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Morphology and ITS sequences provide insights into the phylogeny of Tongoloa (Apiaceae) from China

BMC Ecology and Evolution volume 24, Article number: 103 (2024) Cite this article

Abstract

Background

Tongoloa is a genus comprising approximately 20 species, primarily distributed in the mountainous regions of southwest China. The insufficiency of specimen materials and morphological similarities among species render it a taxonomically challenging genus within the Apiaceae family. To elucidate the phylogenetic relationships and taxonomy of Chinese Tongoloa, this study utilized a total of 115 nrITS sequences, including 47 recently obtained sequences, for phylogenetic reconstruction.

Results

Phylogenetic relationships reconstructed from ITS sequences indicate that the East Asia Clade and the Komarovia Clade are sister groups, and Tongoloa belongs to the East Asia Clade. Species of Tongoloa are subdivided into 3 distinct groups, all sharing similar fruit morphologies and are clearly differentiated from related taxa. Several Tongoloa-like members classified under other genera are interpreted to be closely related to Tongoloa. Morphological and molecular data indicate that Tongoloa, Sinolimprichtia subclade and Chinese Trachydium subclade are separate yet genetically contiguous taxa. It is confirmed that Tongoloa zhongdianensis belongs to the Hymenidium Clade, while Sinocarum is classified within the Acronema Clade. Two new taxa are found in the Hengduan Mountains.

Conclusion

Tongoloa is a genus within the East Asia Clade of Apiaceae, and the phylogeny reconstructed based on ITS sequences divides it into 3 main groups. By integrating fruit morphology and molecular phylogenetic analyses, we preliminary clarified the intricate taxonomic relationships among Tongoloa and related taxa. These results provide valuable opportunities for a deeper understanding of the phylogeny of Tongoloa.

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Background

Tongoloa H.Wolff [1] is a genus including about 20 species distributed in the mountainous regions of East Asia [2], especially in southwest China [3, 4]. Most species are confined to alpine meadows, grasslands, shrubs, and forests ranging from 2000 to 4500 m [1, 3, 5,6,7]. Owing to their distribution in remote mountainous areas with limited accessibility, few of the previously collected specimens have contained mature fruits, which are considered to hold crucial taxonomic value in Apiaceae species [8,9,10,11,12]. Insufficient specimen materials and similar morphologies make this genus taxonomically challenging [13]. Pan and Watson stated that the current taxonomic treatment of Tongoloa should be considered tentative [3]. Most Chinese species are still not fully recognized, and the general delimitation between Meeboldia, Sinoodielsia, Tongoloa and Vicatia has been problematic and controversial [14].

Over the past 20 years, scholars have endeavored to reconstruct the phylogenetic framework of Apiaceae using molecular methods, aiming to establish a more objective and robust classification system [15,16,17,18,19]. Valiejo-Roman et al. reconstructed the phylogeny of Apioideae (Apiaceae) from the Himalayas by incorporating 13 newly obtained nrITS (nuclear ribosomal internal transcribed spacer) sequences, and the findings indicated that Tongoloa is related to Trachydium simplicifolium W. W. Smith and Hansenia Turcz. [20]. After enriching samples and molecular makers of Apioideae from China, Zhou et al. proposed that Tongoloa exhibits a close relationship with specific species of Sinocarum H. Wolff ex R. H. Shan & F. T. Pu, Pimpinella L., Trachydium Lindl. and Sinolimprichtia H. Wolff, all residing within the East Asia Clade [21, 22]. Nevertheless, Xiao et al. argued that the genus Sinocarum does not belong to the East Asia Clade [23]. Pimpinella purpurea (Franch.) H. Boissieu was molecularly closely related to members of Tongoloa [24]. It was found that T. zhongdianensis S.L. Liou may related to Hymenidium Lindl. [25]. Unfortunately, previous molecular analyses often lacked convincing morphological evidence. Examining the vouchers of previous molecular data is rather difficult, as they are usually scattered across herbariums in different regions.

China is the primary distribution area of Tongoloa, with all known species and a diverse range of morphological types [3, 26]. Recent field surveys in the Hengduan Mountains have led to a new understanding of species diversity of this genus [27, 28]. However, a comprehensive phylogenetic analysis of Tongoloa has yet to be conducted. Owing to its moderate length and rapid mutation rate, the ITS sequence serves as an efficient tool for analysis and validates the accuracy of species morphological taxonomy [17, 21, 29,30,31,32]. Additionally, the ITS sequence boasts a remarkable sequencing success rate and offers a cost-effective solution, significantly contributing to the accumulation of extensive species sequences in the current public database. In this study, we adopted extensive sampling and selected the ITS sequence as the molecular marker for phylogenetic reconstruction. Furthermore, we integrated observing and comparative analyses of fruit morphology to determine a more rational phylogenetic relationship for this genus.

Materials and methods

Taxon sampling

To examine the morphological variations of Tongoloa species, a comprehensive study was conducted on a diverse range of specimens preserved in herbariums A, B, CDBI, E, G, GB, GH, HNWP, K, KUN, LD, MW, NAS, NY, P, PE, SZ, US, W, and WUK. Samples were preferentially collected from or near the type localities of the specimens to ensure accurate and reliable identification. The species identifcation was undertaken by Lingjian Gui and Xingjin He (Sichuan University, Chengdu, China). The nomenclature of taxa was primarily based on Flora of China [3] and the updated checklist of Chinese Apiaceae [26]. Voucher specimens were stored in the herbarium of Sichuan University (SZ) in Chengdu, China (Table 1). The fruits of Tongoloa and related taxa were observed and photographed using a Nikon SMZ25 stereomicroscope (Japan).

Table 1 Voucher of Tongoloa and related taxa collected by us
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DNA extraction and sequencing

Total DNA was extracted from silica-dried leaves using the plant genomic DNA kit (CWBIO, Beijing, China). The extracted total DNA was stored in a refrigerator at -20°C. The primers ITS4: 5’-TCC TCC GCT TAT TGA TAT GC-3’ and ITS5: 5’-GGA AGT AAA AGT CGT AAC AAG G-3’, designed by White et al., were selected for PCR amplification of the target sequence [33]. PCR was performed in a 30 µl amplification system, comprising 2 µL total DNA, 9 µl water, 1.5 µL forward primer, 1.5 µL reverse primer and 15 µl Taq PCR Master mix (CWBIO, Beijing, China). All PCR amplifications were performed on a Geneamp PCR System 9700 (USA). The ITS amplification protocol involved an initial denaturation step for 2 min at 95 °C, followed by 35 cycles of 60 s at 94 °C, 45 s at 52.5 °C, and 60 s at 72 °C, and a final extension step of 7 min at 72 °C. Bidirectional sequences were assembled using SeqMan in DNASTAR 5.01 [34], with subsequent necessary manual inspection. The completed ITS sequences and the boundaries of ITS1 and ITS2 were determined by comparison with the ITS sequences of Tongoloa available on NCBI.

Sequence alignment and phylogenetic analysis

A total of 115 ITS sequences of Apiaceae were used for constructing the phylogenetic tree. 47 ITS sequences are recently obtained and the voucher information is presented in Table 1, while the remaining 68 sequences are downloaded from NCBI (Table S1). The ITS dataset includes 48 species from the East Asia Clade, 7 species from the Komarovia Clade, 14 species from the Acronema Clade, 4 species from the Hymenidium Clade (Sinodielsia Clade), and 5 species from Pleurospermeae [17]. All sequences were aligned using MAFFT [35]. The aligned sequences were manually inspected and the phylogenetic analysis was conducted using maximum likelihood (ML) and bayesian inference (BI) methods. The optimal DNA model was selected in Mega7 [36], and a ML tree with 500 bootstraps was subsequently constructed. Mrbayes v3.2 [37] was employed to infer a BI tree with 5 million generations using the GTR + G model. The first 25% of the trees were discarded, and the remaining trees were utilized to calculate posterior probability (PP) statistics. Bootstrap (BS) and PP values were annotated on the branches of a ML tree. Jalview2 [38] was used to visualize sequence base variations.

Results

Fruit characteristics

The fruit morphology and anatomical features of Tongoloa were studied. We found that these fruits are primarily wide oval with a cordate base, exhibiting filiform ribs, 3 vittae in each furrow, and 4 vittae on the commissure. The stylopodium is depressed, while the ventral surface of the endosperm is flat-concave (Fig. 1). We subsequently examined the fruit morphologies of genera that have recently been considered closely related to Tongoloa in molecular phylogeny [21, 22]. The results revealed that the fruits of Pimpinella purpurea (Fig. S2) and Trachydium variabile (Fig. S3) exhibit a cordate base, 5 filiform ribs, 3 vittae in each furrow, and 4 vittae on the commissure. Additionally, they possess a depressed stylopodium, and a ventrally flat-concave endosperm, featuring closely align with those observed in common Tongoloa species. The morphological results support the conclusion that Pimpinella purpurea and Trachydium variabile may be closely related to Tongoloa [22]. However, in contrast, the fruits of Sinolimprichtia alpina exhibit a long obovate shape with a narrowed base and a fluffy peel, and possess 6 vittae on the commissures (Fig. S1 A). These features are significantly different from those of Tongoloa. Similarly, the fruits of Trachydium souliei are obovate with a narrowed base and wing-like ribs, and also display 6 vittae on the commissures (Fig. S1 D), distinguishing them from Tongoloa fruits. The mature fruits of T. zhongdianensis are distinctly dissimilar to those of the genus Tongoloa (Fig. S4).

Fig. 1
figure 1

Fruit morphology of Tongoloa species. (A) T. tagongensis (GLJ18092101 & GLJ18102401); (B) T. elata (GLJ18101802); (C) T. filicaudicis (GLJ18102003); (D) T. loloensis (GLJ18103002); (E) T. arguta (GLJ19100602); (F) T. silaifolia (rt20092320); (G) T. taeniophylla (GLJ18082902); (H) T. gracilis (GLJ18092102). (1) Lateral view of the cremocarp; (2) Cross-section view of the mericarp. The items within the parentheses are voucher specimens

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Phylogenetic analysis

The ML consensus tree and BI tree have similar topological structures. The East Asia Clade and the Komarovia Clade are sister groups with strong support. Phylogenetic analysis using both ML and BI methods divides Tongoloa into three clustered groups (Fig. 2), although some branches require additional reinforcement for stronger phylogenetic support. Group I includes only one species, T. gracilis. Group II comprises at least 6 species, including Pimpinella purpurea, Physospermopsis cuneata H. Wolff, Tongoloa rubronervis, and 2 potential new species (T. spathulata nov. sp. and T. xinlongensis nov. sp.). Group III has at least 10 species, including T. tagongensis, T. loloensis, T. fortunatii, T. silaifolia, T. taeniophylla, T. elata, T. napifera, T. arguta, Trachydium variabile and Hymenidium virgatum. Notably, while the Tongoloa groups, the Sinolimprichtia subclade and the Chinese Trachydium subclade are each recognized as separate taxa on the phylogenetic tree, their branching patterns reveal remarkably close affinities, suggesting complex speciation or recent divergence within the broader evolutionary context. Tongoloa zhongdianensis is not located within the East Asia Clade, but it exhibits a significant association with taxa belonging to the Hymenidium Clade.

Fig. 2
figure 2

Phylogram obtained through maximum likelihood analysis based on ITS sequences. Tongoloa is positioned within East Asia Clade and is divided into 3 groups. The BS/PP values are indicated on the branches. When both values are at their maximum, they are marked with an asterisk (*). Values below 50% and 0.5 are typically omitted

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Sequence comparative analysis

A comparative analysis was performed on the ITS sequences of 13 Tongoloa species and 14 other closely related species from the East Asia Clade. The ITS sequence of Pimpinella purpurea exhibits identical specific base variations at positions 457–460 bp, 533 bp, 537 bp, and 610 bp with 4 closest Tongoloa or Tongoloa-like species (Fig. 3A). The ITS sequence of Trachydium variabile shows a strong similarity to that of closely related Tongoloa species, yet it displays notable variations at loci 108 bp, 484 bp, 500 bp and 605 bp (Fig. 3B).

Fig. 3
figure 3

Comparison of aligned ITS sequences of 24 taxa from East Asia Clade. (A) Pimpinella purpurea shares common variations at loci 457–460 bp, 533 bp, 537 bp and 610 bp with other species in Tongoloa group II (indicated by purple font and black boxes); (B) The specific variations of Trachydium variabile, relative to closely related species, are found at loci 108 bp, 484 bp, 500 bp and 605 bp (indicated by blue font and red boxes)

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Discussion

Phylogeny and taxonomy of Tongoloa

As of now, ITS sequences remain the principal criterion for defining the major clades and tribes of the Apiaceae family [17]. Furthermore, the processing of precursor rRNA transcripts is a remarkably conserved biological mechanism. The secondary structure of the ITS in nuclear rRNA transcripts is widely documented across eukaryotes, and this feature has been employed to facilitate phylogenetic comparisons [39].

Based on the most extensive molecular sampling of Tongoloa and its closest relatives to date, it has been confirmed that Tongoloa belongs to the East Asia Clade of Apiaceae [17]. The internal taxa of Tongoloa have been tentatively divided into three groups, including common Tongoloa species and some Tongoloa-like species, with the exception of T. zhongdianensis. Pimpinella purpurea and Physospermopsis cuneata are classified under Tongoloa group II, while Trachydium variabile falls under Tongoloa group III (Fig. 2). Morphological studies reveal that most of the species within the three groups, including those temporarily listed in other genera, share similar characteristics. These features include conical roots, slender stems, 3-ternate/pinnate leaves, depressed stylopodium, flat petals, fruits with a broadly ovoid shape and cordate bases, 5 filiform ribs, 3 vittae in each furrow and 4 vittae on the commissure. Additionally, the seed faces exhibit a flat-concave appearance. These shared characteristics support the classification of Tongoloa as an independent genus.

Previous studies have revealed that the phylogenetic relationship between Tongoloa, Sinolimprichtia alpina, Trachydium simplicifolium, and Trachydium souliei remains uncertain [21, 22]. In the phylogenetic tree of this study, although the Tongoloa groups, the Sinolimprichtia subclade, and the Chinese Trachydium subclade are almost independent of each other, they are located within the same major clade (Fig. 2), indicating a close genetic relationship. We have newly added fruit morphology data and found that the fruits of the Sinolimprichtia subclade and Chinese Trachydium subclade usually possess distinctive wing-like ribs and a higher number of vittae on the commissure (Fig. S1), which are noticeably distinct from the features of Tongoloa. In recent studies on chloroplast genomes and ribosomal DNA, Sinolimprichtia alpina was identified as a likely ancient hybrid taxon [40]. The potential hybridization may introduce ambiguity to the boundaries between Tongoloa and its closely related taxa. Furthermore, the ubiquity radiation of plants in the Hengduan Mountains presents challenges to the the division of many generic boundaries [41, 42]. In light of molecular phylogenetic analyses and distinctive fruit morphological traits, acknowledging Tongoloa as a separate genus appears to be a justifiable classification strategy. This approach not only reflects the unique evolutionary lineage of Tongoloa but also respects its discernible morphological identity, thereby contributing to a more accurate and refined understanding of its taxonomic placement within the family.

Despite our diligent efforts in this study, we are unable to obtain molecular sequences for three dubious Tongoloa taxa. The species T. pauciradiata H. Wolff, identifiable only through its type specimen collected in Lhasa, Tibet (barcode K000075377), exhibits fruit and plant morphologies similar to advanced Apioideae taxa, diverging from the primitive characteristics of basal lineages such as the East Asia Clade. Tongoloa rockii H. Wolff is represented solely by type specimens (barcodes E000000521 and GH00077982) from Yulong Snow Mountain in Yunnan. This species, characterized by its short and slender plants, thick root and 4-pinnate/pinnatifid leaves, is acknowledged as a good species by Fading Pu [43]. After analyzing the morphological features and ITS sequences of similar species, it is evident that its phylogenetic position belongs to the Tongoloa group III. The species T. smithii H. Wolff (barcode GB-0048829) is exclusively found in its type locality, Maerkang, Sichuan. The distribution of this species lies within 200 km of T. elata’s type locality in Huangshengguan, Songpan, Sichuan. Despite lacking fruits on its type specimen, T. smithii exhibits morphological similarities in its flowers, leaves, and overall plant structure to T. elata, indicating a possible close relationship or even species identity.

Zhou et al. provisionally referred to the Pimpinella species in the East Asia Clade as the Chinese Pimpinella subclade [21, 22]. However, the findings of this study have underscored the imperative need to further categorize these Pimpinella taxa into two separate subclades, temporarily designated as Chinese Pimpinella subclade A and Chinese Pimpinella subclade B (Fig. 2). Subclade A is phylogenetically distant from Tongoloa and exhibits a close relationship with Keraymonia. Currently known species include Pimpinella henryi Diels and P. rhomboidea Diels. The fruit bases of this subclade are cordate, and have 5 filiform ribs, exhibiting some similarity to Tongoloa. However, the stylopodium shape, style length, and the number of vittae in the fruit significantly differ from Tongoloa. Chinese Pimpinella subclade B stands as a pivotal groups in this study. Currently, the confirmed species primarily include P. purpurea, whose ITS sequences exhibit a close clustering with species of Tongoloa group II. Moreover, morphologically, Pimpinella purpurea exhibits an ovoid fruit shape with a cordate base, filiform ribs, 4 commissural vittae, and a flattened stylopodium (Fig. S2), all of which align closely with the morphological features of Tongoloa.

Trachydium variabile is presently a species lacking extensive morphological and molecular studies, and is not included in Flora of China [43]. We verified the original literature, the type specimens, as well as fresh samples collected from the type locality. The newly collected samples exhibited a remarkable consistency with the type specimens. Additionally, we enhanced the population sample by incorporating specimens from the vicinity of Longriba, Hongyuan, Sichuan. The sequencing results reveal a congruency in the ITS sequences of both populations. The phylogenetic tree constructed on the basis of ITS sequences positioned T. variabile within Tongoloa group III. In terms of morphology, the flower, fruit, and leaf characteristics of this species are similar to those of the typical species of Tongoloa group III (Fig. S3). Analysis revealed that the ITS of Trachydium variabile had undergone significant genetic variation compared with related taxa, particularly with four unique base variations at the 108 bp, 484 bp, 500 bp, and 605 bp loci (Fig. 3B). These results hint that Trachydium variabile is an independent species in Tongoloa Group III.

Also noteworthy, the results of molecular analysis support the suggestion made by Pan and Watson in Flora of China, to classify Physospermopsis cuneata as belonging to Tongoloa. However, it is unfortunate that we failed to collect samples with mature fruits from the type locality of Physospermopsis cuneata in Yulong Snow Mountain, Yunnan. Therefore, it is crucial to supplement the collection materials for future research purposes.

The mature fruits of Tongoloa zhongdianensis collected from the type locality exhibit significant morphological differences from Tongoloa species (Fig. S4). ITS molecular markers indicate that the systematic position of this species falls within the Hymenidium Clade (Sinodielsia Clade), possibly having a closer relationship with some species of Meeboldia, while being distant from the East Asia Clade where Tongoloa resides. Currently, this taxon is still considered an independent species, but its precise taxonomic treatment requires further determination.

Sinocarum was previously considered to be closely related to Tongoloa [14]. Valiejo-Roman et al. conducted a sampling and phylogenetic study of Apiaceae from the Himalayan region, involving one representative taxon of Sinocarum and another representative taxon of Tongoloa [20]. The result indicates that despite the morphological similarities between Sinocarum and Tongoloa, they do not share a close genetic relationship. Recent research, bolstered by our findings, confirms that the majority of Sinocarum species, particularly S. coloratum, S. cruciatum (Franch.) H. Wolff, and S. vaginatum H. Wolff, all belonging to the Acronema Clade, exhibit significant genetic divergence from Tongoloa [23, 44]. The morphological similarity between these two genera may be attributed to convergent evolution resulting from selective pressures in high-altitude environments rather than genetic relationships. Indeed, there are significant morphological differences between the mature fruits of Sinocarum [23] and Tongoloa. Unfortunately, most of the previously gathered specimens of Sinocarum, including type specimens, were devoid of mature fruits, potentially leading to erroneous identifications in the past.

Two new species of Tongoloa

The morphological variation of Tongoloa species distributed in the Hengduan Mountains is complex, and specimens are easily misidentified when morphological characteristics are not sufficient [23, 27, 45]. We found a questionable specimen [T. N. Ho (HNWP), B. Bartholomew (CAS), M. Watson (E) and M. Gilbert (MO at BM) 2685] preserved in the Qinghai-Tibetan Plateau Museum of Biology (Xining, Qinghai, China). It was identified as Tongoloa gracilis. Nevertheless, the petal tips of this particular plant are blunt instead of pointed, which is not aligned with the typical features of T. gracilis with incurved petal tips. In 2019, guided by sampling records, we conducted a field investigation to Jiangxi Forest Farm in Jiangxigou, Yushu, Qinghai Province, and collected the fresh materials of this unusual species on the grassy slope under Picea trees. The ITS phylogenetic analysis reveals that this species occupies a distinct branch within Tongoloa group II (Fig. 2). We conducted a thorough examination of all available specimens and literature, with a particular emphasis on the taxa from the Qinghai-Tibet Plateau. After a thorough analysis encompassing morphology, nrITS sequence, and geographic distribution, we have determined that this plant belongs to an undescribed species. Given the distinctive morphological characteristics of its petals, we have designated it as T. spathulata.

Xinlong County, situated in the Hengduan Mountains of Sichuan Province, boasts an altitude ranging from about 2,700 m to 5,900 m. The continuous mountains here are separated by several river valleys. There have been scant records of Apiaceae specimens in Xinlong County and its neighboring regions (at least until recently). In 2020, during our survey of plant resource in this County, we discovered an special species of Tongoloa. The plants preferentially thrive in humid places at altitudes of 3200–3800 m, growing among shrubs or in herb communities. Combined with the geographical distribution, we examined the morphology and molecular marker (ITS) of the newly collected samples (Figs. 2 and 3), and confirmed that this species is closely related to T. spathulata, which is distributed in Qinghai. However, the petals, fruits and ITS sequences of this species are significantly different from those of similar taxa, and it cannot be placed in any known Tongoloa or other Apiaceae species. Here we formally describe it as a new species, T. xinlongensis.

Taxonomic treatment

Tongoloa spathulata L.J. Gui & X.J. He, sp. nov

Diagnosis. The new species is morphologically similar to T. taeniophylla (H. Boissieu) H. Wolff, but distinguished by having spatulate petals (vs. narrow linear petals).

Type. CHINA, Qinghai: Jiangxi Forest Farm, Jiangxigou, Yushu. East of Jiangxi Forest Farm Station, 3640 m, 32°4′ N, 97°4′ E, 22 Sept. 2019, Ling-jian Gui & Chang Peng, GLJ19092201 (Holotype: SZ!; isotype: PE!); Jiangxigou, Yushu. E of Jiangxi Forest Station, on E side of the Zi Qu and SE of Mozhong. 3640 m, 32°5′ N, 97°4′ E, 29 Aug 1996, T.N. Ho et al. 2685 (Paratype: HNWP!, PE!).

Description. Plants 25–80 cm. Root conic, 2–6 × 0.4–1 cm. Stem striate, green to purplish, glabrous, branched. Lower leaves long-petiolate, petioles sheaths inflated, membranous; upper leaves sessile, sheath membranous. Blades triangular, 3-ternate/pinnate; ultimate segments lanceolate, 2–6 × 0.8–2 mm; Terminal peduncle 4–15 cm, lateral peduncle 2–9 cm. Umbels 4–12 cm across; bracts absent; bracteoles absent or 1–6; rays 7–16, 2–7 cm, unequal; umbellules 8–15-flowered, 2–4 mm; Calyx teeth minute, acute; Petals spatulate, white to pinkish, 1–1.5 × 0.4–0.8 mm, apex obtuse-rounded; stylopodium pressed, dark purple; styles short, reflexed outward. Fruit broad–ovoid, ca. 2.2 × 1.8 mm, base cordate; ribs 5, filiform to inconspicuous; vittae 3 in each furrow, ca. 4 on commissure. Seed face flat-concave (Figs. 4 and 5).

Fig. 4
figure 4

Tongoloa spathulata sp. nov. (A) Plant and habitat; (B) Basal leaf; (C) Ultimate segments of basal leaf; (D) Upper leaf with sheath-like petiole; (E) Root; (F) Petal; (G) Umbellule; (H) Fruit, top view; (I) Fruit, Lateral view; (J) Mericarp, cross section view

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Fig. 5
figure 5

Holotype specimen of Tongoloa spathulata

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Etymology. The species is named after the spatulate petals of this plant.

Distribution and habitat.Tongoloa spathulata is currently found in Yushu, located on the eastern Qinghai-Tibet Plateau. It grows on slope, at an altitude of 3600–3900 m. Fl. and Fr. Aug.– Oct.

Tongoloa xinlongensis L.J. Gui & X.J. He, sp. nov

Type. CHINA. Sichuan: Xinlong County. North of Pica Township, in shrubs and herbs near the damp place, 3573 m, 30°49′ N, 100°3′ E, 12 July 2020, Ling-jian Gui, GLJ20071201 (holotype: SZ!).

Diagnosis.Tongoloa xinlongensis sp. nov. is similar to T. spathulata. However, this new species can be distinguished from the latter by its petals broad obovate, 2–2.2 × 1–1.2 mm (vs. spatulate, 1–1.5 × 0.4–0.8 mm), and the fruits are obviously larger (2.6–3 × 2.3–3 mm vs. 2.2 × 1.8).

Description. Plants glabrous, 30–115 cm. Root conic. Stems ribbed and little branched, lower part purplish. Leaf sheaths membranous; blade triangular in outline, 5–14 × 4–10 cm, 2–3-ternate/pinnate, ultimate segments lanceolate-ovate, 3–15 × 2.5–4.5 mm, apex rounded or acute, margin sometiomes pinnate slightly; up petioles sheath-shaped, green. Umbels 3–15 cm across; bracts absent; rays 13–18; bracteoles usually absent or 1–3, linear, slightly longer than pedicels; pedicels unequal; umbellules 15–25-flowered. Calyx teeth minute and acute. petals obovate, apex usually obtuse-rounded, or with tips, white to purplish, ca. 2–2.2 × 1–1.2 mm. Anthers purple to greenlish. Stylopodium depressed, usually slanted inward in fruit period, greenlish to lavender; styles very short, reflexed. Fruit ovoid, base cordate, 2.6–3 × 2.3–3 mm, ribs filiform to inconspicuous; vittae 3 in each furrow, 4 on commissure. Seed face flat-concave (Figs. 6 and 7).

Fig. 6
figure 6

Tongoloa xinlongensis sp. nov. (A) Habitat; (B) Plant; (C) Root; (D–E) Membranous sheaths at the base of petioles; (F) Umbel; (G–H) Flowers; (I) Bracteoles; (J) Lateral and cross section view of mericarpes

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Fig. 7
figure 7

Scientific Illustration of Tongoloa xinlongensis sp. nov. (A) Plant; (B) Basal leaf; (C) Membranous sheath of petiole; (D–G) Flower. The apex of the petals is usually obtuse, but is pointed in a few individuals. Drawn by Bing-Yan Chen. Authorized for written publication

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Etymology. The species epithet “xinlongensis” was named after Xinlong County, where the type materials were found.

Phenology. The species was observed flowering from July to September and fruiting from September to October.

Distribution and habitat.Tongoloa xinlongensis grows in shrubs and herbs from 3200 m to 3800 m.

Conservation status.Tongoloa xinlongensis is currently known to only occur in its type locality Xinlong County, Sichuan Province with several populations. We categorise it as Data Defcient (DD) according to IUCN (2019).

Conclusion

Tongoloa is a genus within the East Asia Clade of Apiaceae, and the phylogeny reconstructed based on ITS sequences divides it into 3 main groups. These groups have similar fruit morphology. By integrating fruit morphology and molecular phylogenetic analyses, we preliminary clarified the intricate taxonomic relationships among Tongoloa, Sinocarum, Chinese Pimpinella subclade, Sinolimprichtia subclade, and Chinese Trachydium subclade. Furthermore, we proposed 2 new species, thereby suggesting a potentially richer species diversity of Apiaceae in the Hengduan Mountains.

Data availability

The ITS sequences can be downloaded from NCBI (https://www.ncbi.nlm.nih.gov) with accession numbers: OP422507-OP422516, MN630613-MN630619, MT124598-MT124620 and MZ054145-MZ054154.

References

  1. Wolff H. Neue umbelliferen-gattungen aus ostasien. notizblatt des Botanischen Gartens und Mus Zu Berlin-Dahlem. 1925;84:275–80.

    Article  Google Scholar 

  2. Watson MF. Tongoloa. In: Flora of Bhutan Edited by Grierson AJC, Long DG, vol. 2–2.: Royal Botanic Garden Edinburgh and Royal Government of Bhutan; 1999: 478–479.

  3. Pan ZH, Watson MF. Tongoloa. In: Flora of China Edited by Wu ZY, Raven PH, Hong DY, vol. 14: Science Press and Missouri Botanical Garden Press; 2005: 34–37.

  4. Nie ZL, Wen J, Gu ZJ, Boufford DE, Sun H. Polyploidy in the flora of the Hengduan Mountains hotspot, southwestern China. Ann Mo Bot Gard 2005:275–306.

  5. Wolff H. Umbelliferae Asiatiae novae relictae. II. Repertorium Specierum Novarum Regni Vegetabilis BerlinDahle. 1929;27(9–15):179–92.

    Article  Google Scholar 

  6. Liu SL. Two new species of Tongoloa Wolff (Umbelliferae) from China. Acta Phytotaxonomica Sinica. 1989;27(1):68–70.

    Google Scholar 

  7. Farille M, Cauwet-Marc A, Malla SB. Apiaceae himalayenses. III. Candollea; 1985.

  8. Bani B, Karakaya MA, Çeter T. Fruit micromorphological characters of the genus Grammosciadium DC.(Apiaceae) in Turkey. Phytotaxa. 2016;246(2):184–91.

    Article  Google Scholar 

  9. Liu M, Downie SR. The phylogenetic significance of fruit anatomical and micromorphological structures in Chinese Heracleum species and related taxa (Apiaceae). Syst Bot. 2017;42(2):313–25.

    Article  Google Scholar 

  10. Morison R. Plantarum Umbelliferarum distributio nova. Oxford; 1672.

  11. Tseng CC. Anatomical studies of flower and fruit in the Hydrocotyloideae (Umbelliferae). Univ Calif Publications Bot. 1967;42:1–58.

    Google Scholar 

  12. Pu GZ, Liu QX. Micromorphological features of pericarp surface of Tongoloa (Apiaceae) in China and its taxonomic significance. Guihaia. 2008;28(5):576–9.

    Google Scholar 

  13. Wang PL, Fu FT. Pollen morphology of Tongoloa, a endemic genus in China and its taxonomic significance. Acta Bot Yunnanica. 1994;16(4):357–61.

    Google Scholar 

  14. Pimenov M, Mukherjee P, Kljuykov E, Leonov M. Notes on the genera Vicatia DC., Sinodielsia H. Wolff and Tongoloa K. Wolff (Umbelliferae). Feddes Repertorium. 1991;102(5–6):375–84.

    Article  Google Scholar 

  15. Calviño CI, Downie SR. Circumscription and phylogeny of Apiaceae subfamily Saniculoideae based on chloroplast DNA sequences. Mol Phylogenet Evol. 2007;44(1):175–91.

    Article  PubMed  Google Scholar 

  16. Clarkson JJ, Zuntini AR, Maurin O, Downie SR, Plunkett GM, Nicolas AN, Smith JF, Feist MAE, Gutierrez K, Malakasi P. A higher-level nuclear phylogenomic study of the carrot family (Apiaceae). Am J Bot. 2021;108(7):1252–69.

    Article  PubMed  Google Scholar 

  17. Downie SR, Spalik K, Katz-Downie DS, Reduron J-P. Major clades within Apiaceae subfamily Apioideae as inferred by phylogenetic analysis of nrDNA ITS sequences. Plant Divers Evol. 2010;128(1–2):111–36.

    Article  Google Scholar 

  18. Nicolas AN, Plunkett GM. The demise of subfamily Hydrocotyloideae (Apiaceae) and the re-alignment of its genera across the entire order Apiales. Mol Phylogenet Evol. 2009;53(1):134–51.

    Article  CAS  PubMed  Google Scholar 

  19. Plunkett GM, Chandler GT, Lowry IIP, Pinney S, Sprenkle T, Van Wyk B-E, Tilney P. Recent advances in understanding Apiales and a revised classification. South Afr J Bot. 2004;70(3):371–81.

    Article  Google Scholar 

  20. Valiejo-Roman CM, Terentieva EI, Samigullin TH, Pimenov MG. nrDNA ITS sequences and affinities of sino-himalayan Apioideae (Umbelliferae). Taxon. 2002;51(4):685–701.

    Google Scholar 

  21. Zhou J, Peng H, Downie SR, Liu ZW, Gong X. A molecular phylogeny of Chinese Apiaceae subfamily apioideae inferred from nuclear ribosomal DNA internal transcribed spacer sequences. Taxon. 2008;57(2):402–16.

    Google Scholar 

  22. Zhou J, Gong X, Downie SR, Peng H. Towards a more robust molecular phylogeny of Chinese Apiaceae subfamily Apioideae: additional evidence from nrDNA ITS and cpDNA intron (rpl16 and rps16) sequences. Mol Phylogenet Evol. 2009;53(1):56–68.

    Article  CAS  PubMed  Google Scholar 

  23. Xiao YP, Guo XL, Price M, Gou W, Zhou SD, He XJ. New insights into the phylogeny of Sinocarum (Apiaceae, Apioideae) based on morphological and molecular data. PhytoKeys. 2021;175:13.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Wang ZX, Downie SR, Tan JB, Liao CY, Yu Y, He XJ. Molecular phylogenetics of Pimpinella and allied genera (Apiaceae), with emphasis on Chinese native species, inferred from nrDNA ITS and cpDNA intron sequence data. Nord J Bot. 2014;32(5):642–57.

    Article  Google Scholar 

  25. Gou W, Guo XL, Zhou SD, He XJ. Phylogeny and taxonomy of Meeboldia, Sinodielsia and their relatives (Apiaceae: Apioideae) inferred from nrDNA ITS, plastid DNA intron (rpl16 and rps16) sequences and morphological characters. Phytotaxa. 2021;482(2):121–42.

    Article  Google Scholar 

  26. Pimenov MG. Updated checklist of Chinese Umbelliferae: nomenclature, synonymy, typification, distribution. Turczaninowia. 2017;20(2):106–239.

    Article  Google Scholar 

  27. Gui LJ, Jia SB, Guo XL, Price M, Zhou SD, He XJ. Tongoloa tagongensis (Apiaceae), a new species from the Hengduan Mountains, China. Phytotaxa. 2020;461(1):12–20.

    Article  Google Scholar 

  28. Gui LJ, Wen J, Xiao YP, Ren T, He XJ. Tongoloa arguta (Apiaceae), a new species from southwest China. PhytoKeys. 2020;164(2):11–9.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Banasiak Ł, Piwczyński M, Uliński T, Downie SR, Watson MF, Shakya B, Spalik K. Dispersal patterns in space and time: a case study of Apiaceae subfamily Apioideae. J Biogeogr. 2013;40(7):1324–35.

    Article  Google Scholar 

  30. Valiejo-Roman CM, Terentieva EI, Pimenov MG, Kljuykov EV, Samigullin TH, Tilney PM. Broad polyphyly in Pleurospermum s.l. (Umbelliferae-Apioideae) as inferred from nrDNA ITS and chloroplast sequences. Syst Bot. 2012;37(2):573–81.

    Article  Google Scholar 

  31. Wang CB, Ma XG, He XJ. A taxonomic re-assessment in the Chinese Bupleurum (Apiaceae): insights from morphology, nuclear ribosomal internal transcribed spacer, and chloroplast (trnH‐psbA, matK) sequences. J Syst Evol. 2011;49(6):558–89.

    Article  Google Scholar 

  32. Yang J, Feng L, Yue M, He YL, Zhao GF, Li ZH. Species delimitation and interspecific relationships of the endangered herb genus Notopterygium inferred from multilocus variations. Mol Phylogenet Evol. 2019;133:142–51.

    Article  CAS  PubMed  Google Scholar 

  33. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: Guide Methods Appl. 1990;18(1):315–22.

    Google Scholar 

  34. Burland TG. DNASTAR’s lasergene sequence analysis software. Bioinformatics methods and protocols. Springer; 2000. pp. 71–91.

  35. Katoh K, Misawa K, Kuma Ki, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30(14):3059–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. MrBayes 3.2: efficient bayesian phylogenetic inference and model choice across a large Model Space. Syst Biol. 2012;61(3):539–42.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25(9):1189–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wolf M. ITS so much more. Trends Genet. 2015;31(4):175–6.

    Article  CAS  PubMed  Google Scholar 

  40. Gui LJ, Xie DF, Peng C, Ren T, Yu LY, Zhou SD, He XJ. Chloroplast genomes and ribosomal DNA provide insights into divergence and morphological evolution of alpine Tongoloa. J Syst Evol. 2023;00(00):1–13.

    Google Scholar 

  41. Ma Y, Mao X, Wang J, Zhang L, Jiang Y, Geng Y, Ma T, Cai L, Huang S, Hollingsworth P. Pervasive hybridization during evolutionary radiation of Rhododendron Subgenus Hymenanthes in mountains of southwest China. Natl Sci Rev. 2022;9(12):nwac276.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Morales-Briones DF, Liston A, Tank DC. Phylogenomic analyses reveal a deep history of hybridization and polyploidy in the neotropical genus Lachemilla (Rosaceae). New Phytol. 2018;218(4):1668–84.

    Article  PubMed  Google Scholar 

  43. She ML, Pu FD, Pan ZH, Watson MF, Cannon JFM, Ingrid H-S, Kljuykov EV, Phillippe LR, Pimenov MG. Apiaceae. In: Flora of China Edited by Wu ZY, Raven PH, Hong DY, vol. 14: Science Press and Missouri Botanical Garden Press; 2005: 1-205.

  44. Gui LJ, Xiao YP, He XJ. Sinocarum muliense (Apiaceae), a new species from Sichuan, China. Nord J Bot 2021:e03027.

  45. Xiao QY, Hu HY, Tong F, Li MJ, He XJ. Semenovia torilifolia is conspecific with S. malcolmii (Apiaceae) based on morphology and molecular data. Phytotaxa. 2017;321(3):225–37.

    Article  Google Scholar 

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Acknowledgements

We are grateful to Jun Wen and Dengfeng Xie for their help in the collection of plant materials, and Bingyan Chen for her drawing.

Funding

This work was supported by the National Natural Science Foundation of China (Grant Nos. 31872647, 32070221), National Specimen Information Infrastructure, Educational Specimen Sub-Platform (Grant No. 2005DKA21403-JK), the fourth national survey of traditional Chinese medicine resources (Grant No. 2019PC002).

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Authors and Affiliations

  1. Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China

    Lingjian Gui, Chang Peng, Lijia Liu, Songdong Zhou & Xingjin He

  2. Guangxi Key Laboratory of High-Quality Formation and Utilization of Dao-di Herbs, Guangxi Botanical Garden of Medicinal Plants, Nanning, 530023, China

    Lingjian Gui, Shugen Wei & Zhigang Yan

  3. Guangxi Botanical Garden of Medicinal Plants, Nanning, 530023, China

    Liying Yu & Xiaomei Zhang

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  1. Lingjian Gui
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  2. Chang Peng
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Contributions

LG and XH conceived and designed the work. CP provided some field samples. LG, LY, and LL performed bioinformatics analyses of the sequence data. LG drafted the manuscript. SW, ZY and SZ revised the manuscript. XZ revised the English text. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Xingjin He.

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The authors declare no competing interests.

Ethics and consent to participate

Our plant materials were collected from the wild and are not listed among the nationally protected plant species. The collection site is not a designated natural reserve. Moreover, the sampling process adhered strictly to the national guidelines.

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Gui, L., Peng, C., Yu, L. et al. Morphology and ITS sequences provide insights into the phylogeny of Tongoloa (Apiaceae) from China. BMC Ecol Evo 24, 103 (2024). https://doi.org/10.1186/s12862-024-02292-5

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BMC Ecology and Evolution

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