Phylogenetic and biogeographical analyses of Thismia (Thismiaceae) support T. malipoensis as the eighth species in China

Abstract: Thismia Griff. (Thismiaceae) is a holo-mycoheterotrophic genus with more than 100 species. In this study, phylogenetic analyses supported that T. malipoensis from Yunnan is a new species in T. sect. Glaziocharis. Morphologically, this new species can be distinguished from its phylogenetic sister species T. abei by having the annulus of the flower expanded and modified into a cucullate (hood-like) structure with zygomorphic symmetry with one opening on one side. Biogeographical inference showed that SW China to Indo-Burma and the Sunda Shelf region was suggested as the ancestral distribution region of Thismia s.s., then eastward to SE China and Japan, and southward to New Guinea to Australia, respectively. The Chinese species should have originated from at least two different ancestral sources, and geographical isolation caused the divergence between T. malipoensis and T. abei at 17.47 Mya. Citation: Ya J.-D. Chen H.-Y. Zhang W. Zhu R.-B. Cai J. & Yu W.-B. 2024: Phylogenetic and biogeographical analyses of Thismia (Thismiaceae) support T. malipoensis as the eighth species in China. – Willdenowia 54: 47–63. Version of record first published online on 28 February 2024 ahead of inclusion in April 2024 issue.


Introduction
Heterotrophic plants exhibit distinct mechanisms for nutrient acquisition when compared to autotrophic plants.To date, more than 880 mycoheterotrophic plants that rely upon associations with fungi for their supply of organic nutrients have been documented (Merckx & al. 2013b).Due to their highly reduced leaves and lack of chlorophyll (Cameron & Leake 2007;Leake & Cameron 2010), holomycoheterotrophic are only visible during brief periods of flowering and fruiting.As large-scale expeditions are conducted across different seasons and remote areas, numerous leafless holo-mycoheterotrophic species have been discovered and classified as new to the field of science.Currently, most of these species are only found in small populations within one or a few locations, making them susceptible to being categorized as Critically Endangered (CR).Therefore, efforts to conserve these species are crucial (Merckx & al. 2013a).
Thismia Griff. is a holo-mycoheterotrophic genus with remarkable flowers in its peculiar appearance and exceptionally complicated and quite diverse floral morphology (Kumar & al. 2017;Shepeleva & al. 2020;Nuraliev & al. 2021).Traditionally, Thismia was treated as a member of the tribe Thismieae in the family Burmanniaceae (Maas-van de Kamer 1998; Merckx 2008;Li & al. 2020), with four other holo-mycoheterotrophic genera, i.e.Afrothismia Schltr., Haplothismia Airy Shaw, Oxygyne Schltr.and Tiputinia P. E. Berry & C. L. Woodw.Recent phylogenetic studies have supported the tribe Thismieae as a separate family Thismiaceae, because Thismiaceae is sister to the family Taccaceae and rather distantly related to the family Burmanniaceae s.s.(Merckx & al. 2006;Lam & al. 2016;Shepeleva & al. 2020).The most recent phylogenetic study showed that Thismia species are polyphyletic in that the Old World species formed five well-supported groups, and the neotropical species are clustered with Tiputinia, which probably represents a separate genus (Shepeleva & al. 2020), i.e.Ophiomeris Miers.In this study, we tentatively followed the traditional delimitation of Thismia s.l., which is characterized by having a prominent hypanthium (also called a flower tube or flower chamber) that bears six tepals and six stamens.To date, Thismia includes about 105 species and one variety (IPNI 2023;POWO 2023) (Supplementary appendix S1), which are distributed mainly in tropical and subtropical Asia to temperate Australia and tropical America (Shepeleva & al. 2020).
Thismia taiwanensis Sheng Z. Yang & al. was the first species to be described from China until 2002 (Yang & al. 2002).To date, seven species have been recognized in China (Yang & al. 2002;Ho & al. 2009;Chiang & Hsieh 2011;Li & Bi 2013;Mar & Saunders 2015;Xu & al. 2020;Li & al. 2023), and T. guangdongensis X.J. Li & al. was most recently described in 2023.Of these, Guangdong, Hong Kong and Taiwan have recorded two species, and Hainan and Yunnan have one species each, with T. hongkongensis Mar & R. M. K. Saunders found in Guangdong and Hong Kong.During botanical surveys in SE Yunnan in 2019, a unique flowering species of Thismia was found and collected (Fig. 1-4).After investigations on morphological characters and herbarium specimens from China and adjacent regions, as well as a literature review, we confirmed that this species was new to science, which was also supported by phylogenetic analyses using two nuclear ribosomal DNA (nrDNA) and two mitochondrial DNA (mtDNA) sequence datasets.Herein, this new species is named as T. malipoensis J. D. Ya & W. B. Yu by providing a morphological description, type specimen, colour photographs (Fig. 1-3) and line drawings (Fig. 4).Moreover, the phylogenetic and biogeographic significance of this new species is inferred and discussed, and conservation suggestions and recommendations are proposed.

Morphological observation
Voucher specimens of Thismia malipoensis were collected from the Laoshan Provincial Nature Reserve of Mali po County during a field expedition in 2019.The morphological characters of this new species were observed, measured and photographed based on living individuals.Fresh flowering specimens were identified by checking herbarium specimens and reviewing the relevant literature (Yang & al. 2002;Larsen & Averyanov 2007;Ho & al. 2009;Chiang & Hsieh 2011;Li & Bi 2013;Mar & Saunders 2015;Kumar & al. 2017;Dančák & al. 2020a;Nuraliev & al. 2020;Xu & al. 2020;Siti-Munirah & al. 2021;Li & al. 2023).DNA tissues of T. malipoensis were collected from flowering shoots, then dried using silica-gel.The type specimen was deposited at the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN; herbarium code following Thiers 2023+).

Phylogenetic analyses
In order to elucidate the phylogenetic association between Thismia malipoensis and other species within the Thismiaceae, phylogenetic analyses were conducted using sequences of nrDNA 18S and ITS, as well as mtDNA atpA and matR.A comprehensive analysis was performed on a total of 48 taxa, encompassing four genera of Thismiaceae, with Tacca palmatifida Baker (Taccaceae) as the outgroup.Appendix 1 provides voucher information for the GenBank accession numbers associated with the sequences generated or obtained from the studies of Dančák & al. (2020bDančák & al. ( , 2020c)), Merckx & al. (2017), Shepeleva & al. (2020) and Sochor & al. (2018) for this study.
Genomic DNA of the new species was isolated from silica-gel dried shoot tissues of the type collection using the CTAB method (Doyle & Doyle 1987).Library preparation for the Next Generation Sequencing followed the procedure of Zeng & al. (2018).Around 500 pg purified genomic DNA was fragmented to approximately 350 bp in size by sonication, then built into a blunt-end DNA library using the NEBNext Ultra II DNA library prep kit for Illumina (New England BioLabs) following the standard protocol.The 150 bp pair-end reads were generated using the Illumina Sequencing Platforms.Raw data were assembled de novo for the mitochondrial and nuclear ribosome DNA contigs/sequences using the Get-Organelle toolkit (Jin & al. 2020).Assembled mitochondrial and nuclear ribosome DNA sequences of the new species were annotated in Geneious (Kearse & al. 2012) using nrDNA 18S and nrITS, and mtDNA atpA and matR sequences of Thismia species from NCBI as references, then four targeted DNA regions were extracted from the annotated sequences, respectively.
Four DNA regions were aligned separately using MAFFT 7.505 (Katoh & Standley 2013).Then the four regions were concatenated into a supermatrix (Supplementary appendix S2) using SequenceMatrix package of TaxonDNA 1.78 (Meier & al. 2006).Bayesian inference (BI) and maximum likelihood (ML) methods were used to reconstruct phylogenies.The BI analysis was performed using MrBayes 3.26 (Ronquist & al. 2012).The total dataset was partitioned, and the DNA substitution model of the Bayesian information criterion (BIC) for two DNA regions was estimated using jModeltest 2 (Darriba & al. 2012;Darriba & al. 2019).Markov chain Monte Carlo (MCMC) analysis was performed using Mr-Bayes for 10,000,000 generations for the total dataset, with two simultaneous runs, each run comprising four incrementally heated chains.The BI analysis was started with a random tree and sampled every 1000 generations.The ML analysis was conducted with RAxML 8.2.10 (Stamatakis 2014) using the GTR substitution model with gamma (Γ) distribution rate heterogeneity among sites and the proportion of invariable sites estimated from the data.Support values for nodes/clades were estimated from 1000 bootstrap replicates.

Divergence time estimation and ancestral area reconstruction
The concatenated dataset of 18S rDNA, ITS, atpA and matR from 49 taxa was used to estimate the divergence time among Thismia species.According to the previous studies (Merckx & Smets 2014), we chose the secondary calibration time for two nodes: (1) 63-96 Ma for the root node between Thismiaceae s.s. and Tacca J. R. Forst.& G. Forst.; and (2) 54-86 Ma for the crown node of Thismiaceae age (Merckx 2008).The divergence time was estimated using the MCMCTree program in PAML 4.10.5 (Yang 2007).Using the independent rate model (clock = 2) and the GTR + Γ model (model = 7).We ran two separate MCMC iterations for 400,000 generations with sampling every 10 iterations, the first 160,000 iterations were discarded as burn-in, and the mcmcfile was used to check ESS values and convergence in Tracer 1.7.2 (Rambaut & al. 2018).
Distribution information of 44 taxa of Thismiaceae and the outgroup Tacca palmatifida was collected from POWO (2023), and that of four accessions without name just using source information of the voucher (Shepeleva & al. 2020).Ten biogeographical areas of distribution range were classified according to previous biogeographical studies (Thomas & al. 2012;Sirichamorn & al. 2014;Liu & al. 2021): (A) Japan; (B) SE China; (C) SW China to Indo-Burma (excluding the Malay Peninsula); (D) India and Sri Lanka; (E) the Sunda Shelf region; (F) Philippines; (G) Wallacea; (H) New Guinea to Australia; (I) South America; and (J) Africa (Fig. 6).The dispersal-extinction cladogenesis (DEC) model (Ree & Smith 2008), dispersal-vicariance analyses (DIVA) model (Ronquist 1997), and BAYAREALIKE model (Landis & al. 2013), as well as the other three model with "+J" parameter (founder event speciation) in the R package BioGeoBEARS (Matzke 2018) that was used to reconstruct ancestral areas of Thismia.The maximum number of regions was set to 2 and the lowest corrected Akaike information criterion (AICc) was considered the "best" model.

Phylogenetic analyses
The nrDNA 18S matrix was 1639 bp in length including 384 variable sites and 247 parsimony-informative sites.The nrDNA ITS matrix was 1012 bp in length including 721 variable sites and 613 parsimony-informative sites.The mtDNA atpA matrix was 1112 bp in length including 146 variable sites and 79 parsimony-informative sites.The mtDNA matR matrix was 1065 bp in length includ-ing 419 variable sites and 190 parsimony-informative sites.The best-fit BIC model of 18S, mrITS, atpA and matR datasets was GTR+G11, TPM1uf+G, TPM1+G11, and GTR+I+G, respectively.The major-rule consensus tree of ML analysis with support values of both ML and BI analyses is shown in Fig. 5, which is consistent with the topology in the study by Shepeleva & al. (2020).Five well-supported clades were recovered in Thismia s.s., and each clade can correspond to four revised sections and one subsection in the subgenus division of Thismia proposed by Kumar & al. (2017).In addition, six species were not placed into any clades of the five recognized clades, with two Chinese species, T. hongkongensis and T. tentaculata K. Larsen & Aver., which are well supported as a monophyletic clade (MLBS/BIPP=98/1.00).
According to the study by Shepeleva & al. (2020), the modified Thismia sect.Glaziocharis (Taub.ex Warm.)Hatus. is a monophyletic group corresponding to Clade 1, which consists of five species from E Asia (China, Japan) and mainland SE Asia (Thailand, Laos).Herein, our phylogenetic analyses supported T. malipoensis as a member of T. sect.Glaziocharis, and allied to T. abei (Akasawa) Hatus., which are both putatively as the sister to T. gongshanensis Hong Qing Li & Y. K. Bi with weak support by ML analysis (MLBS = 53).Of the six sampled Chinese species, four species were fell in this clade, with the exception of T. hongkongensis and T. tentaculata.

Morphological comparisons and species delimitation
Morphologically, Thismia malipoensis stands out significantly from the other six species in T. sect.Glaziocharis of the Clade 1 (Fig. 5), as well as other four recognized Chinese species by having a cucullate (hood-like) and zygomorphic annulus structure of the flower that covering the apical floral tube with an opening on one side.As the phylogenetic sister species of T. malipoensis, T. abei is characterized by having connected inner tepals as a loose dome, a ring-like and actinomorphic annulus structure, short tepal appendages (5-6 mm) and glabrous stigma.To date, the cucullate annulus of has been reported only in T. belumensis Siti-Munirah & Suhaimi-Miloko and T. labiata J. J. Sm., which were supposed to be members of T. sect.Glaziocharis (Siti-Munirah & al. 2021).Thismia malipoensis is distinguished from T. belumensis in characters of the tepal appendages: both outer and inner tepal appendages are spreading; inner tepal appendages are shorter than 2 cm and equal to the outer ones; and inner tepal appendages have a fimbriate base and a subulate tip.Geographical information is also important for species delimitation between T. malipoensis and T. belumensis.Thismia malipoensis is distributed in Malipo, SE Yunnan, China, and grows under evergreen broad-leaved forests in limestone mountains at an elevation of 1100 m, whereas T. belumensis is endemic to Perak, Peninsular Malaysia, under the shade of lowland dipterocarp forests at an elevation of 260-290 m (Siti-Munirah & al. 2021).Due to the lack of molecular data for T. belumensis, it is not clear whether T. malipoensis and T. belumensis are sister species or are sharing similar floral forms and structures by convergent evolution.

Divergence time estimation and ancestral area reconstruction
The crown age of Thismiaceae was estimated to be 75.84Mya (95 % HDP: 42.85-94.53Mya), and the diversification time of Thismia s.s.started at 62.06 Mya (95 % HDP: 34.75-79.37Mya) in tropical and subtropical Asia (Fig. 6).For the five major clades or sections, the diversification times varied from 14.26 Mya (95 % HDP: 6. 15 Ancestral area reconstruction indicated that the ancestral distribution region of Thismiaceae was not clear, because the earliest divergent Oxygyne species are found in Africa and Japan of E Asia, and the following clade Haplothismia exannulata-Thismia panamensis is mainly distributed in Central to South America, as well as the only species Haplothismia exannulata Airy Shaw is in India of S Asia (Fig. 6).Thismia s.s. is restricted in E Asia to S and SE Asia.The Sunda Shelf and SW China to Indo-Burma regions were suggested as the ancestral distribution region of Thismia s.s., and its common ancestor might be originated from the Sunda Shelf region at 64.95 Mya.The back-and-forth migration and dispersal between SW China to Indo-Burma (excluding the Malay Peninsula) and the Sunda Shelf region have happened at least five to six times in Clades 1, 4 and 5, which are highly associated with land-bridge connection between the SE Asia mainland and the Sunda Shelf region before 5.0 Mya (Hall 2009(Hall , 2012(Hall , 2013;;Tan & al. 2020).The common ancestor of T. sect.Glaziocharis and T. sect.Sarcosiphon (Blume) Jonker should have originated in the SW China to Indo-Burma region at 38.91 Mya and 16.87 Mya, respectively, then spread eastward to SE China at 22.45 Mya and Japan at 17.47 Mya in the section Glaziocharis (Clade 1) and in situ diversification in T. sect.Sarcosiphon (Clade 4) started at 16.87 Mya.Thismia sect.Rodwaya (Schltr.)Jonker from New Guinea to Australia might have originated from the Sunda Shelf region at 43.50 Mya, then in situ diversification started at 14.26 Mya as New Guinea to Australia isolated from the Sunda Shelf region (Hall 2012;Zhang & al. 2023).The common ancestor of the clade T. gardneriana + T. hongkongensis + T. tentaculata should be from the Sunda Shelf region, but the ancestral region of T. gardneriana + T. hongkongensis + T. tentaculata was ambiguous, which might be caused by incomplete sampling and an unresolved relationship between T. gardneriana and T. hongkongensis + T. tentaculata.In addition, long-distance dispersal cannot be excluded for T. gardneriana spreading to India.

Biodiversity and conservation of extremely rare Thismia species
Thismia species almost exclusively distributed in tropical and subtropical areas of Asia and America, with the exception that T. americana N. Pfeiff. in the C United States, which has been supposed to be extinct.Since the genus was established in 1845, species discovery progressed very slowly with only 40 species discovered until 1999 (Supplementary appendix S1).After 2011, at least one new species per year was described, and seven and 15 new species were described in 2017 and 2018, respectively.To date, there are 106 species and one variety recognized (Supplementary appendix S1).However, most Thismia species are found or collected only once or a few times with small population sizes (Dančák & al. 2020a;Nuraliev & al. 2020).As holo-mycoheterotrophic herbs, Thismia species are highly reliant on the specialized fungal host, so the distribution range might be restricted by the availability of host fungi (Merckx & Bidartondo 2008;Gomes & al. 2017;Merckx & al. 2017).Meanwhile, Thismia plants are usually very small, inconspicuous and of ephemeral emergence in the flowering and fruiting seasons.To date, only a few Thismia species have been observed in more than three locations; c. 65 % of Thismia species are known only from the type localities and adjacent areas; c. 45 % of them were found only by their discoverers; and some species had just a single individual (Dančák & al. 2020a;Nuraliev & al. 2020).Therefore, most Thismia species could be locally endemic, rare and critically endangered (CR) in accordance with the IUCN Red List categories and criteria (IUCN Standards and Petitions Committee 2022).
It is concerning that in the current geographic distribution range of eight Thismia species in China, six species are known only from the type locality, and only two species, T. hongkongensis and T. tentaculata, have been recorded in two or more populations.In China, T. hongkongensis and T. tentaculata were originally found in Hong Kong, the range of T. hongkongensis extended to Guangdong and the type locality of T. tentaculata was Quang Tri Province, Vietnam, but the total number of individuals was few.Moreover, T. jianfenglingensis Han Shu & al. is endemic to the Jianfengling National Nature Reserve, Ledong County, Hainan Island, and had only six flowering individuals when found for the first time in 2017, whereas no flowering individuals were found in 2018.Herein, T. malipoensis is known only from Malipo County, SW Yunnan, where only ten flowering individu-  margin, subulate at apex.Annulus expanded and modified into a cucullate (hood-like) structure, covering apical part of floral tube and forming a lateral floral aperture; cucullate outer surface white with 3 brown lines, glabrous; cucullate inner surface light green, covered with numerous white trichomes.Stamens 6, light yellow, pendent from apical part of floral tube; each connective c. 3.5 mm long, outer surface with 2 linear to filiform thecae, each c. 2 mm long, inner surface smooth, outer surface and apex covered with transparent trichomes; supraconnective apex rounded and curved outward; filaments short, connected to floral tube and annulus.Ovary obovoid, c. 3.5 × 4.8 mm, 1-loculed, with 3 parietal placentae; style c. 0.6 mm long; stigma 3-lobed, lobes triangular-pyramidal, c. 1.2 mm long, apex truncate and densely papillate.Etymology -The specific epithet "malipoensis" refers to the county name Malipo of Wenshan Prefecture, the type locality of the new species.
Conservation status -We evaluated the conservation status of Thismia malipoensis according to the IUCN Red List categories and criteria (IUCN Standards and Petitions Committee 2022).The new species is known only from the type locality, our field surveys identified only one population with about ten individuals.The conservation status of the species is therefore considered to be Critically Endangered (CR A2acd; B1ab(i,iii,v)+2ab(i,iii,v); C2a(i); D). als were found in 2019.New populations of some species will be found in further investigations.However, most Thismia species have extremely small population sizes, which may be more sensitive to environmental changes and more vulnerable to extinction.As mentioned above, holo-mycoheterotrophic Thismia species are highly reliant on the specialized fungal host for seed germination and seeding development, and those fungi should be associated with the forest and soil ecosystem.Therefore, the most effective strategies are in situ conservation.Diagnosis -Thismia malipoensis belongs to T. sect.Glaziocharis and can be distinguished from its phylogenetic sister species T. abei by having the annulus of the flower expanded and modified into a cucullate (hood-like) structure with zygomorphic symmetry with one opening on one side.Moreover, T. malipoensis can be distinguished from the morphologically similar T. belumensis, in that the outer and inner tepal appendages are spreading (vs erect) and equal in length by less than 2 cm (vs outer tepal appendage 1.5-2.5 cm and inner tepal appendage c. 3 cm), the inner tepal base is fimbriate at the margin (vs entire) and the inner tepal tip is subulate (vs clavate).
Fruit cup-shaped; seeds brown.Phenology -Observed flowering in August, fruiting in September.Distribution and habitat -Thismia malipoensis is currently known only from the type locality in Laoshan Provincial Nature Reserve of Malipo County, SE Yunnan.The population consists of about ten individuals under evergreen broad-leaved forest on limestone mountains at an elevation of 1191 m a.s.l.The forest includes the Lithocarpus tephrocarpus (Drake) A. Camus (Fagaceae), Alseodaphnopsis andersonii (King ex Hook.f.) H. W. Li & J. Li and Litsea yunnanensis Y. C. Yang & P. H. Huang (both Lauraceae) and the bamboo Melocalamus arrectus T. P. Yi.

Fig. 5 .
Fig. 5. Phylogeny of Thismiaceae inferred from the concatenated two nrDNA and two mitochondrial DNA sequences dataset.Maximum likelihood bootstrap (MLBS) and Bayesian inference posterior probability (BIPP) values are presented above the branches.The bottom scale bar represents the number of substitutions per site.