Abstract
The family Thismiaceae, known as “fairy lanterns” for their urn- or bell-shaped flowers with basally fused tepals, consists of non-photosynthetic flowering monocots mainly in tropical regions, extending into subtropical and temperate areas. Here, we propose a new mycoheterotrophic genus, Relictithismia Suetsugu & Tagane (Thismiaceae), with its monotypic species Relictithismia kimotsukiensis Suetsugu, Yas.Nakam. & Tagane from Kimotsuki Mountains in the Osumi Peninsula, Kagoshima Prefecture, Kyushu Island, southern Japan. Relictithismia resembles Haplothismia Airy Shaw in having a cluster of tuberous roots, a feature previously observed only in this genus within the family Thismiaceae. However, it differs in having solitary flowers (vs. 2–6-flowered pseudo-raceme in Haplothismia), anther thecae largely separated (vs. connate), and the presence of an annulus (vs. absent). Additionally, Relictithismia differs from the geographically overlapping genus Thismia Griff. in its stamen structure and the position of the annulus. In Relictithismia, the stamens lack connectives, and its free filaments arise from the annulus located inside the perianth mouth, while in Thismia, the stamens typically have connate connectives, forming a staminal tube pendulous from the annulus located at the mouth of the floral tube. Our morphological and phylogenetic data indicated that R. kimotsukiensis holds an early-diverging position within the family, situated outside the Old World Thismia clade. This paper offers an extensive description and color photographs of R. kimotsukiensis, complemented by notes on its phylogenetic relationship and evolutionary history.
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Introduction
The family Thismiaceae J.Agardh, commonly referred to as “fairy lanterns” for their vibrant, urn- or bell-shaped flowers with basally fused tepals, consists of non-photosynthetic flowering monocots primarily found in tropical regions, with extensions into subtropical and temperate areas (Garrett et al. 2023; Merckx and Smets 2014; Thorogood and Mat Yunoh 2021). Most studies considered that this family encompasses the monotypic genera Haplothismia Airy Shaw and Tiputinia P.E.Berry & C.L.Woodw., Oxygyne Schltr. with six species, Thismia Griff. with about 100 species, and Afrothismia Schltr., thought to have around 16 species (Merckx et al. 2006, 2009; POWO 2024; Shepeleva et al. 2020), although recent phylogenetic research indicates Afrothismia probably does not belong to the family Thismiaceae and is proposed to be an independent family (Cheek et al. 2023).
Mycoheterotrophic plants, which typically obtain their carbon indirectly from photosynthetic plants through shared mycorrhizal networks, depend heavily on the symbiotic interactions between fungi and trees (Gomes et al. 2017; Suetsugu et al. 2022b). This complex dependency renders them particularly susceptible to environmental disturbances, often leading to their rarity and endangered status. Members of the family Thismiaceae are especially notable for their exceptional scarcity and highly localized habitats (Suetsugu et al. 2023; Thorogood 2019; Thorogood and Mat Yunoh 2021). For instance, out of the approximately 100 described Thismia species, at least 55 are known only from their original discovery locations, and at least 38 have been documented based on a single discovery instance (Dančák et al. 2020).
The taxonomy of mycoheterotrophic plants has been a subject of considerable debate, largely due to the significant reduction in vegetative organs, their rarity, and the high rates of molecular evolution. The classification of Thismiaceae, in particular, has been contested. The APG group placed Thismiaceae within Burmanniaceae Blume (The Angiosperm Phylogeny Group 2016), a decision that may have been influenced by contaminant sequences (Lam 2016). Other studies using Sanger sequencing of specific mitochondrial and nuclear genes have underscored the distinctiveness of Thismiaceae from Burmanniaceae (Merckx et al. 2006, 2009; Shepeleva et al. 2020). These comprehensive analyses have positioned Thismiaceae s.s. (specifically, Thismia, Tiputinia, Haplothismia, and Oxygyne) as a sister group to Taccaceae Dumort., placing them in a separate subclade from Burmanniaceae within the order Dioscoreales. The recognition of Thismiaceae as a distinct family is further supported by recent phylogenomic studies that focus on high-throughput plastid and mitochondrial data (Garrett et al. 2023; Lam et al. 2018; Lin et al. 2022), and are complemented by morphological data (Cheek et al. 2018; Merckx et al. 2006).
Moreover, several phylogenetic studies indicated that Afrothismia, typically classified under Thismiaceae, is not monophyletic with the other four genera but is instead a sister clade with photosynthetic Taccaceae and other Thismiaceae species (Lin et al. 2022; Merckx et al. 2009). Morphologically, Afrothismia is uniquely characterized by its underground system comprising short rhizome bearing clusters of globose bulbils, each with a terminal rootlet, distinct from the coralloid, vermiform, or tuberous roots typical of other Thismiaceae species (Cheek et al. 2023; Merckx et al. 2009). Furthermore, the ovary in Afrothismia is characterized by having a single stalked placenta (Cheek et al. 2023). This feature distinguishes it from other species in the family Thismiaceae, which have three placentae. These insights have led to proposals for reclassifying Afrothismia as a distinct family (Cheek et al. 2023). An alternate approach of grouping Afrothismia (alongside Taccaceae and Thismiaceae) within a broadly defined Dioscoreaceae R.Br. may mask the significant morphological diversity among these families. Classifying a wide range of morphologies under Dioscoreaceae could make it undiagnosable due to high internal variation (Cheek et al. 2023). Thus, recognizing Afrothismia at the family level seems to be justifiable. However, due to their apparent morphological similarities, Afrothismia has been included in a morphological comparison with our newly described genus in this paper.
The Japanese archipelago, extending from boreal to subtropical zones, is a crucial part of the continental islands in East Asia (Kubota et al. 2017). It has been proposed as a refuge for Tertiary relict floras, harboring ancient plant species (Milne and Abbott 2002). The present biotas of the archipelago are shaped by a history of migrations, extinctions, and speciations, influenced by geographical isolation and connections (Millien-Parra and Jaeger 1999). This unique configuration makes the Japanese archipelago an ideal region for studying the impact of geohistorical processes on floristic diversification (Kubota et al. 2017). Furthermore, Japanese flora is notably rich in arbuscular mycorrhizal (AM) mycoheterotrophic plants (Suetsugu and Nishioka 2017; Suetsugu et al. 2023). To date, over 20 AM mycoheterotrophic species from four different families (Petrosaviaceae Hutch., Triuridaceae Gardner, Burmanniaceae, and Thismiaceae) have been documented. The Japanese species often represent the northernmost members of their genera or families (Suetsugu et al. 2023). Thus, the Japanese AM mycoheterotrophic plants provide essential insights into the biogeography and evolutionary history of these elusive plants.
During a floristic survey on Osumi Peninsula, Kagoshima Prefecture, Kyushu Island, southern Japan in 2022, the second author YN collected an unknown plant belonging to the family Thismiaceae in a secondary evergreen broad-leaved forest (Fig. 1). It differed from the species of the two genera of Thismiaceae, Oxygyne and Thismia, distributed in East Asia, in having six stamens (vs. three in Oxygyne) and free stamens touching the stigma during anthesis, a trait absent in Thismia (Cheek et al. 2018; Nuraliev et al. 2021; Shepeleva et al. 2020). The characteristic of free stamens touching the stigma is reminiscent of Afrothismia, a genus exclusively found in Africa (Cheek et al. 2023). However, it differs from Afrothismia in having tuberous roots, an actinomorphic flower, and a non-segmented tube. So far, a cluster of tuberous roots has been known only in the monotypic genus Haplothismia found in India. However, the unknown plant also differs from H. exannulata Airy Shaw in having one or two solitary flowers, free thecae, and the presence of an annulus (Airy-Shaw 1952; Sasidharan and Sujanapal 2000; Shepeleva et al. 2020). Furthermore, our phylogenetic analysis showed that the unknown plant exhibits significant genetic divergence from members of both Old World Thismiaceae (Haplothismia, Oxygyne, and Thismia, subg. Thismia) and New World Thismiaceae (Thismia subg. Ophiomeris and Tiputinia), suggesting it represents a distinct lineage.
Consequently, we propose a new genus, Relictithismia Suetsugu & Tagane, along with the newly found species Relictithismia kimotsukiensis Suetsugu, Yas.Nakam. & Tagane. This paper includes a detailed description, illustrations, preliminary conservation status, notes on the phylogenetic relationship, evolutionary history, and conservation assessment of R. kimotsukiensis.
Materials and methods
Morphological observations
An individual of Relictithismia kimotsukiensis was collected from Kimotsuki-cho, Kagoshima Prefecture (its type locality) on 3 June 2022, and three additional R. kimotsukiensis individuals were also collected at the same locality on 9 June 2023. Given that precise identification of Thismiaceae species often hinges on the analysis of floral characteristics within the floral tube, their flowers were carefully dissected to examine their inner floral morphology. Morphological characteristics were observed under a Leica M165C and MZ95 stereomicroscope and measured using a digital caliper. Additionally, the dissected flowering specimens were photographed with a Leica MC170 HD digital camera attached to the same microscope or an OLYMPUS TG-3.
To investigate the morphological characteristics of other Thismiaceae species, we also reviewed relevant literature and online digitized plant collections, including JSTOR Global Plants (http://plants.jstor.org/). Furthermore, we examined herbarium specimens deposited at FOF, HYO, K, KAG, KYO, RYU, TI, TNS, and VNM. These herbarium acronyms follow the Index Herbariorum (Thiers 2023).
DNA extraction, PCR amplification, and sequencing
Genomic DNA was extracted from the scale leaves of two Relictithismia kimotsukiensis individuals collected on 9 June 2023, using the CTAB method (Doyle and Doyle 1990). The primer sets used for the PCR and cycle sequencing (CS) reactions in this study were as follows: for 18S rDNA (18S), NS1 and NS2 (PCR and CS), NS3 and NS4 (PCR and CS), as well as NS5 and NS8 (PCR and CS) (White et al. 1990) and for ATP synthase subunit A (atpA), ATPA-F1 and ATPA-B1 (PCR and CS) (Eyre-Walker and Gaut 1997), with ATPA-F5 and ATPA-B5 (CS only) (Davis et al. 1998). The PCR reaction and DNA sequencing were performed using the modified methods mentioned in Nakano (2012), with the aid of a T100 Thermal Cycler (Bio-Rad, Hercules, USA).
The PCR reactions were initiated with an initial heating step at 95 °C for 5 min, followed by 35 cycles of amplification. For the 18S primer sets, each cycle involved three stages: denaturation at 94 °C for 10 s, annealing at 52 °C for 20 s, and extension at 72 °C for 42 s. For the atpA primer set, the cycles included stages at 94 °C for 10 s, annealing at 54 °C for 20 s, and extension at 72 °C for 73 s. Subsequently, the sequencing mixtures were subjected to an initial heating step at 96 °C for 2 min, followed by 40 cycles of sequencing, with each cycle consisting of denaturation at 96 °C for 10 s, annealing at 50 °C for 5 s, and extension at 60 °C for 48 s. The obtained sequences were edited using DNA BASER (Heracle Biosoft S.R.L., Piteşti, Romania). The four DNA sequences newly obtained in this study have been deposited with the International Nucleotide Sequence Databases (INSD) through the DNA Data Bank of Japan (Table S1).
Sequence alignment and phylogenetic analysis
In addition to the newly obtained sequences of Relictithismia kimotsukiensis, 21 sequences representing nine species of Thismiaceae, and two species of Tacca (outgroup) were retrieved from the INSD based on the selection criteria following Shepeleva et al. (2020), and were included in the present dataset (Table S1). Given the fact that the respective 18S and atpA sequences of the two R. kimotsukiensis individuals were completely identical to each other, the duplicate sequences were not included in the present dataset. The sequences of 18S were aligned using MAFFT Server v. 7.511 Q-INS-i (Katoh and Toh 2008; Katoh and Standley 2013; Kuraku et al. 2013) with the McCaskill routine from the Vienna RNA package (Hofacker et al. 2002) and MXSCARNA (Tabei et al. 2008), while the sequences of atpA were aligned using MAFFT v. 7.520 L-INS-i. The aligned 18S and atpA sequences were of lengths 1758 and 1275 bp, respectively, resulting in a concatenated alignment of 3033 bp.
Phylogenetic trees were reconstructed using maximum likelihood (ML) and Bayesian inference (BI). The best-fit partition scheme and models were identified based on the Bayesian information criterion using ModelFinder (Kalyaanamoorthy et al. 2017) implemented in IQ-Tree v. 2.2.2.6 (Minh et al. 2020). The selected partition scheme and models for each genetic marker were as follows: for 18S, GTR + I + G; for atpA 1st position, GTR + I; and for atpA 2nd and 3rd positions, F81 + I. The ML tree was calculated using IQ-Tree v. 2.2.2.6 with non-parametric bootstrapping (BS) conducted with 1000 replicates. The BI tree and Bayesian posterior probabilities (PP) were estimated using MrBayes v. 3.2.7a (Ronquist et al. 2012). Two independent runs for four Markov chains were conducted for 4 million generations, and the BI tree was sampled every 100 generations. The parameter estimates and convergence were checked using Tracer v. 1.7.1 (Rambaut et al. 2018), and then the first 10,001 trees were discarded based on the results.
Results and discussion
Morphological comparison of Relictithismia and other Thismiaceae genera
The thorough review and examination of herbarium specimens, protologues, and living plants revealed a distinct set of morphological characteristics that consistently distinguish Relictithismia from the previously known five genera of Thismiaceae s.l. (Afrothismia, Haplothismia, Oxygyne, Thismia, and Tiputinia).
In East Asia, two genera Oxygyne and Thismia within Thismiaceae have been known, both of which occur in Japan (Cheek et al. 2018). Upon detailed examination, we concluded that R. kimotsukiensis did not correspond to either Oxygyne or Thismia. Relictithismia is characterized by possessing six stamens (Fig. 2), setting it apart from Oxygyne, which has three stamens (Cheek et al. 2018; Shepeleva et al. 2020; Suetsugu et al. 2019). Notably, its stamens are free from each other, pendulous, and touch the stigma during anthesis (Figs. 2g, 3c). This is in stark contrast to Thismia, which is known for stamens with dilated connectives, forming a staminal tube (except in the paraphyletic Neotropical subgenus Ophiomeris (Miers) Maas & H.Maas), and never touching the stigma (Nuraliev et al. 2021; Shepeleva et al. 2020). It is also noteworthy that although some members of the Neotropical subgenus Ophiomeris also feature stamens without dilated connectives, Relictithismia differs from Ophiomeris in having a cluster of tuberous roots (vs. a single globose tuber with numerous filiform roots or vermiform roots in Ophiomeris) and the annulus located inside the perianth mouth (vs. the annulus located at the mouth of the floral tube) (Maas et al. 1986; Shepeleva et al. 2020).
Relictithismia resembles the monotypic genus Haplothismia, known from India, in having a cluster of tuberous roots and homomorphic outer and inner perianth lobes with long filiform appendages (Figs. 1, 2 and 3). Notably, so far, a cluster of tuberous roots has been known only in Haplothismia within the Thismiaceae (Airy-Shaw 1952; Sasidharan and Sujanapal 2000; Shepeleva et al. 2020). Several recent studies have suggested that, although the outer floral appearance may have little phylogenetic significance in Thismiaceae classification possibly due to convergent evolution based on pollinator preferences (Shepeleva et al. 2020), the structure of the underground organs is a stable trait reflecting phylogenetic information (Feller et al. 2022; Imhof et al. 2020). Thus, R. kimotsukiensis might be most closely related to Haplothismia. However, it can be distinguished from Haplothismia by the following several key features: solitary flowers (vs. 2–6-flowered pseudo-raceme in Haplothismia), largely separate anther thecae (vs. connate), the presence of an annulus (vs. absent), and stamens touching the stigma (vs. apart from the stigma) (Airy-Shaw 1952; Sasidharan and Sujanapal 2000; Shepeleva et al. 2020).
The anther touching the stigma on R. kimotsukiensis is reminiscent of the African genus Afrothismia, as this feature was previously observed only in this genus within Thismiaceae s.l. (Cheek et al. 2023). However, in contrast to Afrothismia, which features an underground system composed of short rhizome bearing clusters of globose bulbils, each with a terminal rootlet, a zygomorphic flower, and a two-chambered floral tube (Cheek et al. 2023), our specimen exhibits a cluster of tuberous roots, an actinomorphic flower, and a non-segmented floral tube. Additionally, not only Relictithismia but also Haplothismia, Oxygyne, Thismia, and Tiputinia differ from Afrothismia in having the ovary with three placentas (vs. ovary with a single placenta) (Fig. 2l). Given that Afrothismia is not monophyletic with other Thismiaceae species (Lin et al. 2022; Merckx et al. 2009), the free stamens touching the stigma likely represent an example of convergent evolution for facilitating self-pollination. Notably, autonomous self-pollination is advantageous for mycoheterotrophic plants in dark, shaded forest understories where pollinators are scarce (Suetsugu 2013, 2015, 2022). Consequently, these morphological traits can be regarded as mechanisms providing reproductive assurance.
Finally, Relictithismia also differs from the monotypic genus Tiputinia, found in Ecuador of South America in having a cluster of tuberous roots (vs. vertical cylindric sympodially branched rhizome in Tiputinia), stamens without filament appendages and interstaminal lobes (vs. stamens with five or six pairs of filament appendages and two globoid interstaminal lobes), the presence of an annulus (vs. absent), and stamens touching the stigma (vs. free, never touching the stigma) (Woodward et al. 2007). Overall, these morphological data indicated that our specimen represents an isolated lineage under the family Thismiaceae.
Phylogenetic comparison of Relictithismia and other Thismiaceae genera
Both the BI (Fig. 4) and ML (Fig. S1) trees exhibited nearly identical topologies, largely consistent with the findings of Shepeleva et al. (2020). The monophyly of the Thismiaceae received strong support in both analyses (BS = 100%, PP = 1.0).
Echoing recent studies (Merckx et al. 2006, 2009; Merckx and Smets 2014; Shepeleva et al. 2020; Yokoyama et al. 2008), our findings confirm that Thismia is polyphyletic. Currently, Thismia is split into two subgenera: the subgenus Thismia, which includes all Old World species and T. americana, and Ophiomeris, which encompasses Neotropical species (Kumar et al. 2017; Maas et al. 1986; Merckx and Smets 2014). The Old World Thismia clade, which aligns with the subgenus Thismia, was confidently retrieved (BS = 100%, PP = 1.0), while the subgenus Ophiomeris, represented by Thismia panamensis (Standl.) Jonker of the New World, is distantly related to the subgenus Thismia. Given the morphological distinction where Ophiomeris stamens are free from each other, in contrast to the connate or connivent stamens forming a staminal tube in the subgenus Thismia (Shepeleva et al. 2020; Yang et al. 2010), these two are differentiated based on both morphological and molecular evidence. It might be reasonable to classify Ophiomeris at the genus level. However, considering the morphological variability of Neotropical Thismia (Maas et al. 1986), there is a question as to whether all of them are closely related, forming a single monophyletic genus. Regrettably, molecular datasets for other Ophiomeris species are currently unavailable. Therefore, acquiring molecular data from additional Ophiomeris species is crucial to substantiate their phylogenetic relationships.
Our findings also indicated that Th. panamensis, Tiputinia foetida P.E.Berry & C.L.Woodw., Haplothismia exannulata, and Relictithismia kimotsukiensis occupy an early-diverging position in this family, positioned outside the Old World Thismia clade (the subgenus Thismia). Notably, these species have less specialized perianths than those of the subgenus Thismia, possessing identical inner and outer perianth whorls and free stamens (Airy-Shaw 1952; Cheek et al. 2018; Shepeleva et al. 2020; Woodward et al. 2007). Thismia panamensis, Ti. foetida, H. exannulata, and R. kimotsukiensis formed a relatively well-supported monophyletic lineage in the BI analysis (PP = 0.94), while this clade did not receive support in the ML analysis (BS = 48%). Additionally, the current phylogenies were unable to resolve robust relationships among these four species, although the monophyly of Th. panamensis and Ti. foetida was well-supported in both analyses (BS = 84%, PP = 0.96).
The cluster of tuberous roots of R. kimotsukiensis suggests some affinity with Haplothismia. On the other hand, the presence of pendulous stamens from the annulus can be found only in R. kimotsukiensis and Thismia. Thus, Relictithismia could potentially act as a connecting link between other early-diverging genera in Thismiaceae (particularly Haplothismia) and Thismia. However, the precise phylogenetic relationships among Relictithismia, Haplothismia, Ophiomeris, and Tiputinia remain uncertain. It is crucial to employ high-throughput sequencing technology for a more detailed understanding of their phylogenetic relationships and morphological evolution. Even if a monophyletic lineage between H. exannulata and R. kimotsukiensis is confirmed, it is noteworthy that sufficient genetic divergence between these two taxa shown in our phylogenetic analysis is comparable to that between other genera.
Overall, the available phylogenetic data, along with the morphological distinctness, strongly support the conclusion that Relictithismia kimotsukiensis does not align with any existing genera. Therefore, we propose a new genus for this taxon, which is described in detail in the following sections.
Taxonomic treatments
Relictithismia Suetsugu & Tagane, gen. nov.
Diagnosis Relictithismia resembles Haplothismia in possessing a cluster of tuberous roots, yet it is clearly distinguished by its solitary flowers (vs. 2–6-flowered pseudo-raceme in Haplothismia), largely separate anther thecae (vs. connate), and the presence of an annulus (vs. absence).
Type species Relictithismia kimotsukiensis Suetsugu, Yas.Nakam. & Tagane.
Description Achlorophyllous herbs. Underground part consisting of a short rhizomatous stem and a cluster of tuberous roots. Stems 1–2 per plant, endogenous, terminating aboveground in a single flower. Leaves spirally arranged, simple, sessile, reduced to scales, appressed on the stem. Floral bracts usually 3 in one whorl, not equal in size, erect, subtending the flower, ovate-triangular to ovate-oblong. Flower solitary, actinomorphic, epigynous; floral tube obpyriform lacking distinctive ridges; perianth lobes 6, equal, patent or ascending, filiform to lanceolate with a callus-like cluster of round cells on the adaxial surface at base; annulus present, inside the floral tube. Stamens 6, pendulous from the annulus, arching inward and touching the stigma; filaments slightly dorsiventrally flattened, adorned with some long hairs; anther thecae 2, largely separated, elliptic, dehiscing longitudinally towards the abaxial side. Ovary inferior, 1-locular with 3 parietal placentae; ovules numerous; style cylindrical; stigma conical, apically 3-lobed.
Etymology The name of the new genus, Relictithismia, is a combination of the Latin word “relictus”, meaning “left behind”, and the generic name Thismia. The name reflects its morphological characteristics that serve as a bridge between the early-diverging genera in Thismiaceae and the genus Thismia, showcasing the putative ancestral condition of these plant groups.
Relictithismia kimotsukiensis Suetsugu, Yas.Nakam. & Tagane, sp. nov. (Figs. 1–3).
Type JAPAN. Kagoshima Prefecture, Kimotsuki-gun, Kimotsuki-cho, 9 June 2023, Y. Nakamura & S. Tagane 23060901 (holotype TNS [TNS VS-8001405 in spirit!], isotypes KAG [KAG183094!], KYO [KYO00027080 in spirit!]).
Description Achlorophyllous herbs. Roots clustered, radiating from the stem base, up to 28, 5.3–20.0 mm long, 1.3–2.2 mm in diam., light brown, tuberous, warty. Rhizome not elongated. Stems 1–2 per plant, white, simple, erect, 5–16 mm long at anthesis, elongated to 21 mm long at immature fruiting stage. Leaves scale-like, spirally arranged, 8–11, appressed on the stem, ovate-triangular to narrowly ovate-oblong, 1.5–7.8 × 1.3–3.8 mm, white, apex acute to obtuse. Floral bracts 3(–4) in one whorl, not equal in size, erect, subtending the flower, ovate-triangular to ovate-oblong, 5.1–8.9 × 3.2–4.5 mm, white, apex obtuse. Flower white, solitary, actinomorphic, epigynous. Perianth basally fused, forming an obpyriform floral tube with 6 free perianth lobes. Floral tube 10.5–12.0 mm long, 6.2–7.0 mm in diam., outer surface glabrous, inner surface without transverse bars, densely hairy at lower 2/3, with an annulus 1.9–2.1 mm below the mouth; mouth circular, 3.4–4.8 mm in diam. Perianth lobes 6, equal, filiform to lanceolate, 9–12 mm long, slightly recurved near apex, with a callus-like cluster of round cells on adaxial surface at base; margins of adjacent lobes fused. Stamens 6, free, pendulous from the annulus, arching inward and touching the stigma; filaments slightly dorsiventrally flattened, 0.6–1.0 mm long, sparsely covered with long hairs; anthers 1.5–1.7 × 0.8–1 mm long, slightly papillate, with some long hairs, anther thecae 2, largely separated, oblong, 1.0–1.3 mm in length, dehiscing longitudinally to the abaxial side. Ovary depressed globose, 3.0–3.8 mm long, 3.5–4.6 mm in diam., 1-locular with 3 parietal placentae, ovules numerous. Style cylindrical, 0.3–0.5 mm long, stigma conical, 2.6–2.7 × 1.5–1.7 mm, 3-ridged, densely covered with papillate to subulate trichomes, apically 3-lobed. Immature fruits slightly depressed globose, 5.6–5.8 mm long, 6.1–6.6 mm in diam. Seeds not seen.
Additional specimen examined (paratype) JAPAN. Kagoshima Prefecture, Kimotsuki-gun, Kimotsuki-cho, 3 June 2022, Y. Nakamura 22060301 (KAG [KAG180387-in spirit!]).
Etymology The species epithet refers to the type locality of the new species.
Vernacular name Mujina-no-shokudai (in Japanese). The vernacular name is inspired by its apparent resemblance to and the distinct diffrences from species of Thismia, locally referred to as “tanuki-no-shokudai,” meaning a candlestick used by a raccoon dog. The term “mujina” is an old Japanese name for a badger, known as “anaguma” in Japanese, although historically and regionally, it has sometimes also been associated with raccoon dogs. Therefore, we consider that this name aptly represents the similarity and differences between Thismia and Relictithismia. Moreover, the name “mujina-no-shokudai” (= a candlestick used by a badger) fits its ecology, as the majority of the plant, even during the flowering stage, remains buried underground beneath fallen leaves, reminiscent of a badger burrowing underground.
Distribution Japan, Kagoshima Pref., Kimotsuki-gun, Kimotsuki-cho (currently known only from the type locality).
Habitat The species occurs in the understory of a secondary evergreen broad-leaved forest mixed with a few planted Cryptomeria japonica (L.f.) D.Don (Cupressaceae). The forest is dominated by Ilex integra Thunb. (Aquifoliaceae), Dendropanax trifidus (Thunb.) Makino ex H.Hara (Araliaceae), Daphniphyllum macropodum Miq. subsp. macropodum (Daphniphyllaceae), Callicarpa japonica Thunb. var. luxurians Rehder (Lamiaceae), Cinnamomum yabunikkei H.Ohba, Machilus japonicus Siebold et Zucc. ex Blume, Machilus thunbergii Siebold et Zucc., Neolitsea sericea (Blume) Koidz. (Lauraceae), Ficus erecta Thunb. (Moraceae), Ligustrum japonicum Thunb. (Oleaceae), Illicium anisatum L. (Schisandraceae), Symplocos kuroki Nagam. (Symplocaceae), and Eurya japonica Thunb. (Pentaphylacaceae). In the understory, some shrubs and herbs, including Arachniodes sporadosora (Kunze) Nakaike (Dryopteridaceae), Epipogium roseum (D.Don) Lindl., Goodyera similis Blume (Orchidaceae), Maesa japonica (Thunb.) Moritzi et Zoll. (Primulaceae), and Rubus buergeri Miq. (Rosaceae), are scattered.
Phenology Flowers and immature fruits were observed and collected in early June.
INSD accession no. LC775771 & LC775772 (18S), LC775773 & LC775774 (aptA); obtained from Y. Nakamura & S. Tagane 23060901 (holotype in TNS & isotype in KYO, respectively).
Preliminary conservation assessment Critically Endangered (CR). We assess Relictithismia kimotsukiensis as “Critically Endangered” [B1 & 2a, b(iii) + C2a(i, ii) + D] following the IUCN Red List Categories and Criteria (IUCN 2022). This species is known only from a single location, where the above specimens were collected and fewer than five individuals were observed in total. Initially, it was discovered by YN with a single specimen on 3 June 2022, but subsequent field surveys in early June (by ST and KS) and late June (by YN) of the same year at the type locality did not yield additional findings. However, a reassessment on 9 June 2023 (by YN and ST), resulted in the discovery of four more individuals. The locality, adjacent to a forest road and outside a protected area, features mostly young trees with a diameter at breast height (DBH) of less than 13 cm, indicating recent forest clearing within the past 20–30 years. Despite extensive searches by YN, ST, and KS around the population during the flowering season, this species has not been found in any other areas.
Given its extremely limited distribution and potential habitat threats, R. kimotsukiensis is classified as Critically Endangered. However, due to its cryptic growth habit and ephemeral flowering phenology, R. kimotsukiensis may exist in other, yet undiscovered locations. Therefore, further intensive field surveys and subsequent observations are essential to more fully comprehend its distribution and elusive natural history.
Possible pollination mode Relictithismia kimotsukiensis exhibits floral traits suggestive of sapromyophily, a pollination strategy that involves attracting fly pollinators. Traits such as the filiform appendages of the perianth lobes in R. kimotsukiensis are largely aligned with adaptations for attracting fly pollinators (Suetsugu et al. 2022a). Notably, dipteran pollinators have been observed being temporarily restrained inside the floral tube of some Thismiaceae species (Guo et al. 2019; Suetsugu and Sueyoshi 2021).
Nonetheless, the floral structure of R. kimotsukiensis, particularly the stamens reaching the stigma surface at anthesis, implies an adaptation for self-pollination. Overall, we consider self-pollination to be a plausible predominant mode in R. kimotsukiensis, although the possibility of some contribution from insect-mediated pollination cannot be completely ruled out.
Biogeographical note Japan boasts the highest diversity of genera within the family Thismiaceae, hosting three distinct genera. Intriguingly, the genus Oxygyne, the probable earliest-diverging member of the Thismiaceae, coexists in Japan with both Thismia and Relictithismia. As discussed above, Relictithismia possesses morphological features that bridge the early-diverging genera in Thismiaceae and the genus Thismia. It is also noteworthy that the two Japanese Thismia species, T. abei and T. kobensis, are early-diverging members within the Old World Thismia clade (Shepeleva et al. 2020; Suetsugu et al. 2023). Detailed studies on Japanese Thismiaceae species are likely to provide crucial insights into the biogeography and evolutionary history of the Thismiaceae as a whole.
Key to the genus of Thismiaceae s.l. (modified from Cheek et al. 2018)
1a. Staminal filaments inserted at the mouth of the floral tube, erect and exserted at base, then arching inwards, the anthers held at the level of the tube mouth, or just inside…………. 2
1b. Staminal filaments inserted inside the floral tube, or in the throat of the floral tube,, the anthers held inside the tube…………. 4
2a. Stamens 6, annulus absent…………. 3
2b. Stamens 3, annulus lamellae present…………. Oxygyne
3a. Inflorescences aerial, with multiple branches and flowers; stamens without filament appendages and interstaminal lobes…………. Haplothismia
3b. Inflorescences at ground level, usually 1-flowered; stamens with five or six pairs of filament appendages and two globoid interstaminal lobes…………. Tiputinia
4a. Annulus located inside the floral tube; stamens inserted in the lower floral tube where they are attached to the stigma…………. 5
4b. Annulus located at the mouth of floral tube; stamens free of and distant from the stigma…………. Thismia
5a. Staminal filaments adjacent to annulus; underground system with tuberous roots…………. Relictithismia
5b. Stamens inserted below the annulus; underground system with rhizome bearing clusters of globose bulbils, each with a terminal rootlet…………. Afrothismia
Data availability
The sequences obtained have been deposited in the DDBJ/ENA/GenBank databases under accession numbers LC775771–LC775774. Additional supporting information is available online in the Supporting Information section at the end of the article.
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Acknowledgements
We thank the curators of FOF, HYO, K, KAG, KYO, RYU, TI, TNS, and VNM for allowing access to their herbaria and collection databases. We are also grateful to Takako Shizuka, Kazuma Takizawa and Hidehito Okada for their technical assistance. This study was supported by the PRESTO program (Grant No. JPMJPR21D6, KS) of the Japan Science and Technology Agency, the JSPS KAKENHI (Grant No. 21K06307, ST), and the Environment Research and Technology Development Fund (Grant No. JPMEERF20204001, ST) of the Ministry of the Environment, Japan.
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YN and ST collected the materials. KS and ST obtained the morphological data. TN and KS conducted the molecular experiments. TN and KS performed the molecular analyses. KS and ST wrote the article with contributions from all authors. All authors reviewed, revised the manuscript, and approved the final version for publication.
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Suetsugu, K., Nakamura, Y., Nakano, T. et al. Relictithismia kimotsukiensis, a new genus and species of Thismiaceae from southern Japan with discussions on its phylogenetic relationship. J Plant Res 137, 411–422 (2024). https://doi.org/10.1007/s10265-024-01532-5
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DOI: https://doi.org/10.1007/s10265-024-01532-5