A new Haniffia species (Zingiberaceae) and a new generic record from Sarawak, Malaysian Borneo

Haniffia Holttum is a genus of three described species of terrestrial gingers hitherto restricted to Peninsular Thailand and various localities in Peninsular Malaysia. With generic placement confirmed using nrITS, trn K and mat K plastid sequence data, Haniffia santubongensis S.Y. Wong & P.C. Boyce is described as a taxonomically novel species representing a new generic record for Borneo, to where it is endemic to Mount Santubong, Kuching Division, NW Sarawak, Malaysian Borneo. An identification key to all species is given and H. santubongensis is illustrated from living plants. Haniffia santubongensis is the fourth species of Haniffia so far described, and the first occurring on sandstone.


Background
Haniffia Holttum is a genus of three described species of terrestrial gingers hitherto restricted to Peninsular Thailand and various localities in Peninsular Malaysia. The three described species are all seemingly locally endemic. The type species, H. cyanescens (Ridl.) Holttum, is restricted to Bukit Tanga (Negeri Sembilan, Peninsular Malaysia), with a variety, H. cyanescens var. penangiana C.K. Lim, occurring on Pulau Pinang and Kedah. The most recently recognized species, H. flavescens Y.Y. Sam & Julius (Sam et al. 2009) is known only from Endau Rompin National Park (Johor, Peninsular Malaysia). The sole extra-Malaysian species, H. albiflora K. Larsen & Mood, is confirmed only from Nam Tok Chatwarin, Naratiwat, Thailand. A summary of the taxonomic history of Haniffia Holttum is presented by Larsen and Mood (2000).

Plant material
Fresh leaf material of Haniffia santubongensis was collected from the type locality, Mount Santubong. The type specimen with the spirit material was deposited to SAR.

DNA extraction, amplification and sequencing
Genomic DNA was extracted using a modified CTAB protocol. ITS, trnK intron and matK gene were amplified using the same set of primers as in Leong-Škorničková et al. (2011). PCR products were purified using GenJet PCR purification kit (Thermo Scientific, Vilnius, Lithuania) and sent for sequencing in forward and reverse directions at First BASE Laboratories Sdn. Bhd., Selangor, Malaysia. Sequences were edited, assembled and aligned using MUSCLE (Edgar 2004) as implemented in Geneious Pro v5.6.4 (Biomatters Ltd., Auckland, New Zealand;www. geneious.com;Drummond et al. 2012). Two newly generated sequences were deposited into GenBank under accession numbers KJ452785 (trnK/matK) and KJ452784 (ITS), and combined with sequences included in Leong-Škorničková et al. (2011). When the placement of the new sequences was confirmed to fall within the Kaempferia Clade, then the final data matrix was reduced to include all the species in the Kaempferia Clade with Cautleya gracilis (Sm.) Dandy and Roscoea cautleoides Gagnep. selected as outgroups. Table 1 shows the list of species included for the final data matrix. The data matrix was deposited into TreeBASE (reviewer access URL: http://purl. org/phylo/treebase/phylows/study/TB2:S15361?x-access-code=f78126f9da891d3c6999dd52dfafdf77&format=html).

Phylogenetic analyses
Phylogenetic analyses were performed with PAUP*4.0b10 (Swofford 2002) for maximum parsimony (MP) reconstruction with all characters equally weighted. The most parsimonious trees were obtained with heuristic searches of 1,000 replicates with random stepwise sequences addition, tree bisection-reconnection (TBR) branch swapping, collapse of zero-length branches, with the multiple-tree option in effect, and saving up to 10,000 trees from each random sequence addition.
The most suitable nucleotide substitution model for each of the gene regions was selected in jModeltest ver. 0.1.1 (Posada 2008) using Akaike information criterion (AIC). General time reversible (GTR + I + G) was the nucleotide substitution model selected. Maximum likelihood (ML) analyses were carried out using RAxML 7.2.6 (Stamatakis et al. 2008). Maximum likelihood bootstrap values were obtained by running 10,000 replicates. Bayesian phylogenetic analyses were performed with MrBayes ver. 3.1.2 (Huelsenbeck and Ronquist 2001). Markov chain Monte Carlo (MCMC) was repeated twice to assure parameter convergence. The MCMC algorithm was run for 2,000,000 generations with one cold and three heated chains, starting from random trees and sampling one out of every 100 generations. Convergence was assessed by using the standard deviation of split frequencies as convergence index with values < 0.005 interpreted as indicating good convergence. The first 10% of trees were discarded as burn-in. Remaining trees were used to construct 50% majority-rule consensus trees.

Morphology and biogeography
In overall appearance H. santubongensis is most similar to H. cyanescens, sharing with that species a wide bluish labellum and semi-glossy fruits. However, H. santubongensis is clearly distinct from H. cyanescens by the lateral staminodes with an oblique bifid tip, the labellum distally notched (not deeply divided), and differences in labellum colouration and patterning (most notably the presence of a median yellow callus in H. santubongensis).
Haniffia santubongensis represents a new generic record for Borneo where it is locally endemic to Gunung Santubong. Combined with the three locally endemic species in Peninsular Malaysia and P. Thailand, it provides further compelling evidence that these Haniffia species, along with numerous other examples in families as diverse as the aroids, the palms, Rubiaceae Juss., and the genus Hanguana Blume, represent relictual fragments of the Riau Pocket phytochore (Ashton 2005;Corner 1960).

Molecular analyses
The combined ITS-plastid dataset contained the new species and 19 species in the Kaempferia Clade recognized in Leong-Škorničková et al. (2011) together with two outgroup species. The dataset comprises 2,115 characters: 1,682 characters were constant, 251 variable, but parsimony uninformative, and 182 (8.6%) were parsimony informative. The data analyses produced 32 shortest trees of length 633 steps with a Consistency Index = 0.7615 and Retention Index = 0.7729. The tree topologies obtained from three analyses are similar and consistent with the results from Leong-Škorničková et al. (2011). Figure 1 shows the partial tree obtained from maximum likelihood analysis. The new species is shown to be a sister taxon to H. albiflora and H. flavescens. Haniffia cyanescens is supported basally to the three Haniffia species. The new species of Haniffia differs by nine nucleotide substitutions (two in trnK intron and seven in ITS), an addition of 17 bps (trnK intron), an eight bp substitution (ITS), and seven bp deletion (ITS).

Diagnosis
Haniffia santubongensis most closely resembles H. cyanescens var. cyanescens (Negeri Sembilan) but is readily distinguished by the oblique-tipped, bifid lateral staminodes, and labellum distally notched (not deeply split) with a yellow median band extending from the entrance to the basal spur to the innermost extent of the lip distal division.

Distribution
Haniffia santubongensis is known only from the type locality where it occurs as two separate, dense, populations.

Etymology
The species epithet is derived from the name of the type locality.

Conclusion
Haniffia santubongensis is the fourth species of Haniffia and represents a new generic record for Borneo.