Taxonomy and phylogenetic position of Fimbristylis fusiformis , a new species of Cyperaceae from Thailand

Fimbristylis fusiformis, an unusual new species of Cyperaceae from Thailand, is described and illustrated. This taxon has a single terminal spikelet per culm with a semi-distichous glume arrangement, bisexual flowers that lack perianth parts, and pistil with persistent style whose base is slightly swollen and trigonous nutlets with pubescent ribs. Phylogenetic reconstruction using ITS sequence data places this taxon in Abildgaardieae and sister to the rest of Fimbristylis. The species has a conservation status of Least Concern (LC).


INTRODUCTION
The genus Fimbristylis Vahl, comprising c. 300 species, occurs mostly in the tropics and subtropics with some species occurring in warm temperate regions (Govaerts et al. 2007).The genus is morphologically diverse, with inflorescences ranging from complex umbel-like structures to a solitary spikelet.The glumes are generally spirally arranged in ellipsoid spikelets, more rarely distichously arranged, the bisexual flowers lack perianth parts and their deciduous styles have distinctly thickened base, and the nutlets often have distinct surface patterns.Fimbristylis is currently (e.g., Govaerts et al. 2007) treated to include the segregate genus Abildgaardia Vahl, the latter only differing in having a distichous glume arrangement (Goetghebeur 1998).
The genus was revised for Thailand by Simpson & Koyama (1998), who enumerated 60 species for that country.A study of undetermined material by one of us (DAS) suggested a previously undescribed species was present in eastern Thailand (Map 1).Further herbarium studies, together with associated fieldwork, confirmed that the species was new.We inferred the phylogenetic position of this new species using DNA sequence data, and describe and illustrate it here (Fig. 1-3).

MATERIALS AND METHODS
Specimens were examined from herbaria at BK, BKF, K, KKU and QBG.Fieldwork was undertaken in September 2008 and November 2011.Observations of inflorescences and fruits were made with an Olympus SZ-PT binocular microscope.Mature nutlets from the spikelet of the holotype were collected for scanning electron microscopy.Selected material was mounted on stubs with double-sided adhesive tape and coated with gold using a SC7620 mini sputter-coater (Polaron range).Micro-graphs were generated using a JSM6460LV scanning electron microscope (JEOL Ltd.) Using a silica gel dried sample of the new taxon, DNA was extracted using the CTAB method and the ITS marker amplified and sequenced using standard protocols and primers (Muasya et al. 2014).Contigs of forward and reverse sequences were assembled using the STADEN package (Staden 1996).Additional ITS DNA sequences, primarily taken from studies of Abildgaardieae (Ghamkhar et al. 2007) and Cypereae (Yano et al. 2012), were downloaded from GenBank.The matrix thus assembled (Table 1) included a total of 90 taxa, with representatives of subfamily Cyperoideae, tribes Abildgaardieae, Cypereae, Eleocharideae, Fuireneae, Scirpeae with Cladium as outgroup.The matrix was aligned using Muscle (v. 3.8.31;Edgar 2004), and further manually aligned in BioEdit (v. 7.0.9;Hall 1999).The aligned matrix was analysed using maximum parsimony in PAUP* (Swofford 2002), with heuristic searches using the random-addition-sequence method with 10 000 replicates, Tree-Bisection-Reconnection (TBR) branch-swapping with the Multrees option in effect and no maximum tree number set.Node support was evaluated using bootstrap analyses with 500 replicates, repeating the heuristic search procedure (with 10 replicates) above.

RESULTS AND DISCUSSION
Although the new species was easily identifiable as a member of subfamily Cyperoideae (absence of the spicoid floral structure (Simpson et al. 2003), individual flowers comprising stamens and gynoecium subtended by a glume), its generic placement within Cyperoideae was not immediately obvious.The combination of morphological characters, especially in the structure of the gynoecium, with the scabrous style, the apparent lack of a disjunction between the style base and nutlet, the fusiform shape of the nutlet and the fimbriate hairs at its apex and base (Fig. 1), suggested a genus other than Fimbristylis.Indeed, some aspects of the plant's gross morphology (e.g., a single, terminal spikelet) were superficially similar to genera such as Trichophorum (tribe Scirpeae) whereas the lack of perianth parts and possession of a persistent style with an indistinct base on the nutlet, were reminiscent of features observed in Isolepis (tribe Cypereae).The aligned matrix yielded 837 characters, of which 419 were parsimony-informative, 116 were variable but parsimony-uninformative and 302 were constant.
Parsimony analysis recovered 16 equally parsimonious trees (Fig. 4), and the ITS phylogeny is similar to results previously published using plastid (e.g., Ghamkar et al. 2007, Muasya et al. 2009) and nuclear (e.g., Yano & Hoshino 2006, Ghamkar et al. 2007) markers.These strongly supported the Abildgaardieae clade as having two subclades, one comprising Bulbostylis (including Actinoschoenus and Arthrostylis) and the other comprising Fimbristylis.The new taxon was sister to the rest of Fimbristylis in all the 16 trees generated in our study, but there was a lack of bootstrap support for the backbone topology in Fimbristylis in this study as well as previous studies (e.g., Ghamkhar et al. 2007) using trnL-F and ITS sequence data.
On present evidence, the best placement for the new species is in Fimbristylis.However, we were unable to assign the new taxon to any of the sections in Fimbristylis s.l.recognized by previous researchers (e.g., Kern 1974) and further studies are needed to elucidate the precise relationships of the new taxon.Superficially similar to F. pauciflora R.Br. but distinguished by the glumes 6.5 -8.5 mm long (vs 2.5 -3 mm long in F. pauciflora), nutlets fusiform, fimbriate at apex and base, with 3 longitudinal costae (vs nutlets obovate and glabrous in F. pauciflora).-Type: K. Wangwasit 080927-17 (holo K; iso BK, KKU), Thailand, Ubon Ratchathani, Pha Taem National Park, 27 Sept. 2008.

Fimbristylis fusiformis
Etymology.Named after the shape of the nutlets.
Distribution -Endemic to eastern Thailand.Mostly seen in Pha Taem National Park but also observed in Phu Chong Na Yoi National Park, both in Ubon Ratchathani.
Habitat & Ecology -Open, stony places with scattered tree cover on seasonally wet, sandy soils.Altitude 227 m (Google Earth 2016).
Conservation status -Least Concern (LC; IUCN 2012).The species occurs in protected areas and is locally abundant in these localities.

Fig. 4
Fig. 4 One of 16 maximum parsimony trees, showing the position of Fimbristylis fusiformis in the Abildgaardieae clade (highlighted in the grey box).Nodes observed in the strict consensus tree are marked with an asterisk (*) and bootstrap support values greater than 50 % are indicated above the branches before the nodes.

Table 1
List of taxa sampled for the phylogenetic study.