Thismia ornata and T. coronata (Thismiaceae), two new species from Sarawak, Borneo

Abstract: Thismia ornata and T. coronata (Thismiaceae), two new species from Sarawak (Malaysian Borneo) are described and illustrated. Thismia ornata belongs to T. sect. Thismia subsect. Odoardoa and occurs at several localities in western Sarawak around the city of Kuching. This species has been misidentified as T. aseroe until now, although the two species differ considerably. The species is assigned a preliminary conservation status of VU according to IUCN criteria. Thismia coronata belongs to T. sect. Sarcosiphon and has been found at only one site near the village of Long Tuyo in Lawas district of northern Sarawak. It is related to T. goodii and T. kelabitiana. The species is assigned a preliminary conservation status of CR. Citation: Dančák M., Hroneš M. & Sochor M. 2020: Thismia ornata and T. coronata (Thismiaceae), two new species from Sarawak, Borneo. – Willdenowia 50: 65–76. doi: https://doi.org/10.3372/wi.50.50106 Version of record first published online on 10 March 2020 ahead of inclusion in April 2020 issue.


Introduction
Thismia Griff. (Thismiaceae) is a genus of monocotyledonous plants that typically inhabit the understorey of tropical and subtropical forests. The genus comprises about 85 species Suetsugu & al. 2018b) and is distributed mostly in the tropical regions of Asia, Australia and South America and extending into subtropical and temperate regions of Japan, New Zealand, Australia and the USA (Merckx & al. 2013). Two main centres of Thismia diversity in Southeast Asia are located on the Malay Peninsula and in Borneo, with 16 and 22 recognized species, respectively (Jonker 1948;Chantanaorrapint 2018;Nishioka & al. 2018;Siti-Munirah 2018;Tanaka & al. 2018;Siti-Munirah & Dome 2019;Dančák & al. 2020). The number of described species has been increasing rapidly over the last few years, which is probably one of the highest rates among angiosperms (Stevens 2001+). For example, 14 species were described in 2018 alone (Chantanaorrapint & Suddee 2018;Nishioka & al. 2018;Siti Munirah 2018;Suetsugu & al. 2018aSuetsugu & al. , 2018bSuetsugu & al. , 2018cTanaka & al. 2018).
During our field trip to Sarawak (Malaysian Borneo) in January and February 2019 we found two new species of Thismia and describe them in this article, which brings the number of Bornean species to 24. 66 Dančák & al.: Thismia ornata and T. coronata from Sarawak

Material and methods
This study is based on material collected during January and February 2019 in Limbang and Kuching Divisions of Sarawak. Morphological characters were studied using a hand lens (30 -60× magnification), stereo microscope and macro photography. Collected specimens were thoroughly compared with original drawings and descriptions given in protologues of representatives of Thismia sect. Thismia and T. sect. Sarcosiphon (Blume) Jonker. Herbarium vouchers for this study are deposited in SAR and OL. Because Thismia populations are sometimes composed of a few or even a single reproductive individual, voucher specimens were not always collected to prevent unnecessary harm to the population. In these cases only DNA samples were taken because for DNA analysis substantially less material is needed than for useful herbarium specimens. Also, herbarium vouchers were not collected if the only plants available were not in full bloom. In both cases plants were documented by photographs. Additionally, herbarium material deposited in K and SAR was studied (herbarium codes according to Thiers 2019+).
DNA was extracted by the CTAB method (Doyle & Doyle 1987) from a silica gel-dried piece of tissue from one individual per population. Sequence data were generated for three nuclear and two mitochondrial loci. The small subunit of ribosomal DNA (SSU rDNA) was amplified and sequenced with primers NS1 and NS6, internal transcribed spacers of ribosomal DNA (ITS) with primers ITS1 and ITS4 (White & al. 1990) and large subunit of ribosomal DNA (LSU rDNA) by primers N-nc26S6 and 2134rev (Kuzoff & al. 1998). The mitochondrial genes atpA and matR were amplified and sequenced with primers developed by Eyre-Walker & Gaut (1997) and primers 26F and 1002R (Meng & al. 2002, respectively. All PCRs were performed with Kapa polymerase (Kapa Biosystems) following a standard protocol with 37 to 40 cycles and annealing temperature of 56°C (rDNA, atpA) or 47 °C (matR). The PCR products were purified by precipitation with polyethylene glycol (10 % PEG 6000 and 1.25 M NaCl in the precipitation mixture) and sequenced in both directions by Sanger method at Macrogene Europe. The most variable locus, ITS, was sequenced in all collections to screen variation, whereas the other loci were only analysed in selected specimens.
Sequences were edited and aligned in Geneious 8 (Biomatters) and deposited in NCBI GenBank under accession numbers MN067232 -MN067237, MN067250 -MN067259, MN067281 -MN067283, MN067288, MN067290, MN067300, MN067302, MN067303, MN067307, MN067309, MN067318 -MN067320, MN067327 and MN067328. The newly generated sequences were added to the dataset from  and Dančák & al. (2020), which included nine species whose sequences were downloaded from NCBI GenBank. The alignments are provided as supplemental content online. Bayesian phylogeny inference from concatenated data from the five loci (or four in the Gen-Bank accessions) was computed in MrBayes (ver. 3.2.4; Ronquist & al. 2012) with 2×10 7 generations, sampling every 3000 th generation, in two independent runs, each with four chains; the first 10 7 generations (50 %) were excluded as burn-in. The substitution model for each locus was used as determined by ITS1, ITS2 and 5.8S rDNA were treated as separate partitions).

Results and Discussion
Thismia ornata Dančák, Hroneš & Sochor, sp Diagnosis -Thismia ornata differs from the most similar known species, T. filiformis Chantanaorr., by flower size (to c. 10 cm in diam. vs to c. 3 cm including tepal appendages), inner surface of floral tube (with very fine bright orange reticulum inside vs lacking any reticulum), length of tepal appendages (to 35 mm vs to 8 mm), appendages on apical margin of connective (five of three different shapes vs three of two different shapes) and shape of lateral appendage (with small horn-shaped projection arising from each side of the lateral appendage vs lacking any horn-shaped projections).
Distribution -Thismia ornata occurs in western Sarawak in a number of locations surrounding Ku ching, including Kubah National Park, Santubong National Park, Dered Krian National Park and Fairy Cave Nature Reserve (Fig. 3). Habitat -Thismia ornata inhabits a wide range of tropical lowland rain forest habitats with an altitudinal range from 40 m to c. 300 m a.s.l. It is known from rather dry limestone outcrops, lowland mixed dipterocarp forests, riverine forests including forests with some anthropogenic disturbance.
Conservation status -Thismia ornata is endemic to Borneo. Most, if not all, known populations occur within national parks and other protected areas. While the extent of occurrence (EOO) is c. 270 km 2 , its minimal area of occupancy (AOO) could be estimated to be 30 km 2 . It is known from several populations, which represent three locations (sensu IUCN 2012). Thismia ornata is therefore assigned a preliminary conservation status of VU (D1+2) according to the IUCN Red List categories and criteria (IUCN 2012).
Etymology -The specific epithet is the feminine form of the Latin adjective ornatus (ornate or decorated), whichreflects the colourful flowers and very fine bright orange reticulum inside the floral tube that resembles lace.
Remarks -Thismia ornata is not completely unknown to the scientific community; it has been collected several times (see Additional material examined) and many of its photographs circulate on the internet, especially from limestone outcrops around Bau town (e.g. Fehland 2019). However, it has been misidentified as T. aseroe Becc., a superficially morphologically similar but not closely related species (unpublished data). Thismia ornata was also studied under the name of T. aseroe in an extensive study of floral morphology and development in Diosco reales (Caddick & al. 2000) and is adequately described and illustrated in that work. Correspondingly, the DNA sequence entries in GenBank that are based on the specimen Caddick 349 (K, SAR!) and stored under the name T. aseroe belong to T. ornata (see Fig. 4).
Thismia ornata belongs to T. sect. Thismia subsect. Odoardoa Schltr. on account of its having six free tepals of the same size and shape. Relationships of T. ornata within this section however are unclear. Its morphology is rather unique although it superficially resembles T. filiformis (and its relatives). The inner morphology of the flower is different. The bright orange reticulum in-   (Dančák & al. 2013;Tsukaya & al. 2017). Although it has been frequently misidentified as T. aseroe, the two species are different. Thismia aseroe has a prominently raised annulus and small teeth alternating with the tepals, which make it appear different to the naked eye. The structure of the connectives is also strikingly different between the two species (for comparison see Groom 1895 and Fig. 2). Although no variation was detected among the seven studied specimens (six of them from Kubah) at the ITS locus (see also Fig. 4B), one SNP was detected at each of the SSU and matR loci in the Santubong population (Fig. 4A). Such a small difference, of course, cannot have consequences in taxonomy, but it implies that the species exhibits some small geographical genetic variation. Nevertheless, no significant variability in phenotype was observed. Diagnosis -Thismia coronata differs from T. kela bitiana by the shape of outer tepals (entire with a single tooth in the middle vs deeply divided into several acute lobes), mitre apex (without tetrahedral depressions on upper surface vs with tetrahedral depressions on upper surface), smaller size of flower (1.8 -2.3 cm vs 2.6 -2.8 cm long) and flower colour (dark yellow to orange across whole flower vs white floral tube and bright yellow upper parts).
Distribution -Thismia coronata is known only from the type locality and its close surroundings in the Paya Maga mountain range in northern Sarawak (Fig. 3). At least 40 individuals were recorded at the locality.
Habitat -Thismia coronata occurs in lower montane primary tropical rainforest at an altitude around 1300 m a.s.l. It was found in relatively humid forest with dense understorey (Fig 7C). A variety of other mycoheterotrophic species were abundant at the type locality and surroundings, including Burmannia lutescens Becc. agg.  Conservation status -Thismia coronata is endemic to Borneo and the only known population occurs outside national parks and other protected areas. The number of mature individuals observed is fewer than 50. The extent of occurrence cannot be estimated because the species is known only from the type locality and its area of occupancy (AOO) is estimated to be only 4 km 2 . Thismia coronata is therefore assigned a preliminary status of CR (B2ab(iii); D) according to the IUCN Red List categories and criteria (IUCN 2012).
Etymology -The specific epithet is the feminine form of the Latin adjective coronatus (crowned), which reflects the shape and colour of the tepals resembling a royal crown. Remarks -Thismia coronata superficially resembles T. kelabitiana mostly by its colour, which is, nevertheless, darker and nearly homogeneous across the flower. However, the flower shape is similar to T. goodii Kiew, which differs in colour (blue tepals and white floral tube with a green tinge), smaller mitre and hairless apex of the connective. These three species (T. coronata, T. goodii and T. kelabitiana) are clearly closely related (see also Fig.  4), forming a morphologically distinct group within T. sect. Sarcosiphon. They share the well-developed outer tepals that are wider than long, trilobed apex of the connective and a prominent central rib along the inner side of the connective. Thismia goodii is almost sympatric with T. coronata: it is known from a nearby site only 1.5 km away (Ampeng & al. 2013). All three species occur in the highlands of northern Borneo and have no apparent relatives outside this region. Interestingly, this group bears some similarities to the group of T. cla vigera (Becc.) F. Muell. (see also . Although both groups are superficially rather different, primarily due to the three mitre appendages present in the T. clavigera group, the structure of the stamens is somewhat similar in both groups because the filaments reach the floral opening (which has no annulus) and the connectives have a prominent central rib along their inner side.