001), which is equal to the percentage of phylogenetic trees sampled when a given clade was resolved. Only PP scores above 50 % are shown.

Phylogenetic analyses
Th e ITS datasets for 82 individuals with 29 taxa of Scaphochlamys and 6 taxa of Borneocola contained 786 characters after alignment, which decreased to 769 after gaps were deleted; 319 of these were parsimony-informative. Likelihood analysis resulted in a ML tree with -lnL = 10438.212. Parsimony analysis produced three parsimonious trees with 1865 steps, a consistency index (CI) of 0.391 and retention index (RI) of 0.609. Th e ML, MP and Bayesian trees had similar topology; the ML tree is shown with bootstrap (BS) and MP-BS, and Bayesian Posterior Probability (PP) support in Figure 1 below. Scaphochlamys formed a well supported clade (ML-BS/MP-BS/BA-PP support, 84/87/1.00). Each Myxochlamys and Borneocola consisted of a well supported subclade and became sisters to each other and they also became sistersto the Scaphochlamys clade. Distichochlamys species formed a well supported subclade and became sister to the Myxochlamys + Scaphochlamys + Borneocola clade (ML-BS/MP-BS/BA-PP 99/100/1.00).
Th e matK datasets for 78 individuals including 25 taxa of Scaphochlamys and 7 taxa of Borneocola contained 1,599 characters after alignment; 182 of these were parsimony-informative. Likelihood analysis resulted in a ML tree with -lnL = 5952.438. Parsimony analysis produced ten parsimonious trees with 557 steps, a consistency index (CI) of 0.613 and retention index (RI) of 0.080. Th e ML, MP and Bayesian trees had similar topology; the ML tree is shown with BS and MP-BS, PP support in Figure  2 below.
Each of the two Myxochlamys species and seven Borneocola species formed a strongly supported subclade and became sisters to each other. Scaphochlamys became sister to them, but bootstrap or probability support was weak. Th e Distichochlamys species formed a well supported subclade, but all the genera that belong to subfamily Zingiberoideae became sisters to Scaphochlamys + Myxochlamys + Borneocola clade and not only to Distichochlamys.
Th e combined ITS and matK datasets for 61 individuals including 13 taxa of Scaphochlamys and 6 taxa of Borneocola, resulted in 2,336 characters, 488 of these were parsimony-informative (Figure 3 below). Likelihood analysis resulted in a ML tree with -lnL = 16671.531. Parsimony analysis produced the most parsimonious trees with 2247 steps, a CI of 0.440 and a RI of 0.635. Th e ML, MP strict consensus and Bayesian trees had almost the same topology; the ML tree is shown with MP-BS, ML-BS and BA/PP support in Figure 3.
Two Myxochlamys species and six Borneocola species formed a strongly supported subclade each and became sisters to each other. Scaphochlamys became sister to them and the bootstrap or posterior probability support was moderate. Distichochlamys species formed a well supported subclade and became sister to Scaphochlamys + Myxochlamys + Borneocola clade.

Morphology
Th e Borneocola and Scaphochlamys species look similar in their vegetative morphologies. Th ey are mostly small-sized gingers without the conspicuous pseudostem, with one to several leaves arranged spirally and tightly on a very short stem at the base. So far, all the Borneocola species examined are unifoliate. Similarly, most of the Scaphochlamys species also bear one leaf except for several species which have leafy shoots composed of multiple leaves, for example, S. grandis, S. lanceolata, S. kunstleri, S. malaccana and S. minutifl ora. Th e basal part of the leaves is covered with a few bladeless sheaths which are rather diff erent for both groups in terms of their texture and colour. For Scaphochlamys, the sheaths are coriaceous, green, green with a red tinge or red and mostly persistent until the end of fl owering ( Figure 4A, B). On the other hand, the sheaths of Borneocola are thinner in texture with a lighter shade of green or brown. Th e thin sheaths normally dry up early ( Figure 4C) and sometimes they are completely shredded during the time of fl owering.
Th e infl orescences of Borneocola and Scaphochlamys are terminal, stalked and consisted of few to many fl oral bracts. Th e diff erences lie in the characteristics of the fl oral bracts and fl owers. Borneocola species have thin, translucent, early decaying and marcescent fl oral bracts. Th e colours of the bracts can be pink, pale brown, pale or light green ( Figure 5A). On the contrary, the bracts of Scaphochlamys are coriaceous and sometimes hard in texture. Th ey are usually green, green tinged red, red or reddish brown and remain fresh throughout the fl owering ( Figure 5B, C).
Besides the characteristics of the fl oral bracts, the variegation on the labellum can give a quick guide to the two genera. Most Scaphochlamys have white fl owers with a yellow median band and lilac, purple, red streaks or patches fl anking the band on the labellum ( Figure 5D, E). However, there is no such variegation on the labellum of Borneocola ( Figure 5F). Th e whole labellum of Borneocola is pale pink, lilac, violet or white with a light yellow or greenish yellow median band.
Both Borneocola and Scaphochlamys have a long slender fl oral tube which is mostly puberulent externally in Borneocola (except for B. calcicola) but glabrous for Scaphochlamys. Another marked diff erence observed is in the stigma shape. Scaphochlamys has a funnel-shaped or beak-like stigma ( Figure 6A, B) while it is almost oblate with two dorsal knobs in Borneocola ( Figure 6C).    Description. Terrestrial rhizomatous herb, evergreen, rarely exceeding 50 cm in height. Rhizome creeping on the ground, terminal decumbent, rhizome elements short or long; roots fi ne, extensive, not tuberous. Leafy stem unifoliate, enclosed by a few bladeless sheaths at base, bladeless sheaths linear, papery, glabrous to hairy, light green or light brown, decaying early, leaf sheath glabrous or hairy, base swollen, margin thin and narrow; ligule membranous, inconspicuous, decaying early; petiole channelled in cross section, glabrous, lamina narrowly ovate to elliptic, rarely oblong, asymmetric, margin entire, smooth.

Scaphochlamys longifolia FRI47065
Infl orescence fl owering from base to apex; peduncle short, usually hidden within leaf sheath; spike composed of compact rachis and 2-5 (-13) fertile bracts, bracts spirally and closely overlapping (rarely distichous), boat-shaped, 2-keeled, pink, pale brown, pale or light green, thin, translucent, glabrous or hairy, decaying early, marcescent, amplexicaul at the base of the bract, cincinni compact, 2-3 fl owers in each cincinnus. First bracteole directly opposite fl oral bract and enclosing all the fl owers and subsequent bracteoles, linear-shaped, 2-keeled, shorter than bracts, rarely same length. Flowers thin, delicate, ephemeral. Calyx tubular, splitting unilaterally on one side, fl oral tube long slender, usually puberulent externally, inner surface with a groove enveloping the style, corolla lobes 3, triangular ovate, translucent, glabrous, dorsal lobe apex hooded, lateral lobes 2, narrower than dorsal lobe. Staminodes elliptic to narrowly obovate, white, light yellow or green, spreading laterally, lined with translucent veins from base to apex, covered with glandular hairs on adaxial surface. Labellum obovate, fl at, bilobed distally, rarely entire, translucent veins spread from base to apical part, pale white, pink, lilac or violet, median band light yellow or greenish yellow, without coloured streaks or patches beside the band, adaxial surface covered with glandular hairs. Stamen bends forward over labellum, usually white and covered with glandular hairs on abaxial surface, fi lament grooved, short, anther theacae 2, linear, dehiscing along entire length, basal ending with or without short spur, anther crest short, not recurved. Ovary ellipsoid, glabrous or sparsely hairy, unilocular, with basal placentation, less than 10 locules. Stigma small, held at apex of thecae, near oblate, 2 dorsal knobs, ostiole forward facing, ciliate. Epigynous glands 2, fi liform, yellow. Fruit a capsule, ellipsoid or ovoid. Seeds not seen.
Etymology. Th is new genus is named after the island of Borneo and -cola (Latin) means dweller or inhabitant. Th is is to recognise the extremely rich and unique biodiversity that is found in Borneo.
Distribution. Borneo. Th e genus is currently known to occur only in the northwest and possibly central Borneo. Eight species are recorded from Sarawak, Malaysia and many more are undescribed. Notes. It is one of the most distinctive species, either in the fi eld or herbarium sheet. Th is dainty plant has a long creeping rhizome and well spaced leafy shoots, prominently stiff lateral veins raised on its adaxial lamina, both on living plants and dried specimens.

Key to Borneocola species (modifi ed from Ooi and Wong 2014)
Scaphochlamys depressa Mas Izzaty, Ampeng & Meekiong is unmistakably the same as S. argentea with its prominent raised lateral veins, broadly elliptic lamina and well spaced leafy shoots. Meekiong (2015) explained that the infl orescence of S. depressa which exerted from the petiole is diff erent from S. argentea where the infl orescence emerges from the base of the petiole. Th is observation is incorrect as all gingers have terminal infl orescences. ( Notes. Borneocola biru is the most recent species described from Sarawak. It has a deep purplish blue labellum, diff erent from all other Borneocola species which are white or in lighter shades. Notes. Borneocola calcicola is the largest amongst the Borneocola species. Poulsen and Searle (2005) observed that the distichous infl orescence is one of the character-istics of the plant. However, a recent collection of B. calcicola, Sam FRI 50290, from Seromah, Bau, showed spirally arranged fl oral bracts. Th ere was a mixture of spirally and distichously arranged fl oral bracts in its population in Bau, Sarawak. Notes. Borneocola iporii is a small ginger creeping on the humus rich forest fl oor. It is most similar to B. argenteus with both having a unifoliate shoot, leafy shoots far apart, broad lamina, short infl orescence and compact rachis. However, the conspicuously raised lateral veins of B. argenteus can readily distinguish it from B. iporii. Notes. Borneocola petiolatus is distinguished by its long petiole and narrow leaves from the other species. Its lamina length is almost 3 times the width (12-21.5 × 3.1-7.1 cm). Smith (1987) found that B. petiolatus has small infl orescences as in B. argenteus. However, both can be easily separated by their leaf characters. Borneocola petiolatus has much longer petioles compared to B. argentea (12.7-31.5 cm versus 3-6 cm). Borneocola argenteus also has prominently raised lateral veins on the adaxial surface of lamina, more conspicuous on dried specimens than fresh materials. Th is character is lacking in B. petiolatus. Gastrochilus reticosa Ridl., J. Straits Branch Roy. Asiat. Soc. 44: 195 (1905 Meekiong et al. (2011). Th e plants are more of an opportunist growing on humus-rich substrate accumulated on the rocks. Type. MALAYSIA. Sarawak, Kuching Division, Bau, Gunung Buan, 1°33'28.9"N, 10°08'35.2"E, 92 m, 21 Nov 2013, Ooi Im Hin & Jepom ak Tisai OIH74 (holotype: SAR. Type not yet deposited as of 5 May 2016).

Borneocola petiolatus
Note. Borneocola stenophyllus is another new species recently discovered from Sarawak. Its grass-like leaves instantly separate it from other species in the genus.
Kaempferia anomala Hallier f., Bull. Herb. Boissier 6: 357 (1898). Gastrochilus anomalum (Hallier f.) K.Schum. in Engler, Pfl anzenr. IV, 46 (Heft 20): 92 (1904). Boesenbergia anomala (Hallier f.) Schltr., Repert. Spec. Nov. Regni Veg. 12: 315 (1913). Gastrochilus hallieri (Hallier f.)  Notes. Th e type, the only specimen ever collected, was lost. However, Hallier (1898) gave a very detailed description and drawing of the plant and this has convinced Searle (2010) to place it in the genus Scaphochlamys. Th e drawing, which is based on the type specimen and designated by Searle as the lectotype, is the only material that gives a glimpse of the appearance of the species. In the drawing, the fl ower and spirally arranged fl oral bracts are typical of both Scaphochlamys and Borneocola. Until another specimen is collected and is available for close examination, we prefer to retain this imperfectly known species in Scaphochlamys.

Discussion
Th e phylogenetic analyses confi rm the distinctive character of Borneocola and Scaphochlamys and their placement in the tribe Zingibereae (Figures 1, 2, 3). Th e Borneocola species form a monophyletic group which is sister to Myxochlamys. It is surprising to fi nd Borneocola having a closer affi nity to Myxochlamys than to Scaphochlamys, considering it shares more morphological similarities with Scaphochlamys than with Myxochlamys.
Morphologically, Myxochlamys is very diff erent from Borneocola. Th ere are two Myxochlamys species named so far: M. amphiloxa and M. mullerensis (Takano and Nagamasu 2007;Searle and Newman 2010) and a third undescribed species, also from Borneo. All three Myxochlamys species are very robust plants that can attain a height of 70 cm. Most Borneocola species examined so far are small-sized (not more than 50 cm tall), except for B. calcicola which can grow to 60 cm tall. Myxochlamys has 3-10 large leaves (50-60 cm long) in each shoot whereas Borneocola are unifoliate and the leaves are small (less than 20 cm long except for B. calcicola). Th e leaves of Myxochlamys are sessile compared to the conspicuously stalked leaves in Borneocola. Th e most marked diff erence is in the infl orescence structure. Borneocola has small infl orescences consisting of less than 15 fertile bracts but Myxochlamys has large torch-like infl orescences with easily more than 40 bracts. Th e bracts of Borneocola are membranous and marcescent, often measuring less than 2 cm long (except for B. calcicola measuring 2.5-3.2 cm long). By contrast, the fl oral bracts of Myxochlamys are coriaceous, persistent, measuring 2.5-5 cm long and most notably are covered with transparent slimy mucilage. In addition, the unique versatile anthers of Myxochlamys, a rare feature in the Zingiberaceae, are distinct from the adnate anthers in Borneocola and also from all its sister genera. Based on morphological features, Myxochlamys is more similar to Scaphochlamys, the closest being S. grandis. Both have large sessile leaves and decurrent lamina base, large, coriaceous and persistent fl oral bracts,their bracts being concave with refl exed and spreading apices.
Based on morphology, Borneocola is also similar to Distichochlamys. However, Distichochlamys is distinguished from Borneocola, Myxochlamys and Scaphochlamys by its unique tubular bracteoles, fl oral tube without a groove on the inner surface and trilocular ovary (Newman 1995). Other characteristics such as distichous fl oral bracts, 2-keeled bracteoles, thecae without basal spurs have been observed in the three closely allied sister genera in this study (Table 2).
Haplochorema K.Schum. is another small-sized genus endemic to Borneo, which can be mistaken for Borneocola. It has short and few-fl owered infl orescences as in Borneocola but its fl owers appear somewhat quadrate with the labellum and lateral staminodes held fl at, more resembling Kaempferia L. Haplochorema has distichous fl oral bracts, single-fl owered cincinni and the fl owering proceeds from apex to base, to name some of the characters which distinguish it from Borneocola. In fact, the genus is more allied to Boesenbergia Kuntze than Borneocola.
Borneocola is morphologically most similar to Scaphochlamys but both can be distinguished by the texture of the bladeless sheath and fl oral bracts, variegation on the labellum, indumentum on the fl oral tube and the stigma shape. Th e current study recognises eight Borneocola species while S. gracilipes, S. polyphylla B.L.Burtt & R.Sm., S. limiana Meekiong & K.Yazid and S. samunsamensis Meekiong & Hidir from Borneo remain in the genus Scaphochlamys. Th ere are no recent collections of S. gracilipes but the lax infl orescence and persistent fl oral bracts in the type specimens clearly distinguish it from the Borneocola species. Scaphochlamys polyphylla, S. limiana and S. samunsamensis can be readily distinguished from the Borneocola species by their papery bladeless sheath and large, green or green tinged red, coriaceous fl oral bracts. Th is shows that the distinct morphologies that separate Borneocola and Scaphochlamys are signifi cant and are also supported by the phylogenetic analyses (Figures 1, 2 and 3). An anatomical study on the leaves also discovered some characteristics that separate Borneocola from Scaphochlamys (Norhati, pers. comm).
Th e morphology of Borneocola is very similar to Scaphochlamys but, combining both, necessitates synonymising Myxochlamys and possibly Distichochlamys and this will result in a very heterogenous genus. A similar situation is observed in the naming of Newmania N.S. Lý & Škorničk, a genus very similar in morphology to Haniffi a Holttum but appears as its sister group in the molecular phylogenetic analyses. Th e authors decided against placing Newmania under Haniffi a which would create a heterogenous group. Th e current description of Borneocola is further supported by the chromosome number with 2n=10 (Šída et al., unpublished data), diff erent from Distichochlamys (2n=26) and Scaphochlamys (2n=28). Such signifi cant diff erences in molecular data and chromosome number have conclusively supported the circumscription of the new genus Borneocola.