Based on observations of living plants of Hydrocotyle calcicola in the field, together with examination of herbarium specimens and descriptions of both H. calcicola and H. chiangdaoensis (including type material), we demonstrated that H. calcicola is a synonym of H. chiangdaoensis. The species was previously compared with H. sibthorpioides; our phylogenetic analysis revealed that H. chiangdaoensis and H. sibthorpioides belong to different lineages, the former being closely related to the larger-leaved clade.

Hydrocotyle, synonym, taxonomy, Yunnan

The genus Hydrocotyle Tourn. ex L. contains approximately 180 species (Plants of the World Online, http://www.plantsoftheworldonline.org/). The native range of this genus is cosmopolitan, with Australia, South America, and China as three diversity distribution centers. Hydrocotyle was formerly a member of the family Apiaceae, but was later transferred to Araliaceae based on molecular phylogenetic studies (Chandler and Plunkett 2004; Plunkett et al. 2004; Nicolas and Plunkett 2009). Subsequently, Wen et al. (2024) highlighted the independent status of this genus within Apiales based on the analysis of chloroplast genome data. All these analyses have suggested that the genus Hydrocotyle is key to further study on the evolution of Apiales.

We have conducted a comprehensive study of Hydrocotyle in China and found that there are 16 species, 2 varieties, and 2 subspecies in the country, among which Yunnan Province is most species-rich (Sheh et al. 2005; Pimenov 2017). Some species occurring in Yunnan were also found in adjacent areas, such as H. sibthorpioides Lam., H. javanica Thunb., and H. siamica Craib, which were also found in Myanmar and Thailand (Watson and Smith 2004; Pimenov 2017). Studies on Myanmar’s plant diversity have been conducted in recent years. A newly recorded species of Hydrocotyle, H. chiangdaoensis Murata, has been reported by Kang et al. (2018), which was formerly known to be endemic to Thailand. However, the photo of this species was very similar to H. calcicola Y.H.Li, which was endemic to Xishuangbanna, Yunnan Province, China (Sheh et al. 2005). Based on this, we conducted a comparative study between the two species.

Hydrocotyle chiangdaoensis was recognized as a new species by G. Murata in 1973 and was described based on Murata G. et al. T-15040 (holotype KYO 00028951, Fig. 1A; isotypes KYO 00028952, AUU, TI 00083127, and L 0008361, Fig. 2A–D), collected in Doi Chiang Dao, Northern Chiang Mai, Thailand (Murata 1973). In the protologue, Murata emphasized four distinguishing features of H. chiangdaoensis – palmate-dissected stipules, annual habit, not emitting any roots from the nodes, and limestone area endemic. Specifically, the annual habit was speculated because the stem and branches have terminal inflorescences (Murata 1973).

Figure 1. 

Holotype sheets of Hydrocotyle chiangdaoensis (A) and H. calcicola (B). The arrows indicate the roots of the species.

Figure 2. 

Isotype sheets of Hydrocotyle chiangdaoensis. The arrows indicate the roots of the species.

A collection of this species from Doi Chiang Dao (M. Tagawa & K. Iwatsuki T-4389, L.2583137) was reported by Hiroe as H. siamensis H. Wolff (1921) (Hiroe 1967). The latter name is a synonym of H. siamica Craib (Craib 1911), based on the same gatherings (Hosseus no. 192; E00000016, P00757659, L.2595136, and M0168575). Hydrocotyle siamica was described with long peduncled umbels (Umbellae axillarea, solitariae, pedunculo ad 10.5 cm. longo brunneo-puberulo suffultae) and large leaves (Folia pentagona, vix vel fere ad medium lobata, ad 9 cm. longa et 10 cm). The diagnostic characters of the specimen L.2583137 (subsequently identified as H. chiangdaoensis) are quite different from H. siamica, with short peduncled umbels and small leaves.

The oldest specimen of this species we found was collected by Kerr in 1922 (Kerr 6530, K005513556) from Doi Chiang Dao in Northern Chiang Mai, Thailand. For nearly 100 years since then, this species has only been seen in Doi Chiang Dao. Kang et al. (2018) made a new distribution record of this species in Ywangan Township, Southern Shan State, Myanmar, when they conducted joint floristic surveys of this area in 2017. Since then, it has not been seen anywhere else.

Hydrocotyle calcicola Y.H.Li was described on the basis of G.D.Tao 13671 (holotype HITBC0037397, Fig. 1B; and isotype KUN0467704, Fig. 3) from Jiluo Shan (according to textual research, it should be Jinuo Mountain), Jinghong City, Yunnan, China (Li and Zhang 1989). In the protologue, Li and Zhang highlighted the features that set this species apart from H. sibthorpioides Lam.: membranous leaves conspicuously covered with sparse spinous hairs, umbels of cymes usually terminal with opposite leaves, short and slender peduncles, umbels of 2–5 flowers, and mature fruits not purplish-spotted. Liou (1997) treated H. calcicola as a variety of H. sibthorpioides due to the weak difference only focused on flowers and fruit. This treatment has not been accepted by Flora of China (Sheh et al. 2005), because the habitat of H. calcicola was very special. It was noted in the protologue that the habitat of H. calcicola was also limestone, and the stipules were irregular, i.e. very similar to H. chiangdaoensis. Upon comparing the original descriptions of the two species, we found that many features of both were similar, with only minor differences in the descriptions of their life forms, roots, and fruit. Hydrocotyle chiangdaoensis was described as “not emitting any roots from the nodes”, with “annual habit” and “fruit broadly ovate, papillose-setulous, truncate or subcordate at the base”; but H. calcicola was described as “perennial herbs”, “rooting at nodes”, and “fruits subcordate, smooth on the outside, ribs conspicuously convex”. However, the annual habit of H. chiangdaoensis was speculated on the basis that the stem and branches have terminal inflorescences, which were also a feature of H. calcicola (Murata 1973; Li and Zhang 1989). We suggested that H. calcicola might be a synonym for H. chiangdaoensis based on their similar traits and the order of publication years.

Figure 3. 

Isotype sheet of Hydrocotyle calcicola. The arrows indicate fruit (a) and root (b) of the species.

This study aimed to determine the identity of H. calcicola and elucidate the distribution and morphological variation of H. chiangdaoensis. Our research was conducted mainly on observations of herbarium specimens (including the type material) and living plants in the field. The phylogenetic tree was reconstructed to determine the phylogenetic placement of H. chiangdaoensis.

The type materials of Hydrocotyle chiangdaoensis (Figs 1A, 2) are small erect herbs, leaves cordate-orbiculate, gradually smaller above, slightly hairy above towards the veins, glabrous beneath, palmately 7-lobed, lobes usually 3-toothed, teeth obtuse, blades (0.6)1–2(2.5) cm long, (0.7)1.5–2.5(3) cm wide; petioles (0.7)1.5–3.5(5) cm long; stipules membranous flabellate-orbiculate, 1–5 mm wide, palmately dissected; umbels terminal or axillary of the branch or opposite the leaves of the branches, terminal cymose umbels 2–3, thin peduncles 0.5–6 mm long, umbel 2–4 flowers, pedicels very short, membranous bracts minute ovate-lanceolate; petals ovate-lanceolate; fruit broadly ovate, 1–1.2 mm long, papillose-setulous, base truncate or subcordate, stylopodia shortly conoid, short styles reflexed at the end. The root system of this species was described as “not emitting any roots from the nodes” in the protologue. However, we found that some individuals have roots growing from two or three nodes at the base (Fig. 2A–C).

Type specimens of H. calcicola (Figs 1B, 3) have shown that this species was rooting only at nodes of the basal stem and never elsewhere, the same as H. chiangdaoensis. The holotype of H. calcicola has membranous leaves subrounded or cordate, conspicuously covered with sparse spiny hairs above, glabrous beneath, cordate at the base, 5–7-lobed at the apex, lobes broadly obovate, crenate at the margin, 6–8 palmate nerves, 0.5–1.5 cm long, 0.7–2.5 cm wide; petiole 0.7–3 cm long, glabrous; stipules small, kidney-shaped, thinly branched, irregularly; cymose umbels usually terminal with opposite leaves, umbels 2–3, slender peduncles not quite equilong, laterals about 1 cm long, middle 1–2 mm long, umbel 2–5 flowers, flowers sessile, bracts ovate-lanceolate, about 1 mm long, membranous; petal ovate, about 0.5 mm long, white, filaments equal to or slightly shorter than the petal, anthers ovate; style about 0.2 mm long; fruits subcordate, 1–1.3 mm long, 0.8–1.2 mm wide, flat on both sides, smooth or with papillose setae on the outside, ribs conspicuously convex (Fig. 1B). Fruits with papillose setae were also discovered in the isotype (Fig. 3). These morphological features were corroborated through field observations of living plants in Jinuo Mountain, Jinghong City, Yunnan (Fig. 4), the type locality of H. calcicola. At the same time, after observing live plants in the type locality, we found that the young fruits are sometimes smooth, but most fruits have papillose setae in the furrow. The additional observations of living plants in Pu’er show that most of the traits of this population were consistent with the description of H. calcicola, including habitat, roots, leaves, stipules, terminal umbels, and fruits (Fig. 5). Our analysis added some additional information about this species. Two types of stipules in this population were observed, one irregularly divided and one palmately dissected (Fig. 5B). The latter form is the same as that of H. chiangdaoensis. Two petal numbers (4 and 5) have been observed in this population, marking the first time such numbers have been reported in Hydrocotyle.

Figure 4. 

Hydrocotyle chiangdaoensis in the wild (China, Yunnan, Jinghong, Mountain Jinuo, the type locality of H. calcicola) A habitat B habit, with the arrows indicating stipule (a) and leaf epidermal spines (b) C the terminal inflorescence, with the arrows indicating cymose umbels (c) and fruit (d). Photographed by Ren-Bin Zhu.

Figure 5. 

Hydrocotyle chiangdaoensis in the wild (China, Yunnan, Pu’er) A plant, with the arrow indicating root B stipules C, D flowers E fruit F the terminal inflorescence G leaf H habitat. Photographed by Jun Wen.

In our examination of the type specimens (Figs 1B, 3) and field observation (Figs 4, 5) of H. calcicola, particularly concerning critical diagnostic characters, such as roots, leaves, stipules, inflorescence, and fruits, no significant differences were observed when compared to H. chiangdaoensis. In conclusion, H. calcicola is morphologically indistinguishable from H. chiangdaoensis. Therefore, we propose to synonymise H. calcicola with H. chiangdaoensis.

As previously noted, H. calcicola was compared with H. sibthorpioides by its discoverers (Li and Zhang 1989) and subsequently treated as a variety of the latter (Liou 1997). When we examined the specimens, we also found that the specimens of H. calcicola were often misidentified as H. sibthorpioides by various researchers. Thus, we here provide a detailed morphological comparison between the two species in Table 1, Fig. 6. The results show that there are clear morphological distinctions (especially in roots, inflorescences, and mericarps) between the two species. The phylogenetic analysis based on the chloroplast genome was implemented to clarify the relationship between these two species. Phylogenetic trees reconstructed by BI and ML methods both recovered a stable topology within the genus Hydrocotyle with strong support. Within the genus, H. chiangdaoensis and H. sibthorpioides were located in two different branches (Fig. 7). Two accessions of H. chiangdaoensis were gathered together, forming a sister branch of the larger-leaved clade (Clade I, Wen et al. 2024). The results of the morphological and phylogenetic analyses confirm that H. chiangdaoensis and H. sibthorpioides are different taxonomic entities.

Figure 6. 

Fruits of Hydrocotyle sibthorpioides (China, Hunan, Zhangjiajie, NAS00638796) and H. chiangdaoensis (China, Yunnan, Pu’er) A, B fruits of H. sibthorpioides C, D fruits of H. chiangdaoensis.

Figure 7. 

Phylogenetic tree inferred from 14 whole chloroplast genome sequences. Support values marked above the branches follow the order Bayesian inference (PP, posterior probability) / maximum likelihood (BS, bootstrap support), * represent the best support (100%).

We are grateful to the reviewer Prof. Yan Yu and the editor Alexander Sennikov for their valuable comments on the manuscript. We thank Qi-Zhi Wang, Xu-Dong Ma, Xiao-Fang Chu, Shi-Kui Peng, Hong-Bo Ding, Jing Li, Qun Liu, and Prof. Yun-Hong Tan for their help during our fieldwork and the curators of AAU, HITBC, K, KUN, KYO, L, NAS, P, PE, and TI for allowing us to use their scanned images of specimens and for research facilities. Thanks are due to Dr. Wei Zhou for his kind help with the suggestions for improving the manuscript.