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. 2026 Apr 22;16(4):e73498. doi: 10.1002/ece3.73498

Rediscovery of Craspedosorus and Plastome‐Based Evidence for Its Synonymy With Leptogramma (Thelypteridaceae)

Jing Zhao 1, Jian‐Jun Yang 1, Zhong‐Yang Li 1,2, Chuang‐Jie Huang 1, Ting Chen 3, Yong‐Li Zhao 3, Zhao‐Rong He 4, Chao‐Shan Gu 3,✉, Xin‐Mao Zhou 1,✉
PMCID: PMC13103283  PMID: 42040860

ABSTRACT

Yunnan Province is the richest region in China for lycophytes and ferns. Its unique geological history and climatic conditions have nurtured numerous endemic and rare plant species. Craspedosorus, a monotypic genus of Thelypteridaceae containing only Craspedosorus sinensis, is a genus endemic to Yunnan, China. It was first discovered and described in the 1970s. However, since its initial discovery in 1973, there have been no subsequent field observations or collection records. Moreover, due to a lack of molecular data, Craspedosorus remains the only fern genus in China without such data and has long had a controversial systematic position. This study rediscovered a population of only 15 mature individuals in Yongshan County, Zhaotong City, Yunnan Province, 52 years after its initial discovery. Using flow cytometry, it was confirmed that Craspedosorus sinensis is diploid, with a genome size of approximately 5.12 Gb. Molecular phylogenetic analyses further determined that C. sinensis belongs to Section Haplogramma (K. Iwatsuki) L.Y. Kuo & Y.H. Chang of the genus Leptogramma, and Craspedosorus therefore should be treated as a synonym of Leptogramma. This research is significant for understanding the taxonomy, distribution, and phylogeny of Leptogramma.

Keywords: Leptogramma, rare species, rediscovery, Stegnogramma, Thelypteridaceae

Habitat and morphology of Craspedosorus sinensis.

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1. Introduction

The Chinese endemic monotypic genus Craspedosorus Ching et W.M. Chu was first described by Prof. R.C. Ching (Ching 1978) based on C. sinensis Ching et W.M. Chu (voucher: W.M. Chu 4938; Figure 1). C. sinensis was firstly collected by Prof. W.M. Chu in 1973 in Suijiang County, Yunnan Province, China, at an elevation of approximately 1500 m. However, whether Craspedosorus should be considered an independent genus or a synonym of Stegnogramma sensu lato (s.l.) has remained controversial since its establishment. Craspedosorus has been recognized by the Flora of China (FOC; Lin et al. 2013) and Flora Reipublicae Popularis Sinicae (FRPS; Shing 1999) as closely related to Leptogramma. However, A.R. Smith, a co‐author of the FOC, argued that the diagnostic characters of Craspedosorus are insufficient to distinguish it from Leptogramma. Zhang (2012) proposed that Craspedosorus should be classified as a member of Stegnogramma Blume s.l. based on morphological data. Kuo et al. (2020) divided Stegnogramma s.l. into two genera: Stegnogramma sensu strict (s.s.) and Leptogramma, with the latter including two sections (L. sect. Leptogramma and L. sect. Haplogramma), instead of three genera (Ching 1963) or one genus with four sections as suggested by Iwatsuki (1963). In their study, C. sinensis was transferred into Leptogramma, and based on geographical distribution, the presence of multicellular hairs on stipes, and other morphological evidence, C. sinensis was further assigned to L. sect. Haplogramma. Fawcett and Smith (2021) also advocated merging Craspedosorus into Leptogramma J. Sm. based on morphological evidence. Morphologically, C. sinensis is mostly similar to species of Leptogramma J. Sm in laminar outline, segment shape, venation, and sori (He and Zhang 2012; Kuo et al. 2020). However, it differs from Leptogramma by its taller habit (up to 1.1 m), more pinnae (ca. 25 pairs), and pinnae that are separated from the rachis except at the apex (Ching 1978; Chu 2006).

FIGURE 1.

FIGURE 1

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Habitat and morphology of Craspedosorus sinensis. (a) Habitat. (b) Habit. (c) Dorsal view of portion of fertile frond. (d) Ventral view portion of fertile frond. (e) dorsal view of laminae. (f) Short rough hairs on rachises. (g) Ventral view of laminae. (h) Sporangia. (i, j) Rhizomes. (k) Stipe. (l) Spore. Photos taken by Chuan‐Jie Huang (a–e, g, i–k), Zhong‐Yang Li (f, h), and Dan‐Ni Ma (l).

The difficulty of using micromorphological features to distinguish genera within Thelypteridaceae is evident, and molecular evidence offers a more objective basis for the treatment of genera, especially for morphologically diverse taxa (Ching 1936, 1940; Morton 1963; Pichi Sermolli 1970; Smith and Cranfill 2002; Almeida et al. 2016; PPG I 2016; Patel et al. 2019; Liu et al. 2020; Fawcett and Smith 2021; Fawcett et al. 2021; Zhou and He 2025). The phylogenetic position of Craspedosorus remains controversial, and substantial evidence to clarify its placement is still lacking. The primary reason for the uncertain phylogenetic position of C. sinensis is the extreme rarity of this species. Since the initial discovery of Craspedosorus, there have been no further records of C. sinensis in the field, including at the type locality. China's vast land area encompasses diverse vegetation types, allowing ferns to adapt widely across various flora throughout the country (Qian et al. 2021). Over the past 20 years, research advancements have led to changes in the number of fern families, genera, and species distributed in China. According to FOC (38 families, 177 genera, approximately 2219 species) (Wu et al. 2013), FRPS (61 families, 231 genera, approximately 2600 species) (Wu and Chen 2004), and the Catalogue of Life China (42 families, 191 genera, approximately 2693 species) (Liu et al. 2025), a combined total of 68 families and 280 genera have been recorded in China. Among the pteridophyte genera recorded in China, only one genus, Craspedosorus, lacks molecular data. The addition of molecular data from Craspedosorus will complete the final piece of the puzzle regarding pteridophyte genetic diversity at the genus level and will enhance the study of pteridophyte phylogeny in China.

In this study, we successfully obtained the plastome of Craspedosorus sinensis using next‐generation sequencing. Simultaneously, we carefully examined the morphological characteristics of the type specimens and collected detailed morphological data. Our objectives are to (1) explore the phylogenetic position of Craspedosorus; (2) compare the morphological differences between C. sinensis and closely related species based on character state reconstruction results; and (3) infer the evolution of major anatomical, macromorphological, and spore features in Craspedosorus.

2. Materials and Methods

2.1. Investigation and Morphological Observations

Two extensive field investigations were conducted in 2024 and 2025 in both type locality and adjacent areas. Craspedosorus sinensis was rediscovered (elev. 1784 m, 28.243 N, 103.948 E) in Xisha Town, Yongshan County, Yunnan Province, China. Voucher specimens were deposited at PYU (herbarium acronyms follow Index Herbariorum by Thiers 2024). Macromorphological data were obtained from field observations, herbarium specimens, and literature (e.g., Ching 1978; Chu 2006). Gross morphology was examined, and photographs were taken using an SMZ1270 stereo microscope (Nikon, Japan). Spores of the species were coated with gold using the BAL‐TEC SCD 005 Cool Sputter Coater (BAL‐TEC AG., Liechtenstein) and imaged with a QUANTA 200 Scanning Electron Microscope (SEM; FEI Co., USA) at 25 kV at Yunnan University, Kunming, China.

2.2. Genome Size Assessment

Fresh and healthy leaves (ca. 5 g) were collected from living plants introduced from the field and frozen at −80°C. Flow cytometry was performed using a BD FACScalibur. Tender leaves of Pisum sativum L. (genome size = 3.8 Gb) one month after germination served as the reference. Genome sizes were determined based on the sample/standard ratio.

2.3. DNA Extraction and Sequencing

Total genomic DNA was extracted from silica‐dried material using the TIANGEN plant genomic DNA extraction kit (TIANGEN Biotech., Beijing, China) following the manufacturer's protocols. The DNA samples were sent to Biomaker Technology Co. Ltd. (Beijing, China) for library construction and next‐generation sequencing. A paired‐end library with an insert size of 350 bp was constructed, and sequencing was performed using the Illumina Nova 6000 platform. The raw Illumina data generated three gigabases (3 Gb). The raw reads were subsequently trimmed for quality using Fastp v0.23.1 with default parameters (Chen et al. 2018).

2.4. Plastome Assembly and Annotation

The plastome was assembled using GetOrganelle v1.7.5 (Jin et al. 2020) and annotated with CPGAVAS2 (Shi et al. 2019) and GeSeq (Tillich et al. 2017). We then manually adjusted the chloroplast genome data in Geneious Prime 2021.2.2 referring to the published plastome of Stegnogramma sagittifolia (NC035863; Wei et al. 2017). All tRNAs were verified using tRNAscan‐SE v2.0 (Chan and Lowe 2019). Circular genome maps were generated with OmicsSuite v1.3.9 (Miao et al. 2023). Four DNA regions (matK, rbcL, rps16‐matK, and trnL‐F) were extracted from the plastome and used for subsequent phylogenetic analysis.

2.5. Molecular Phylogenetic Analyses

Considering the close relationship between Craspedosorus and Stegnogramma s.l., two data matrices were generated: (1) plastome dataset consisting of 38 samples, and (2) a four plastid genes dataset consisting of 41 samples. Voucher information and GenBank accession numbers for the samples used in this study are listed in Tables 1 and 2. Matrices of the complete plastomes and each plastid region were aligned using Mafft v7.450 (Katoh and Standley 2013) and manually adjusted in BioEdit (Hall 1999). Based on the Akaike information criterion (AIC), ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best‐fitting models for both maximum likelihood (ML) and Bayesian analyses (BI). ML tree searches and ML bootstrapping were conducted using IQ‐tree v2.1.3 (Nguyen et al. 2015) with 5000 rapid bootstrap analyses followed by a search for the best‐scoring tree in a single run. BI was conducted using MrBayes v3.2.2 (Ronquist et al. 2012). Four Markov Chain Monte Carlo (MCMC) (one cold, three heated) were run, starting from a random tree. A total of 10,000,000 generations were executed, with sampling every 1000 generations. Convergence among generations was assessed using Tracer v1.7.1 (Rambaut et al. 2018) and the first 25% of samples from the cold chain were discarded as burn‐in. The remaining 7,500,000 trees were used to calculate a 50% majority‐rule consensus topology and posterior probability (PP) values. Equally weighted maximum parsimony (MP) analyses for each locus and the combined dataset were conducted in PAUP* v4.0b10 (Swofford 2002) using 1000 tree‐bisection reconnection (TBR) searches, with MAXTREES set to increase without limit. Gaps were coded as missing data. Parsimony jackknife (JK) analyses (Farris et al. 1996) were performed using PAUP*, with the removal probability set to approximately 37%, and “jac” resampling emulated. One thousand replicates were conducted, each with 10 TBR searches and a maximum of 100 trees held per search.

TABLE 1.

List of plastomes used in this study.

Species Voucher Herbarium Location GenBank ID Cities
Abacopteris megacuspis (Baker) Ching Wei WQ224 KUN Yunnan, China MT130555 Du et al. (2021)
Abacopteris nudata (Roxb.) S.E. Fawc. & A.R. Sm. Wei et al. FB374 KUN Yunnan, China MT130561 Du et al. (2021)
Amauropelta beddomei (Baker) Y.H. Chang Cheng et al. FB475 KUN Yunnan, China MT130660 Du et al. (2021)
Ampelopteris prolifera (Retz.) Copel. Cheng et al. FB040 KUN Yunnan, China MT130611 Du et al. (2021)
Ampelopteris prolifera (Retz.) Copel. WR0326 PE Yunnan, China NC035835 Wei et al. (2017)
Asplenium nidus L. Liu 2020 SYS Cult. (SCBG) NC045119 Cui et al. (2019)
Christella acuminata (Houtt.) Holttum Unknown Unknown Unknown NC070299 Unknown
Christella appendiculata (Wall. ex C. Presl) Holttum WR035 PE Yunnan, China NC035842 Wei et al. (2017)
Christella arida (D. Don) Holttum Unknown Unknown Unknown NC070302 Unknown
Christella dentata (Forssk.) Brownsey & Jermy JXJLS0001234 The Biological Herbarium of Jiangxi Provincial Management Bureau for Jiulian Mountain National Natural Reserve Jiangxi, China OM001014 Xu et al. (2023)
Christella latipinna (Benth.) H. Lév. Unknown Unknown Unknown NC070300 Unknown
Christella parasitica H. Lév. Cheng et al. FB049 KUN Yunnan, China MT130695 Du et al. (2021)
Christella parasitica H. Lév. Unknown Unknown Unknown NC070301 Unknown
Christella sp. Cheng et al. FB215 KUN Yunnan, China MT130565 Du et al. (2021)
Coryphopteris japonica (Baker) L.J. He & X.C. Zhang Cheng et al. FB226 KUN Yunnan, China MT130553 Du et al. (2021)
Craspedosorus sinensis Ching & W.M. Chu Zhou et al. YUS14506 PYU Yunnan, China C_AA133102 This study
Cyclogramma auriculata (J. Sm.) Ching Wei WQ331 KUN Yunnan, China MT130552 Du et al. (2021)
Cyclosorus interruptus (Willd.) H. Itô NIBRVP0000627878 National Institute of Biological Resources, Incheon, Korea Jejudo Island, Korea NC057240 Ramekar et al. (2020)
Glaphyropteridopsis erubescens (Wall. ex Hook.) Ching Unknown Unknown Cult. (WBG) MN623355 Liu et al. (2020)
Glaphyropteridopsis erubescens (Wall. ex Hook.) Ching Wei WQ287 KUN Yunnan, China MT130562 Du et al. (2021)
Grypothrix triphylla (Sw.) S.E. Fawc. & A.R. Sm. Unknown Unknown Cult. (FLBG) MN623361 Liu et al. (2020)
Macrothelypteris oligophlebia (Baker) Ching Lu Lu649 KUN Zhejiang, China MT130591 Du et al. (2021)
Macrothelypteris torresiana (Gaudich.) Ching Liu. 201618 SYS Cult. (SCBG) MH500230 Zhou et al. (2018)
Macrothelypteris torresiana (Gaudich.) Ching 7471 PE Guizhou, China NC035858 Wei et al. (2017)
Menisciopsis penangiana (Hook.) S.E. Fawc. & A.R. Sm. Cheng et al. FB227 KUN Yunnan, China MT130694 Du et al. (2021)
Mesopteris tonkinensis (C. Chr.) Ching Liu 201617 SYS Cult. (SCBG) NC041428 Ding et al. (2018)
Onoclea sensibilis L. WR0327 PE Beijing, China NC035860 Wei et al. (2017)
Phegopteris decursive‐pinnata (H.C. Hall) Fée Unknown Unknown Cult. (FLBG) MN623353 Liu et al. (2020)
Phegopteris decursive‐pinnata (H.C. Hall) Fée Cheng et al. FB170 KUN Yunnan, China MT130548 Du et al. (2021)
Pseudocyclosorus esquirolii (C. Chr.) Ching Cheng et al. FB527 KUN Yunnan, China MT130607 Du et al. (2021)
Pseudophegopteris aurita (Hook.) Ching WR0326 PE Jiangxi, China KY427355 Wei et al. (2017)
Pseudophegopteris pyrrhorhachis (Kunze) Ching Cheng et al. FB462 KUN Yunnan, China MT130575 Du et al. (2021)
Pseudophegopteris yunkweiensis (Ching) Ching Cheng et al. FB127 KUN Yunnan, China MT130680 Du et al. (2021)
Stegnogramma griffithii (T. Moore) K. Iwats. Cheng et al. FB137 KUN Yunnan, China MT130604 Du et al. (2021)
Stegnogramma sagittifolia (Ching) L.J. He & X.C. Zhang 7486 PE Guizhou, China NC035863 Wei et al. (2017)
Trigonospora ciliata (Wall. ex Benth.) Holttum Wei et al. FB754 KUN Hainan, China MT130659 Du et al. (2021)
Woodsia macrochlaena Mett. ex Kuhn Wu126 PE Heilongjiang, China NC035864 Wei et al. (2017)
Woodwardia japonica (L. f.) Sm. NIBRVP0000524323 National Institute of Biological Resources, Incheon, Korea Jejudo Island, Korea NC050356 Ramekar et al. (2019)
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TABLE 2.

Vouchers and GenBank accession information for each species included in this study. A dash (—) indicates missing data.

Species Voucher Herbarium Location trnL‐F rbcL rps16‐matK matK
Abacopteris gymnopteridifrons (Hayata) Ching Kuo 857 TAIF Taiwan, China MN159524 MN159456 MN159488 MN159424
Ampelopteris prolifera (Retz.) Copel. WR0326 PE Yunnan, China KY427329 KY427329 KY427329 KY427329
Christella dentata (Forssk.) Brownsey & Jermy Kuo 960 TAIF Taiwan, China MN159523 MN159455 MN159487 MN159423
Craspedosorus sinensis Ching & W.M. Chu Zhou et al. YUS14506 PYU Yunnan, China C_AA133102 C_AA133102 C_AA133102 C_AA133102
Cyclogramma auriculata (J. Sm.) Ching Zhang 4455 PE Yunnan, China — JN572338 — —
Goniopteris tetragona (Sw.) C. Presl Testo 791 VT Heredia, Costa Rica — MN159459 MN159492 MN159428
Leptogramma amabilis Tagawa Nakato 2705 TNS Okinawa, Japan MN159511 MN159445 MN159476 MN159411
Leptogramma burksiorum (J.E. Watkins & Farrar) Y.H. Chang & L.Y. Kuo Eddie s.n. Unknown Alabama, America MN159505 MN159439 MN159470 MN159405
Leptogramma centrochinensis Ching ex Y.X. Lin Sanxia Exped. 1795 PE Hubei, China JN572303 JN572391 — —
Leptogramma chandrae (Fraser‐Jenk.) Y.H. Chang & L.Y. Kuo 319749 TAIF Meghalaya, India MN159521 MN159454 MN159485 MN159421
Leptogramma cyrtomioides (C. Chr.) Y.H. Chang & L.Y. Kuo Kuo 2126 TAIF Sichuan, China MN159517 MN159450 MN159481 MN159417
Leptogramma dissitifolia (Holttum) Y.H. Chang & L.Y. Kuo Kuo 3566 TAIF Mindano, Philippines MN159506 MN159440 MN159471 MN159406
Leptogramma himalaica Ching Liu 9476 TAIF Yunnan, China MN159520 MN159453 MN159484 MN159420
Leptogramma intermedia Ching ex Y.H. Chang & L.Y. Kuo CYH20140714043 TAIF Guizhou, China MN159512 MN159446 MN159477 MN159412
Leptogramma latipinna (Ching ex Y.X. Lin) Y.H. Chang & L.Y. Kuo Liu 9463B TAIF Yunnan, China MN159518 MN159451 MN159482 MN159418
Leptogramma leptogrammoides (K. Iwats.) Y.H. Chang & L.Y. Kuo Kuo 1426 TAIF Yunnan, China MN159519 MN159452 MN159483 MN159419
Leptogramma mollissima (Fisch. ex Kunze) Ching Tagane & Tsujita TF592 TNS Fukuoka, Japan MN159510 MN159444 MN159475 MN159410
Leptogramma mollissima (Fisch. ex Kunze) Ching Kuo 1009 TAIF Tsukuba, Japan MN159513 MN159447 MN159478 MN159413
Leptogramma mollissima (Fisch. ex Kunze) Ching 357631 TAIF Tamil Nadu, India MN159507 MN159441 MN159472 MN159407
Leptogramma mollissima (Fisch. ex Kunze) Ching Kuo 110 TAI Taiwan, China MN159509 MN159443 MN159474 MN159409
Leptogramma petiolata Ching 000531335 BM Sri Lanka MN159508 MN159442 MN159473 MN159408
Leptogramma pilosa (M. Martens & Galeotti) Underw. Pringle 2589 VT Puebla, Mexico MN159504 MN159438 — —
Leptogramma pilosa var. major (E. Fourn.) Y.H. Chang & L.Y. Kuo Testo 1070 VT Oaxaca, Mexico MN159503 MN159437 MN159469 MN159404
Leptogramma pozoi (Lag.) Heywood LGQ 1095 MA Coruna, Spain MN159501 MN159435 MN159467 MN159402
Leptogramma scallanii (Christ) Ching CYH20140712024 TAIF Guizhou, China MN159515 MN159448 MN159479 MN159415
Leptogramma sp. Kuo 2238 TAIF Sichuan, China MN159516 MN159449 MN159480 MN159416
Leptogramma totta (Schltdl.) J. Sm. 00312042 P La Convalescence, Comores MN159514 — — MN159414
Leptogramma tottoides Hayata ex H. Itô Kuo 3818 TAIF Taiwan, China MN159502 MN159436 MN159468 MN159403
Macrothelypteris torresiana (Gaudich.) Ching 7471 PE Guizhou, China KY427352 KY427352 KY427352 KY427352
Meniscium reticulatum (L.) Martyn Testo 782 VT Heredia, Costa Rica MN159527 MN159458 MN159491 MN159427
Metathelypteris uraiensis (Rosenst.) Ching Kuo 2347 TAIF Taiwan, China MN159525 MN159457 MN159489 MN159425
Oreopteris quelpaertensis Holub Zhang 3583 PE Jeju Island, Korea — JN572355 — —
Stegnogramma aspidioides Blume Wade 1902 TAIF Java, Indonesia MN159500 MN159434 MN159466 MN159401
Stegnogramma dictyoclinoides Ching 20140531‐1 TAIF Taiwan, China MN159499 MN159433 MN159465 MN159400
Stegnogramma griffithii (T.Moore) K. Iwats. 390559 TAIF Meghalaya, India MN159497 — MN159463 MN159398
Stegnogramma mingchegensis (Ching) X.C. Zhang & L.J. He Kuo 4240 TAIF Fujian, China MN159498 MN159432 MN159464 MN159399
Stegnogramma sagittifolia (Ching) L.J. He & X.C. Zhang CYH20140714044 TAIF Guizhou, China MN159495 MN159430 MN159461 MN159396
Stegnogramma wilfordii (Hook.) Seriz. 20150329‐1 TAIF Taiwan, China MN159496 MN159431 MN159462 MN159397
Steiropteris leprieurii (Hook.) Pic. Serm. Testo 1227 VT Alajuela, Costa Rica — MN159460 MN159493 MN159429
Thelypteris palustris Schott Larsson 16 UPS Uppsala, Sweden — JF832085 — JF832292
Woodsia manchuriensis Hook. Zhang 2398 PE Korea KP226783 — — —
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2.6. Ancestral State Reconstruction

For ancestral state reconstruction (ASR), we followed the same selection of samples and characters as Kuo et al. (2020). A total of 12 morphological characters, including 10 discrete and two continuous, were chosen. These characters are: (1) stipe indumentum, (2) rhizome habit, (3) venation, (4) basal pinnae, (5) lateral pinnules, (6) length of proximal pinnae, (7) maximum areole row number, (8) minimum areole row number, (9) maximum leaf dissection, (10) minimum leaf dissection, (11) maximum proportion of free vein pairs to the leaf margin, and (12) minimum proportion of free vein pairs to the leaf margin. Morphological characteristics of Craspedosorus sinensis were obtained from field observations and specimen studies. ASR analyses were performed in R v4.1.1 (R Development Core Team 2008) using phytools v0.7.80 (Revell 2012). Model selection was performed prior to stochastic character mapping. Three evolution models (Brownian Motion [BM], Ornstein–Uhlenbeck [OU], and Early Burst [EB]), for two continuous characters were fitted with “fitContinuous” command in the R package “geiger” (Pennell et al. 2014), and the best‐fitting model (Table S1) was selected by corrected Akaike information criterion (AICc; Burnham et al. 2002). Three different models (equal‐rates [ER], symmetric [SYM], and all‐rates‐different [ARD]) for ten discrete characters were fitted to the phylogenetic tree with “fitDiscrete” command in the R package “geiger” and best models (Table S2) were selected by AICc.

3. Result

3.1. Genome Size and Plastome Features of Craspedosorus sinensis

Cytologically, the plant exhibited a DNA content of 5.12 Gb and was inferred to be diploid. The plastome of Craspedosorus sinensis sequenced here exhibits a typical quadripartite architecture, consisting of a large single copy (LSC: 81,800 bp), a small single copy (SSC: 21,705 bp), and two inverted repeats (IR: 26,841 bp) regions (Figure 2). The plastome contains 131 genes, including eight ribosomal RNA genes and 35 tRNA genes (Figure 2). The overall GC content of the plastome was 44.00% (Figure 2).

FIGURE 2.

FIGURE 2

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The plastome map of Craspedosorus sinensis. The dark gray track inside the map shows the GC content. Genes on the outside of the map are transcribed clockwise, and genes on the inside are transcribed counter clockwise. Genes belonging to different functional groups are shown in different colors; see the legend for groups.

3.2. Phylogenetic Relationships of Stegnogramma s.l.

MP, ML and BI analyses produced trees sharing the same general topology based on the same datasets (Figures 3 and 4). From our plastome and four plastid region datasets (Figures 3 and 4), Stegnogramma s.l. was consistently divided into two well‐supported clades. The tree topology derived from the plastome dataset fully supported the sister relationship between Craspedosorus sinensis and Stegnogramma s.s. (ML‐BS = 100, BI‐PP = 1.00, MP‐JK = 100; Figure 3). The inferred phylogenetic relationships based on the four plastid regions were highly consistent with the previous study by Kuo et al. (2020). The monophyly of Stegnogramma s.l. is also strongly supported (ML‐BS = 100; BI‐PP = 1.00; MP‐JK = 100) and is sister to Cyclogramma (Figure 4). In addition, Stegnogramma s.l. includes two well‐supported monophyletic clades (ML‐BS ≥ 99, BI‐PP = 1.00, MP‐JK = 100; Figure 4): Stegnogramma s.s. and Leptogramma. The Leptogramma lineage comprises two clades, L. sect. Leptogramma and L. sect. Haplogramma, which differ in the indumentum on the stipe. C. sinensis is well nested within the Leptogramma lineage, and C. sinensis is the sister group to L. chandrae with moderate support (ML‐BS ≥ 81, BI‐PP = 0.65, MP‐JK = 73; Figure 4). This represents the first molecular evidence for the position of Craspedosorus. However, the relationships among some species in L. sect. Haplogramma clade remain unclear, with some support values being moderate or even low (e.g., ML‐BS = 53, BI‐PP < 0.50, MP‐BS < 50; Figure 4).

FIGURE 3.

FIGURE 3

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Maximum likelihood phylogeny of Craspedosorus sinensis based on the plastome dataset. Maximum likelihood bootstrap support (MLBS), maximum parsimony jackknife support (MPJK), and bayesian inference posterior probability (BIPP) are shown above the branches. Support values of 100 or 1.00 are not displayed.

FIGURE 4.

FIGURE 4

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Maximum likelihood phylogeny of Craspedosorus sinensis based on the four plastid markers (matK, rbcL, rps16‐matK, and trnL‐F). Maximum likelihood bootstrap support (MLBS), maximum parsimony jackknife support (MPJK), and bayesian inference posterior probability (BIPP) are shown above the branches. Support values of 100 or 1.00 are not displayed.

3.3. Ancestral State of Morphology

We conducted a detailed morphological observation of Craspedosorus sinensis and performed ancestral state reconstruction. The results for 12 morphological characters are presented in Figure 5. The absence of multicellular hairs on the stipes represents the ancestral state in Stegnogramma s.l., whereas the presence of multicellular hairs on the stipes is a derived character in L. sect. Haplogramma (Figure 5a). Regarding the maximum areole rows, zero maximum areole rows represent the ancestral state in Stegnogramma s.l. while having more than one maximum areole row is an apomorphic character in L. sect. Haplogramma and Stegnogramma (Figure 5j). ASR analyses revealed that C. sinensis can be distinguished from closely related species of L. sect. Haplogramma by free venation, increasing lengths of proximal pinnae, and the absence of areole rows (Figure 5d,f,j).

FIGURE 5.

FIGURE 5

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Ancestral character reconstruction of 12 morphological characters. (a) Stipe indumentum. (b) Rhizome habit. (c) Basal pinnae. (d) Venation. (e) Lateral pinnules. (f) Length of proximal pinnae. (g) Maximum leaf dissection. (h) Minimum leaf dissection. (i) Minimum areole row number. (j) Maximum areole row number. (k) Maximum proportion of free vein pairs to the leaf margin. (l) Minimum proportion of free vein pairs to the leaf margin. The pie charts on nodes summarize results of stochastic character mapping, and the color in each of the tip node shows the state.

4. Discussion

4.1. Phylogenetic Position of Craspedosorus

With the advancements in molecular technology, the publication of new taxon is now often accompanied by molecular data (e.g., Jiang et al. 2025; Zhao, Liang, et al. 2025; Zhao, Ma, et al. 2025; Zhou et al. 2025). Craspedosorus, a monotypic genus described by Ching (1978) based on morphological characters, was the only genus in China lacking molecular data due to limited material and field investigations. Although FRPS (Shing 1999) and FOC (Lin et al. 2013) recognized Craspedosorus as a distinct genus and considered it closely related to Leptogramma, the phylogenetic relationship between Craspedosorus and other genera of Thelypteridaceae remained unclear. Morphologically, Craspedosorus and Leptogramma are very similar in laminar outline, segment shape, venation, and sori. Recently, Kuo et al. (2020) proposed that Craspedosorus should be treated as a synonym of Leptogramma and further classified as a member of L. sect. Haplogramma based on morphological characteristics. Our inferred phylogeny also supports that Craspedosorus belongs to Leptogramma within the subclade L. sect. Haplogramma (Figure 4). The presence of multicellular hairs on the stipes further confirms that C. sinensis belongs to L. sect. Haplogramma (Figure 5a). C. sinensis is closely related to L. chandrae (Figure 4). However, C. sinensis differs from L. chandrae in several aspects: it is a larger plant, reaching nearly 100 cm (vs. up to 60 cm); it has free venation (vs. anastomosing venation with one or two areole rows); and its laminae are pinnate‐bipinnatifid (vs. pinnatifid‐pinnate). Furthermore, C. sinensis , which exhibits free venation, differs from other species of L. sect. Haplogramma in its venation pattern (free vs. anastomosing in L. sect. Haplogramma). Additional characteristics of C. sinensis are as follows: rhizomes erect; scales broad lanceolate, yellowish‐brown, with hairy margins; laminae broad lanceolate, pinnate‐bipinnatifid, not tapering at the bases; leaf margins membranous; basal pinnae sessile; and sori oblong, attached below ends of veinlets and close to margins (Ching 1978; Chu 2006; Figure 1). ASR analyses also indicate that C. sinensis is a relatively distinctive species both morphologically and phylogenetically within Leptogramma (Figure 5). Our results support treating Craspedosorus as a synonym of Leptogramma.

4.2. Morphological Evolution

The two genera, Stegnogramma s.s. and Leptogramma, can be distinguished by their venation patterns and the number of areole rows (Fawcett and Smith 2021; Figure 5). Leptogramma exhibits either free or anastomosing venation. Moreover, when venation is anastomosing in Leptogramma, the number of areole rows is limited to no more than two (Figure 5). Discrete characters such as rhizome habit, venation, leaf dissection, and the number of areole rows may have evolutionary significance related to environmental adaptation, geographical distribution, and other factors (Kuo et al. 2020). These discrete characters can effectively distinguish different clades from one other (Figure 5). The ancestral states of the remaining discrete characters in Stegnogramma s.l. appear to be uncertain, which also implies morphological diversity within the thelypterids (Figure 5).

Regarding continuous characters, the tips of the veinlets do not reach the leaf margin in Craspedosorus sinensis. However, this species, which has highly dissected fronds, exhibits an increased proportion of free vein pairs extending to the leaf margin. This pattern is consistent with the findings of Kuo et al. (2020), which suggest a correlation that it be correlated with latitude. For example, the frond of C. sinensis is pinnate‐bipinnatifid, with all free vein pairs reaching the leaf margin. According to collection records and field surveys, C. sinensis is distributed at high latitudes (Zhaotong, Yunnan, China, ca. N 28°21′ to N 28°40′).

4.3. The Protection of Craspedosorus sinensis

Although Craspedosorus sinensis was successfully identified using molecular evidence based on next‐generation sequencing technology, it has not been observed in the wild since 1973. Recently, we discovered a wild population in Yongshan County with ca. 15 adults. Based on the IUCN red list criterion (IUCN 2024), we classify it as Critically Endangered (CR). According to the Chinese government's policy document, the list of wild plants under special state protection includes a total of 11 families, 16 genera, and ca. 106 species of lycophytes and ferns. These species are either of significant application value or are relatively rare in the wild. With increased awareness of plant conservation in recent decades, more and more ferns have been effectively protected and rediscovered (e.g., rediscovery of Cystoathyrium chinense and Angiopteris tonkinensis) (Wei and Zhang 2014; Wang et al. 2020). This progress has also facilitated more in‐depth studies of many species (e.g., Alsophila spinulosa, Ceratopteris richardii , and Isoetes taiwanensis) (Huang et al. 2022; Marchant et al. 2022; Wickell et al. 2021). Therefore, we advocate that C. sinensis to be designated as a protected species in China in the future.

Author Contributions

Jing Zhao: data curation (lead), formal analysis (lead), investigation (lead), methodology (lead), software (lead), validation (lead), visualization (lead), writing – original draft (lead), writing – review and editing (lead). Jian‐Jun Yang: data curation (lead), formal analysis (lead), investigation (lead), methodology (lead), software (lead), validation (lead), visualization (lead), writing – original draft (lead), writing – review and editing (lead). Zhong‐Yang Li: investigation (lead), resources (lead), software (equal), supervision (equal), validation (equal), writing – original draft (supporting), writing – review and editing (supporting). Chuang‐Jie Huang: investigation (supporting), resources (supporting). Ting Chen: investigation (supporting), resources (supporting). Yong‐Li Zhao: investigation (supporting), resources (supporting). Zhao‐Rong He: investigation (supporting), resources (supporting). Chao‐Shan Gu: funding acquisition (equal), investigation (lead), resources (lead), supervision (supporting), writing – original draft (lead), writing – review and editing (supporting). Xin‐Mao Zhou: conceptualization (lead), funding acquisition (equal), investigation (supporting), project administration (lead), visualization (supporting), writing – review and editing (lead).

Funding

This work was financially supported by the Yunnan Fundamental Research Projects (Grant No. 202301BF070001‐016) and National Science & Technology Fundamental Resources Investigation Program of China (Grant No. 2022FY100201).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Model selection for ancestral state reconstruction based on two continuous characters. The bold font represents the best‐fit model.

Table S2: Model selection for ancestral state reconstruction based on ten discrete characters. The bold font represents the best‐fit model.

ECE3-16-e73498-s001.xlsx (13.5KB, xlsx)

Acknowledgments

The authors gratefully acknowledge the contributions of Miao Luo for assistance with the field survey and Dan‐Ni Ma for assistance with the scanning of the spores.

Contributor Information

Chao‐Shan Gu, Email: wmsguchaoshan@163.com.

Xin‐Mao Zhou, Email: xinmao.zhou@ynu.edu.cn.

Data Availability Statement

The DNA sequences generated in this study have been deposited in the GenBase database of the China National Center for Bioinformation (CNCB). The accession numbers and the information on the voucher specimens are available in Tables 1 and 2.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1: Model selection for ancestral state reconstruction based on two continuous characters. The bold font represents the best‐fit model.

Table S2: Model selection for ancestral state reconstruction based on ten discrete characters. The bold font represents the best‐fit model.

ECE3-16-e73498-s001.xlsx (13.5KB, xlsx)

Data Availability Statement

The DNA sequences generated in this study have been deposited in the GenBase database of the China National Center for Bioinformation (CNCB). The accession numbers and the information on the voucher specimens are available in Tables 1 and 2.

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