Thismia hongkongensis (Thismiaceae): a new mycoheterotrophic species from Hong Kong, China, with observations on floral visitors and seed dispersal

Abstract A new species, Thismia hongkongensis S.S.Mar & R.M.K.Saunders, is described from Hong Kong. It is most closely related to Thismia brunonis Griff. from Myanmar, but differs in the number of flowers per inflorescence, the colour of the perianth tube, the length of the filaments, and the shape of the stigma lobes. We also provide inferences on the pollination ecology and seed dispersal of the new species, based on field observations and interpretations of morphology. The flowers are visited by fungus gnats (Myctophilidae or Sciaridae) and scuttle flies (Phoridae), which are likely to enter the perianth tube via the annulus below the filiform tepal appendages, and exit via small apertures between the filaments of the pendent stamens. The flowers are inferred to be protandrous, and flies visiting late-anthetic (pistillate-phase) flowers are possibly trapped within the flower, increasing chances of pollen deposition on the receptive stigma. The seeds are likely to be dispersed by rain splash.


Introduction
Th ismia Griff . species are small herbaceous plants with a highly reduced vegetative structure. Th ey are invariably mycoheterotrophic, relying on fungal symbionts to obtain nourishment from decaying organic material, and they therefore lack chlorophyll. Individuals remain underground throughout most of the year, only emerging briefl y to fl ower and fruit after periods of heavy rain; as a consequence, Th ismia species are rarely collected and relatively little is known of their taxonomy, distribution and reproductive biology.
Th e fl oral morphology of the genus is complex. Th e tepals are congenitally fused (Caddick et al. 2000) to form a perianth tube with apically free lobes that are arranged in two whorls of three. Th ese tepal lobes are often morphologically very elaborate, sometimes apically coherent and forming a mitre or dome, and often adorned with elongated tentacles that are either free or united. Although the functional signifi cance of these morphologically complex tepals is obscure, they presumably perform a role in pollinator attraction. Th e aperture at the apex of the perianth tube is surrounded by a prominent annulus. Each fl ower has six stamens, the lateral margins of which are often postgenitally connate with each other, forming a ring that is suspended from the annulus. Since the stamens are pendent, the abaxial surface faces the centre of the fl ower, and the adaxial surface, which bears the thecae, faces the inner surface of the perianth tube. Th e fl owers are epigynous, with the fused carpels surrounded by an expansion of the receptacle (Caddick et al. 2000).
Although Th ismia was historically classifi ed in the tribe Th ismieae Miers (Miers 1847) within the Burmanniaceae (e.g., Jonker 1938; Maas-van der Kamer 1998), recent molecular phylogenetic analysis of nuclear and mitochondrial DNA sequences have revealed that it is more closely aligned with the Taccaceae (Merckx et al. 2006(Merckx et al. , 2009. Th e third iteration of the Angiosperm Phylogeny Group classifi cation (APG 2009) adopts a very conservative approach in which Th ismia is retained within the Burmanniaceae; we follow the majority of contemporary taxonomists, however, in separating Th ismia and related genera (Afrothismia Schltr., Haplothismia Airy Shaw and Oxygyne Schltr.) in the Th ismiaceae. Govaerts et al. (2007) listed 41 Th ismia species in their global checklist of the Dioscoreales, of which 24 were recorded from Asia; these statistics increase to 42 and 25, respectively, if Geomitra clavigera Becc. is recognized as a Th ismia species, as suggested by morphological data (Stone 1980) and molecular phylogenetic analysis (Merckx et al. 2006). Th e discovery of new species in the genus has accelerated signifi cantly in recent years, with 10 new Th ismia species described from Asia (Larsen and Averyanov 2007;Chantanaorrapint 2008Chantanaorrapint , 2012Chiang and Hsieh 2011;Tsukaya and Okada 2012;Dančák et al. 2013;Li and Bi 2013;Nuraliev et al. 2014;Truòng et al. 2014) since the publication of the checklist by Govaerts et al. (2007). In addition to these newly described species, there are several reports of signifi cant extensions to distributional ranges in Asia, including T. alba Holttum ex Jonker In this paper we describe a new species, T. hongkongensis, recently collected from Hong Kong. Th is is the fourth species in the genus recorded from China, supplementing earlier reports of T. taiwanensis S.Z.Yang, R.M.K.Saunders & C.J.Hsu from Taiwan (Yang et al. 2002;Wu et al. 2010), T. tentaculata from Hong Kong (Ho et al. 2009;Wu et al. 2010;Zhang and Saunders 2011), and T. gongshanensis H.Q.Li & Y.K.Bi from Yunnan (Li and Bi 2013). We also present some new observational data and inferences on the pollination ecology and seed dispersal mechanism of the new species. Although this information is limited in scope, it is of signifi cance given the paucity of existing data on the reproductive biology of the genus. Diagnosis. Similar to Th ismia brunonis Griff ., but diff ering in bearing a maximum of only three fl owers (with a single fl ower at anthesis), and having a dark red perianth tube with the fi liform appendages on the outer tepals remaining upright and forming a loose mitre over the annulus, longer staminal fi laments with two distinct teeth at connective apex, and rounded stigmas. Description. Small achlorophyllous holomycotrophic herbs. Roots clustered, ± horizontal, vermiform, fl eshy, 1.2-1.3 mm in diameter, cream-coloured. Stem creamcoloured, unbranched, erect, ca. 1.7 cm tall, 1.8-2 mm in diameter, glabrous, terete, with ca. 12 longitudinal ridges. Leaves white, appressed, clasping stem, narrowly triangular with acute apex, scale-like, 3-5.5 mm long, 1.7-2 mm wide; basal leaves smallest, upper leaves (equivalent to fl oral bracts) largest. Flowers in clusters of up to 3, developing sequentially with only one anthetic. Perianth actinomorphic, of 6 fused tepals, forming a perianth tube with free apical lobes. Perianth tube pinkish-white, membranous, urceolate, ca. 6.7 mm long, ca. 6.1 mm in diameter, with 12 dark red vertical ribs, abaxial surface distinctly verrucose; apex of perianth tube fused to form a dark red, rounded-hexagonal annulus, ca. 1.4 mm wide (top, externally), ca. 2.3 mm wide (base, externally) and ca. 1 mm (internal aperture); dark red, inverted V-shaped structures (putative nectaries) at apex of adaxial surface of perianth tube, opposite apertures between staminal fi laments. Outer tepal lobes triangular, ca. 1.8 mm long, ca. 1.5 mm wide at base; inner tepal lobes spathulate, concave adaxially, ca. 3.3 mm long, ca. 1.7 mm wide at widest point, bearing a dark red fi liform appendage on the abaxial surface,  ca. 4 mm long, ca. 0.5 mm in diameter (towards base), 0.4 mm in diameter (towards apex); the three fi liform appendages remain upright and cross each other, forming a persistent mitre. Stamens 6, pendent from the inner margin of perianth annulus, ca. 2.9 mm long, ca. 1.1 mm wide at widest point; fi laments free, ca. 1 mm long; stamens laterally connate, forming an anther tube; individual stamens with two thecae (adaxial, dehiscing towards inner surface of perianth tube), ca. 0.7 mm long; apical connective of stamens ca. 1 mm long, with two distinct teeth, adorned with trichomes, ca. 0.5 mm long. Ovary inferior, obconical, ca. 2.7 mm long, ca. 4 mm wide towards apex; style ca. 0.6 mm long, ca. 0.6 mm in diameter, with three bilobed, rounded stigmas; stigmatic head ca. 1 mm long, ca. 1 mm in diameter. Fruit a capsule ca. 4 mm long, ca. 4.8 mm wide, cup-shaped, carnose, pale orange-brown, dehiscing apically; fruiting peduncle ca. 2.5 mm diameter. Seeds numerous, yellow-brown.

New species description
Phenology. Flowering was observed between May and September. Flower development extends over several weeks (Fig. 1C-H). Based on our fi eld observations, mature fl owers are functional for up to three weeks, with up to three fl owers developing successively in each individual (Fig. 3A). Th e perianth tube abscises after fertilization (Fig. 1I, J), with a clear abscission zone (ab in Fig. 2A); the epidermis on the upper surface of the carpel subsequently disintegrates and the stigma is shed, exposing the seeds (Fig. 3A, D). Fruiting was observed between June and October.
Distribution and habitat. Th ismia hongkongensis is only known from the type locality in Hong Kong. Th e habitat is lowland secondary forest (west-facing slope, ca. 240 m above sea level); the site is not close to a stream and is therefore likely to dry out during periods of low precipitation. Co-occurring species include Adiantum fl abellulatum L. Discussion. Th ismia hongkongensis is most similar to T. brunonis Griff . (1844, 1845; as 'T. brunoniana' in the latter), the type species in the genus. Th ismia brunonis is only known from a single collection from Tenasserim in Myanmar, dating from October 1834. According to Jonker's (1938) supraspecifi c classifi cation of the genus, Figure 3. Fruit structure in Th ismia hongkongensis sp. nov. A Flower (rear right), immature fruit, shortly after fertilization (left), and mature fruit with exposed seeds (front). B Two fruiting individuals, each with three fruits. C Lateral view of fruiting specimen, illustrating elongated fruit stalk. D Mature fruit with exposed seeds. E Dehydrated fruit. F Rehydrated fruit, after rainfall. Photos by S.S. Mar. both species would be included in sect. Th ismia ('Euthismia') subsect. Brunonithismia Jonker as they have unequal and free tepal lobes. Comparison of the two species reveals several signifi cant diff erences, however, including fl ower number per infl orescence. Th e protologue of T. brunonis includes reference to fl owers clustered in a terminal raceme ("Flores pauci, in racemum brevem terminalem dispositi": Griffi th 1845: 341) with the accompanying illustration in the same publication ( Fig. 1 in Pl. XXXIX) showing infl orescences with four and six fl owers; Jonker (1938) subsequently described the species as bearing 3-8 fl owers per raceme. In contrast, T. hongkongensis invariably has fewer fl owers, with a maximum of three per infl orescence, reaching anthesis consecutively. Th e perianth tube of T. brunonis is pale yellow with red ribs (Griffi th 1845), whilst it is pink with red ribs in T. hongkongensis (Figs 1G-J, 2A, B). Th e fi liform appendages on the outer tepals of T. brunonis appear to be spreading at maturity (Griffi th 1845: Pl. XXXIX), whereas those of T. hongkongensis invariably remain upright and cross each other to form a loose mitre over the annulus 2A,B,4A). Th ismia brunonis also diff ers from T. hongkongensis as it has a much shorter fi lament: although a measurement was not included in Griffi th's descriptions of T. brunonis, the accompanying plate (Griffi th 1845: Fig. 7 in Pl. XXXIX) indicates that it is considerably shorter than the rest of the stamen. Th e shape of the stigma lobes also diff ers: it is acute in T. brunonis (Griffi th 1845: Fig. 9 in Pl. XXXIX; Jonker 1938), but rounded in T. hongkongensis (Fig. 4D).
Th ismia hongkongensis is strikingly diff erent from its congener in Hong Kong. Th ismia tentaculata has a white perianth tube with a bright yellow annulus, and three divergent orange-red fi liform appendages on the inner tepals (Ho et al. 2009).

IUCN conservation status. CR D (IUCN 2001
). Only one population is known, consisting of ca. 10 individuals, covering an area of approximately 4-5 m 2 . Th e population is located within the Tai Po Kau Nature Reserve, but is close to a major hiking path and the population is therefore susceptible to human disturbance and vegetation clearance.

Pollination ecology
Although the Burmanniaceae s.l. (inclusive of Th ismia) are reported to be protandrous (Vogel 1998), this inference was based on a paraphyletic circumscription of the family and it is unclear whether protandry has specifi cally been observed in Th ismia. Dissections of late-anthetic fl owers of T. hongkongensis allow tentative confi rmation of protandry, however, as the thecae are completely devoid of pollen (th in Fig. 2C).
Little is known of the pollinators of Th ismia, although several authors have suggested that the fl owers may be visited by small fl ies (Vogel 1962;Stone 1980;Rübsamen 1986). Th ese inferences were based on perianth morphology, the presence of osmophores on the tepals (Vogel 1962), the presence of nectaries either at the base of the perianth (Poulsen 1890) or along the suture between contiguous anthers (e.g., Groom 1895;Pfeiff er 1918;Jonker 1938Jonker , 1948Cribb 1986Cribb , 1995Th iele and Jordan 2002;Ho et al. 2009), and the formation of sticky pollen (Cranwell 1953). Th e only previous observational report of insect visitors to Th ismia fl owers is of small, unidentifi ed fl ies entering the perianth tube of T. gongshanensis (Li & Bi, 2013).
Several researchers have inferred that fungus gnats are likely to pollinate Th ismia fl owers based on structural mimicry (e.g., tepal extensions and reticulate patterning on the inner surface of the perianth tube), perianth colour and the emission of fungus-like fl oral scents (Vogel 1978;Rübsamen 1986;Th iele and Jordan 2002). We retrieved a fungus gnat (belonging to either the Mycetophilidae or Sciaridae) from within the perianth tube of a late-anthetic specimen of T. hongkongensis; unfortunately the poor state of preservation of the fl y precluded further identifi cation. We also retrieved an isolated insect wing from within the fl oral chamber which had the characteristic venation typical of a scuttle fl y (Phoridae). Fungus gnats and scuttle fl ies are generally associated with decaying organic matter and are often reported to feed on fungi (Hill et al. 1982).
Th e pollinators presumably enter the fl oral chamber of T. hongkongensis via the aperture within the annulus (a in Figs 2C, 4C). Assuming that the fl ower is protandrous as suggested, the movements of the pollinators are likely to diff er depending on whether the fl ower is in its early anthetic (staminate) or late anthetic (pistillate) phase. In staminate-phase fl owers, the fl ies are likely to be attracted to the putative nectaries (arrowed in Fig. 2D) located at the apex of the perianth tube, behind the pendent staminal ring. We hypothesize that the irregular surface on the adaxial surface of the perianth tube resulting from the reticulate venation (Fig. 2D) possibly enables the insects to climb and access these nectaries. Th e fl ies are likely to make contact with the thecae (th in Figs 2C, 4C) and inadvertently collect pollen whilst feeding from the nectaries, before departing via the small apertures (ca. 0.5 mm diameter) located between the fi laments (f in Figs 2C, 4C) of adjacent stamens. If the fl ies enter lateanthetic pistillate-phase fl owers, however, it is possible that they might be prevented from accessing the nectaries because of the raised lateral appendages of the stamens (la in Figs 2C, 4C), thereby increasing the time in which contact with the stigma is possible. Th e possible trapping of fl ies may explain the frequency with which fl ies are observed inside the perianth tube of late-anthetic fl owers. Similar interpretations of pollinator movement, involving climbing the inner wall of the perianth tube and exiting via the apertures between the fi laments, has previously been suggested by Maas et al. (1986) and Th iele and Jordan (2002) for other Th ismia species.

Seed dispersal
Several diff erent interpretations of seed dispersal have been proposed for Th ismia, including movement by earthworms with secondary dispersal possible if the worms are eaten by birds (Beccari 1890). Stone (1980) suggested that Th ismia seeds are likely to be dispersed by raindrops that splash seeds out of the fruit-cup. Th e size and shape of the fruit-cups of T. hongkongensis closely resemble functionally similar rain-splash dispersal structures in phylogenetically disparate groups (Nakanishi 2002): the upper surface of the fruit disintegrates at maturity (Fig. 3A), resulting in an upright, cup-like hypanthium (ca. 4.8 mm in diameter) with seeds that are fully exposed (Fig.  3D). Studies of other plant groups indicate dispersal of up to 1 m (Nakanishi 2002), although there are inevitably many variables (including plant height, seed weight, etc.) that aff ect potential dispersal distance; it is perhaps signifi cant that the stem of T. hongkongensis elongates as the fruit matures (Fig. 3C), thereby possibly increasing seed dispersal distance. Brodie (1951) observed that seeds of species that are rain-splash dispersed are often coated with a hydrophilic colloid that rapidly absorbs water, and which may act as a lubricant, facilitating separation of seeds by rain drops. Th ismia hongkongensis seeds are surrounded by a conspicuous mucilage-like substance (Fig. 3D) that may function in this way.
Th e fruits of T. hongkongensis appear to remain functional for several weeks. Although the plants are inevitably subjected to periods of desiccation, the fruits appear to be able to rehydrate eff ectively (Fig. 3E, F), thereby prolonging the period for seed dispersal.