Larsenianthus, a new Asian genus of Gingers (Zingiberaceae) with four species

Abstract Larsenianthus W. J. Kress & Mood, gen. nov. is described with one new combination and three new species. Larsenianthus careyanus (Benth.) W. J. Kress & Mood, comb. nov., is widespread in India and present-day Bangladesh; Larsenianthus wardianus W. J. Kress, Thet Htun & Bordelon, sp. nov., is from upper Myanmar in Kachin State; Larsenianthus assamensis S. Dey, Mood, & S. Choudhury, sp. nov., is restricted to Assam, India; and Larsenianthus arunachalensis M. Sabu, Sanoj & T.Rajesh Kumar, sp. nov., has only been found in Arunachal Pradesh, India. A phylogenetic analysis using the plastid trnK intron and nuclear ITS DNA sequence data indicates that the four species of Larsenianthus form a monophyletic lineage that is sister to Hedychium, a geographically widespread genus of about 50 species in tribe Zingibereae of subfamily Zingiberoideae. A dichotomous key and three-locus DNA barcodes are provided as aids for the identification of the four species of Larsenianthus.


Introduction
Th e classifi cation of the family Zingiberaceae continues to be refi ned (Kress et al. 2002, Harris et al. 2006 and new taxa are still being discovered and described (e.g., Ly et al. 2010). Recent fi eld work in South Asia has not only uncovered taxa new to science, but also provided new insights into our understanding of generic boundaries and species defi nitions (Sabu 2006). Here we clarify the generic placement of a species fi rst recognized over 125 years ago and describe a new genus of gingers with three additional new species.
In 1835 Nathaniel Wallich applied the name Hitchenia glauca to a ginger that he had listed earlier in his catalogue (Wallich 1832) as Curcuma glaucophylla Wall. (no. 6594). Another species listed in his catalogue as Curcuma careyana Wall. (no. 6595) was later moved to the genus Hitchenia by Bentham, who neither described its morphology nor provided any reason for the new generic placement (Bentham and Hooker 1883). A third species, which had previously been listed as Curcuma caulina J.Graham (Graham 1839), was added to the genus Hitchenia as H. caulina (J. Graham) Baker (Baker 1892). In that publication Baker circumscribed the genus Hitchenia and provided more detailed descriptions of the other two species, H. glauca and H. careyana. However, he realized the taxonomic problems that existed and stated that the latter species "..resembles H. glauca in habit, but diff ers so much in structure that probably it should form a diff erent genus…" (Baker 1892).
In Wallich's 1835 original application of the generic name, the genus honored Mr. Th omas Hitchin of Norwich, England, who was a gardener and distributor of rare plants in the 1810-1830s (Noltie 2005). Unfortunately Wallich misspelled Th omas Hitchin's name when he published the new genus as Hitchenia. Horaninow (1862) changed the name back to Hitchinia to correct Wallich's mistake, but this nomenclatural revision was not recognized by later botanists. In fact Bentham and Hooker (1883) mentioned Horaninow's corrected generic spelling as an error and preferred to use Hitchenia.
Since the time of Baker, little progress has been made in understanding the taxonomic status of Hitchenia until the publication of a new classifi cation of the Zingiberaceae by Kress et al. (2002). Hitchenia glauca, which had been once more discovered in Myanmar after many years of obscurity, was included in the molecular phylogenetic study of the family and shown to be closely related to several species of Curcuma. It was tentatively placed in Curcuma Group I (Kress et al. 2002). At about the same time as the publication of the new classifi cation of the family, living specimens of Hitchenia careyana (Plate 1B) were collected near the type locality of Sylhet (formerly Silhet) in Bangladesh (M. Collins, pers. com.). From a comparison by the authors of these living collections to material of Hitchenia glauca, it became clear that these two taxa were only superfi cially similar and probably not at all closely related to each other. For this reason it appeared that a new generic name was needed for H. careyana.
In 2002, as part of a survey of the gingers of Myanmar, one of us (WJK) collected specimens of an unknown ginger near Myitkyina, in Kachin State, which closely re-sembled H. careyana, but were distinctive in the infl orescence color and orientation (Plate 1A). In 2008 another of us (SD) discovered an unusual ginger during fi eld work in southern Assam. Surprisingly, the fl owers closely resembled those of both the former H. careyana and the newly collected material from Myanmar (Plate 1A-C). Finally, only a few weeks later, a fourth species of ginger with the same distinctive infl orescence and fl oral morphology was recognized by the third author (MS) of this paper whose botanical team had just returned from a survey of plants in northeast India (Plate 1D).
After studying both living specimens and preserved collections of all four taxa, the accumulated research data were suffi cient to warrant the circumscription of a new genus. Th e long exserted and arched style (with included fi lament) of the fl ower resembles a similar fl oral structure found in genera such as Hedychium, Globba, and Pommereschea. In order to obtain evidence of the origin of this fl oral characteristic in the new genus, to provide an independent test of the evolutionary relatedness of these four species, and to determine their position in the evolution of the family Zingiberaceae a molecular phylogenetic investigation was undertaken (see below). All data suggested that these four species should be placed in a new genus, which we have named Larsenianthus, allied to Hedychium.
With regards to the remaining species in the genus Hitchenia, Leong-Škorničková et al. (2007) in a study of chromosome number and genome size variation in the gingers demonstrated that one species, H. caulina, would be better placed in the genus Curcuma as initially proposed by Graham (1839). Eventually some taxonomists may prefer to subsume both species of Hitchenia into Curcuma.
Below we provide descriptions of the new genus and the four included species. Plant measurements were for the most part recorded from living material. Methods and results of the molecular phylogenetic analysis, GenBank accession numbers for a three-locus DNA barcode for each species, and assessments of the conservation status of all species of Larsenianthus are also provided.

Phylogenetic evidence and DNA barcode markers
Materials and methods DNA extraction, amplifi cation, and sequencing. Total genomic DNAs were extracted for a representative sample of each species of Larsenianthus described herein (Table  1) using a modifi ed CTAB protocol. Nucleic acid fragments for trnK and ITS as phylogenetic markers were amplifi ed using custom primers and Promega Go Taq Flexi under standard cycling conditions, and cleaned using an abbreviated PEG/NaCl procedure as described in Kress et al. (2002). Amplifi cation of rbcL and the trnH-psbA spacer region as DNA barcoding loci used published primers under standard conditions (Kress and Erickson 2007). Th e matK DNA barcode locus was isolated from the trnK intron sequences that were generated for the phylogenetic analyses. Fragments were fl uorescently labeled using Applied Biosystems (Foster City, California, USA) Big-Dye v3.1 (1/8 concentration) chemistry Terminator Cycle Sequencing Ready Reaction Kit following AB protocol for a 3130xl Automated DNA Sequencer. DNA fragments were compiled and edited in Sequencher 4.9 (Gene Codes Corp., Ann Arbor, Michigan, USA).
Phylogenetic analyses. Newly generated sequences of the trnK intron and ITS were added to a reduced taxon version of the Kress et al. (2002) data matrix, which included representatives of all subfamilies of the Zingiberaceae. Siphonochilus was designated as the outgroup based on results of the prior study (Kress et al. 2002). Th e data matrix was realigned using MUSCLE (Edgar 2004) as implemented in Geneious Pro v4.8.5 (Biomatters Ltd., Auckland, New Zealand; www.geneious.com) followed by minor manual adjustment with indels treated as missing data.
Maximum Parsimony. Separate and combined Fitch parsimony analyses of one thousand random sequence addition replicates with tree bisection and reconstruction (TBR) branch swapping, holding four trees, saving all shortest trees were conducted in PAUP *4.0b10 (Swoff ord 2002). Branch support was calculated based on 1,000 bootstrap pseudoreplicates (BS) of 100 random addition replicates (holding four trees, TBR branch swapping, saving 10 trees per replicate) to maximize the accuracy of the estimation while minimizing analysis time.
Likelihood. Bayesian analyses were conducted in MrBayes (Huelsenbeck andRonquist 2001, Ronquist andHuelsenbeck 2003) to estimate branch support using three replicates of fi ve million generations (sampling every 100 generations), running four chains. Th e trnK dataset was partitioned into three regions (trnK5' IGS, matK, and trnK3'IGS) and the combined dataset was partitioned into four regions (trnK as described above, plus ITS). Appropriate burn-in (number of generations discarded prior to calculation of posterior probability [PP]) for each analysis was determined based on a standard deviation of split frequencies (discarding all trees prior to stabilization below a standard deviation >0.01). Burn-in times for each data matrix are available from the senior author.

ITS.
Analyses of the ITS data matrix produced 23 shortest trees of 1,011 steps based on 268 parsimony informative characters (results not shown). Trees fell into three distinct tree islands, one of 21 trees, and two of one tree only. Diff erences between these three tree islands were minor. In all shortest trees, the four species of Larsenianthus form a monophyletic clade with moderate to strong support (BS=89%; PP=1.00), and are sister to a monophyletic clade of Hedychium (BS=100%; PP=1.00). Th e sister relationship is also supported although less strongly than the reciprocal monophyly of the genera if only parsimony is considered (BS=68%; PP=1.00).
trnK intron. trnK intron analyses produced over 100,000 shortest trees of 552 steps based on 328 parsimony informative characters (results not shown). Relationships were generally less well resolved and less strongly supported than with the ITS data, however there were missing data for part of the 5' trnK sequences for two of the Larsenianthus samples. Monophyly of Hedychium was moderately supported by parsimony (BS=84%) and strongly supported by Bayesian methods (PP=1.00). Th e majority rule consensus tree recovered a monophyletic Larsenianthus, but not the strict consensus tree. Th ree of the four species of Larsenianthus formed a monophyletic group with low to moderate support (BS=65%; PP=0.96). Similarly, a sister relationship between the two genera was not recovered.
DNA barcodes. Th e DNA barcodes that were generated for the three barcode loci, rbcLa, matK and trnH-psbA, (Kress andErickson 2007, CBOL Plant Working Group 2009) provide unique genetic identifi ers for each of the four species of Larsenianthus. GenBank accession numbers for sequences of each DNA barcode locus for each species are provided (Table 1).

Conservation status
Th e overall distribution of the genus covers a large area of sub-Himalayan forests in northeastern Bangladesh, the Indian states of Meghalaya and Assam, the northern border of Arunachal Pradesh, and into upper Myanmar. Larsenianthus careyanus is the most widespread of the species and by IUCN guidelines (IUCN Standards and Petitions Subcommittee 2010) we categorize it as Near Th reatened (NT) because of the gradual decline of the habitats it occupies and the small number of recent collections. Description. Evergreen, rhizomatous, terrestrial herbs, clumping with 10-20 shoots per plant, 1-2.5 m tall, plane of distichy of leaves parallel to rhizome, 2-12 leaves per shoot, alternate, sessile to petiolate. Infl orescence terminal on leafy shoot or basal on leafl ess shoot, pedunculate; bracts basally attached, refl exed or adpressed, spirally arranged and imbricate, 35-80 per infl orescence, not pouched, adventitious plantlets sometimes produced in sterile bracts at base of infl orescence; fl owers mature from base to apex of infl orescence. Bracteoles variable in size, the inner one largest, not tubular. Flowers conspicuous, in cincinni of 2-6 fl owers or rarely reduced to one fl ower; calyx tubular, tri-dentate, shorter than the corolla; fl oral tube long and curved, exserted well beyond the bract, corolla lobes subequal with dorsal lobe slightly larger than laterals, apex opening oblong, bordered on the two lateral sides with a thickened and rounded margin formed by the base of the lateral staminodes and the labellum; lateral staminodes small, bowl-shaped, refl exed; labellum narrow at the base, widening towards the apex, elongate, oblanceolate or spatulate, basal margins thickened, raised with center channeled or 'v' in cross-section, apex dentate or entire; fertile stamen long and arched over the labellum, anther oblong, without a crest, thecae dehiscent for full length; epigynous glands linear; style with stigma protruding beyond anther thecae; ovary trilocular, fruit an oblong capsule, two-layered with the outer splitting into three coiled sections, the inner forming an arillate membrane covering the 1-10 seeds.
Discussion. Larsenianthus is a distinctive genus in the tribe Zingibereae. Some similarities exist with the genera Hedychium, Globba and Pommereschea in the vegetative parts, the bract orientation, and the long fi lament. Th e uniqueness of the genus and its close relationship to Hedychium is confi rmed by phylogenetic analyses of DNA sequence data (Fig. 1). However, the combination of unique features including the narrow, elongate labellum with slightly thicken edges and a deeply channeled center combined with the small, cup-shaped lateral staminodes and strongly arched fi lament clearly distinguish this genus from others in the tribe. Two additional traits that char- acterize at least two of the four species in the genus (L. assamensis and L. careyanus) are the unusual white capsular fruit with a gelatinous fused arillate structure that encases the seeds and the multiple adventitious plantlets that form in the axils of the sterile bracts at the base of the infl orescence.
Etymology. Th is new genus honors Dr. Kai Larsen, Professor of Botany Emeritus, Aarhus University, Denmark, for his many years of dedicated eff orts in the education of botanists worldwide and for his profound achievements in the taxonomy of the family Zingiberaceae, especially in the fl ora of Th ailand. Description. Evergreen herb, medium-size to 125 cm tall; rhizome to 2.0 cm in diameter, fi brous, aromatic, inner color white; tubers present. Leafy shoots 10-20 per plant, erect, densely clumped. Leaves 4-5 per shoot, to 118 cm in total length; basal leaf sheaths reddish and glabrous, ca. 30 cm in length × 5 cm in width; sheathing petioles to 52 cm in length × 1.5 cm in diameter, glabrous, green and clasping stem, margin slightly revolute; ligule small, 10 mm in length × 1 mm in width, not lobed, truncate on petiole, papery and ephemeral disappearing in mature leaves; lamina 67 cm in length × 14 cm in width, ovate, green and glabrous above, lower midrib green and glabrous, base long attenuate onto petiole, slightly subequal, apex acuminate, adaxial surface dark green. Infl orescence basal on leafl ess shoot, erect to 33 cm in height; peduncle 20 cm in length × 1.0-1.5 cm in diameter, glabrous, lower sheaths red, upper sheaths green; spike ovoid, 6-10 cm in length × 4-5 cm in diameter; rachis straight; infl orescence bracts about 40 per infl orescence, 1-2 lower bracts sterile, bracts 3.7 cm in length × 2.4 cm in width at base of infl orescence, 2.0 cm in length × 1.8 cm in width distally, spirally arranged and imbricate, not pouched, held at 40° from vertical axis, glabrous, bright green with reddish apex, margins smooth; bracteoles not tubular, 2.1 cm in length × 1.6 cm in width, glabrous, pale white with reddish apex. Flowers conspicuous, 3-4 per bract; calyx tubular, 1.5 cm long, tri-lobed with central lobe shortest, pale yellow to pink; fl oral tube 3.2 cm in length × 2.0 mm in diameter, reddish pink, externally glabrous with scattered unicellular papillate hairs inside, lobed with each lobe 1.4 cm in length, refl exed; lateral staminodes 3.0 mm in length × 2.0 mm in width, cup-shaped, glabrous, red; labellum 2.2 cm in length × 3.0 mm in width at apex, linear to spatulate, not lobed, red basally with yellow apex, glabrous; fertile stamen with fi lament 2.1 cm long, red at throat of corolla becoming yellow distally, glabrous; anther 3.0 mm in length × 2.0 mm in width, without a crest; pollen pale yellow; epigynous glands two, linear; stigma minute, <1 mm across, white, extending slightly beyond anther; ovary trilocular, 2.0 mm in length × 2.0 mm in width, pubescent, white to cream colored. Fruits and seeds unknown.

Key to species of
Distribution. Known only from the type locality in Kachin State, upper Myanmar. Ecology. Th is species is found in the understory of evergreen forests dominated by members of the family Dipterocarpaceae in the foothills of the Himalayas.
Distribution. Known only from two locations in the Barail Wildlife Sanctuary. Larsenianthus careyanus is also frequently found in this same region.
Distribution. Larsenianthus arunachalensis is narrowly endemic in Arunachal Pradesh, India, and is known only from the type locality. It is highly endangered due to various anthropogenic activities.
Ecology. Th is species grows in sandy soil above 1,400 m in elevation amidst thick clumps of wild species of Musa.
Etymology. Th e specifi c epithet "arunachalaensis" is derived from the name of the state in northeast India from where the type specimen was collected.
Specimens examined. Known only from the type specimen.