Nanocnide simingshanensis (Urticaceae), a New Species from Zhejiang, Eastern China

Abstract

In this paper, Nanocnide simingshanensis is described as a new species based on morphological and molecular analyses, and its taxonomic relationships are discussed. The new species is currently known only from the Siming Mountain region of Yuyao County and the Yaolin National Forest Park of Tonglu County, both in Zhejiang Province, eastern China, where it grows in shrubs at an elevation of about 650 m under deciduous broad-leaved forests and at a limestone cave entrance. Morphological analysis strongly supports that N. simingshanensis should be classified into the genus Nanocnide. This species is distinguished from the related species N. pilosa and N. lobata by having long straight hirsute hairs perpendicular to the stem, petioles and peduncles, longer petioles, larger leaf blades, and female inflorescences developing on branchlets. It differs from N. zhejiangensis and N. japonica by having glomerulate male inflorescences shorter than leaves (vs. cymose inflorescences longer than leaves). Nuclear ribosomal DNA internal transcribed spacer (ITS) phylogenetic analysis demonstrates that N. pilosa and N. lobata are the closest extant relatives to the new species.

1. Introduction

The family Urticaceae comprises approximately 2625 species in 53 genera distributed worldwide, characterized by hairs which are often stinging, as well as unisexual flowers and achene fruits [1]. The genus Nanocnide Blume (1856: 154) [2] belongs to the tribe Urticeae within Urticaceae and is distributed in warm-temperate to tropical regions of east and southeast Asia. The genus comprises four species: Nanocnide japonica Blume [2], Nanocnide lobata Weddell [3], N. pilosa Migo [4], and N. zhejiangensis X.F. Jin & Y. F. Lu [5]. While N. closii H. Léveillé & Vaniot [6] has been established, it does not belong to Nanocnide. Qiu & Gilbert (2008) stated that N. closii is a synonym of Acalypha supera Forsskål (1775: 162) in Euphorbiaceae [7]. Another species, N. dichotoma Chien [8], originally described as being from mainland China, is synonymized under N. japonica [9,10].

The genus Nanocnide is characterized by the following: plants armed with stinging hairs; female flowers without staminodes, leaves alternate, stipules interpetiolar; achene straight, sesile; stigma penicillate-capitate [9]. The four Nanocnide species are distinguished by staminate peduncle length, stem pubescence, and leaf characteristics. N. japonica and N. zhejiangensis both have peduncles exceeding subapical leaf length and glabrous or appressed-hairy stems, but differ in that N. japonica has strigose peduncles and perianths while N. zhejiangensis has glabrous ones. N. lobata and N. pilosa have shorter peduncles not exceeding leaf length and depressed stem hairs, but N. lobata has very short peduncles (<1 mm) and leaves with up to 5 teeth, whereas N. pilosa has peduncles equaling leaf length and leaves with 11–15 teeth [11].

Previous molecular phylogenetic studies on Urticaceae have included limited sampling of Nanocnide species. Wu et al. (2013) [12] first included multiple Nanocnide samples in a family-wide phylogenetic analysis using nuclear ribosomal ITS and chloroplast regions, recovering a strongly supported monophyletic Nanocnide. Kim et al. (2015) [13] included four Nanocnide samples in their study of tribe Urticeae and found Nanocnide to be sister to Laportea cuspidata. More recently, Aoki et al. (2023) [11] conducted the first comprehensive molecular phylogenetic study of the genus using ITS sequences and MIG-seq from all four recognized species, resolving the relationships among species.

During the preparation of the latest edition of the Flora of Zhejiang, botanical expeditions were conducted in Zhejiang Province and neighboring regions in southeast China, resulting in the discovery of an unidentified Nanocnide species by the authors in May 2019. These plants grow in shrubs under deciduous broad-leaved forests at an elevation of about 650 m in the Siming Mountain region, and at a limestone cave entrance in the Yaolin National Forest Park.

After in-depth and careful studies, we were unable to assign these plants to any species described so far. Their populations exhibit a unique combination of morphological features, including erect stems with long straight hirsute hairs perpendicular to the surface (unlike the depressed hairs of N. pilosa and N. lobata or the appressed hairs of N. japonica), large leaf blades (2.5–5 × 2.5–5 cm), and glomerulate male inflorescences with peduncles 0.5–4 cm long, which do not match any of the four recognized species. In order to characterize this putatively new species, we examined its morphological features and performed molecular phylogenetic analyses in order to reconstruct its relationships with other related species, such as N. pilosa, N. lobata, N. japonica, and N. zhejiangensis. As a result, the distinguished geographical distribution, morphological characteristics, and phylogenetic status of the populations allow us to refer to them as a novel species, named Nanocnide simingshanensis.

2. Materials and Methods

2.1. Field Work and Sampling

Collection activities were conducted at two localities within Zhejiang Province: the Siming Mountain region in Yuyao County (geographical position: 29°45′47″ N, 121°2′1″ E, at 650 m above sea level); and the Yaolin National Forest Park situated in Tonglu County (geographical position: 29°56′49.6″ N, 119°32′39.3″ E). Reference materials were archived at the Zhejiang Museum of Natural History’s herbarium facility (ZM). Photographic documentation of morphological features was performed using Nikon (Nikon Corporation, Shinagawa-ku, Tokyo, Japan) digital imaging equipment from Japan. Comparative morphological assessments were undertaken with congeneric taxa including N. japonica, N. zhejiangensis, N. pilosa, and N. lobata. Morphological reference data were derived from multiple sources: Qiu & Gilber (2008) [7], Yateishi (2006) [10], and Jin et al. (2019) [5], along with our direct observations; these are compiled in

Table 1. Main differences among Nanocnide simingshanensis, Nanocnide pilosa, Nanocnide lobata, Nanocnide japonica, and Nanocnide zhejiangensis. Based on Qiu & Gilbert (2008) * [7], Yateishi (2006) ^$ [10], Jin et al. (2019) ^@ [5], and the authors’ own measurements at ZM ^#.

Characteristics	N. simingshanensis #	N. pilosa *,$	N. lobata $	N. japonica *,$	N. zhejiangensis @
Stems	Erect, unfolded hirsute	Diffuse, retrorse hirsute	Diffuse, retrorse hirsute	Erect, antrorse hirsute	Erect, glabrous
Leaf blade (cm)	Broadly ovate or flabellate, 2.5–5 × 2.5–5, margins unequally 5–9-crenate per side	Broadly ovate, triangular-ovate, or subflabellate, 1.5–2 × 1.3–1.8, margins unequally 4–5(-7)-crenate per side	Flabellate to ovate, 0.6–1.5 × 0.6–1.5, margins 1–2-lobes per side, rarely unlobed	Blade triangular-ovate or rhombic-ovate 1.5–4 × 1.3–4, margins deeply 4–7-crenate-dentate per side	Triangular-ovate, broadly ovate or flabellate, 0.8–4.0 × 1.0–3.5, margins deeply 3–9-crenate per side
Petioles (cm)	1.5–5.5	0.8–1.8	0.3–1	1–5	0.7–4.5
Male inflorescences	Glomerules, born in distal axils, shorter than leaves, peduncles 0.5–4 cm	Glomerules, born in distal axils, shorter than leaves, peduncles 0.5–1.2 cm	Glomerules, born in lower 1 or 2 axils, shorter pedunculate	Cymose, born in distal axils, longer than leaves, long pedunculate	Cymose, born in distal axils, longer than leaves, peduncles 3.6–9.0 cm
Female inflorescences	Glomerules, born in distal axils or in proximal leafless nodes or slender flower branches of middle axil, peduncles 0.1–2.2 cm	Glomerules, born in distal axils or in proximal leafless nodes, or sometimes distal part of main, peduncles short or nearly absent	Glomerules, born in 2 or 3 uppermost axils, sessile	Glomerules, born in distal axils, shortly pedunculate	Glomerules, born in distal axils, peduncles 0.3–2.5 cm
Male flowers	5-merous, in bud 2–3 mm in diameter	5-merous, in bud 1.2–1.5 mm in diameter	4(5)-merous, in bud 1 mm in diameter	5-merous, in bud 2–3 mm in diameter	5-merous, in bud ca. 2 mm in diameter

.

2.2. Morphological Analysis

Morphological characteristics of N. simingshanensis were examined using fresh specimens collected from the two known populations. A total of 20 individual plants were examined for morphological measurements, while 4 voucher specimens (including the holotype and paratypes listed in the taxonomic treatment section) were collected and deposited at ZM. All morphological measurements were taken from fresh material in the field or from freshly collected specimens in the laboratory. No obvious morphological differences were observed between the two populations. The following morphological characteristics were measured: stem height; leaf blade dimensions; petiole length; number of marginal teeth per side; stipule size; male inflorescence peduncle length; female inflorescence peduncle length and diameter; male flower diameter; and achene length. Larger structures (stem height, leaf dimensions, petiole length) were measured using a ruler, while smaller structures (stipule size, flower diameter, achene length) were measured using digital calipers. Indumentum type was observed using a stereomicroscope. Photographic documentation was performed using a digital camera (Nikon, Japan). Comparison between fresh and pressed specimens showed no obvious differences in key diagnostic characteristics. For comparison with other Nanocnide species, morphological data for N. japonica, N. lobata, N. pilosa, and N. zhejiangensis were obtained from sources published in the literature: Qiu & Gilber (2008) [7], Yateishi (2006) [10], and Jin et al. (2019) [5]. These literature-based data were compiled and compared with our observations of N. simingshanensis listed in Table 1 above.

2.3. DNA Extraction and Sequencing

Fresh foliar material from specimens discovered at Simingshan Geopark and Yaolin National Forest Park served as the source for genomic DNA extraction, which was accomplished through application of a commercial plant DNA isolation system (Tiangen Biotech, Beijing, China) per provided protocols. Amplification of the ITS locus employed primers ITS-A (forward: 5′-GGAAGGAGAAGTCGTAACAAGG-3′) and ITS-4 (reverse: 5′-TCCTCCGCTTATTGATATGC-3′) based on references [14] and [15]. Thermocycling parameters were as follows: initial heating at 94 °C for 5 min; 35 amplification rounds each consisting of 98 °C for 20 s (denaturation), 58 °C for 30 s (primer binding), and 68 °C for 45 s (elongation); 7 min at 68 °C for final extension. The KODFX enzyme system (KFX-101, TOYOBO, Osaka, Japan) facilitated the amplification process. Amplified fragments were resolved through 1.5% agarose electrophoresis in TAE buffer prior to commercial sequencing at the Beijing Genomics Institute (Shenzhen, China). Resulting sequence data have been accessioned in the NCBI GenBank repository under the identifiers OR852698, OR852699, and OR852700.

2.4. Data Analysis

To ascertain the systematic classification of the newly discovered species, we performed molecular phylogenetic analysis using DNA sequences of all four recognized Nanocnide species (N. japonica, N. lobata, N. pilosa, and N. zhejiangensis), as well as Laportea cuspidata (Wedd.) Friis, Laportea canadensis (L.) Wedd., and Laportea lanceolata (Gagnep.) C.J. Chen as the outgroup taxa. Following the comprehensive sampling approach established by Aoki et al. (2023) [11], we obtained a total of 64 ITS sequences from the NCBI GenBank database to construct a robust phylogenetic tree. Completely identical sequences were excluded from the phylogenetic analysis to avoid unnecessarily reducing the effective space of tree topology exploration. This sampling strategy ensured that the analysis was directly comparable to the most recent and comprehensive phylogenetic study of the genus [11], which studied 44 genera, thus providing a solid framework for determining the evolutionary relationships of this new species within Nanocnide. Assembly and refinement of forward and reverse sequence reads was accomplished via the SeqMan program [16]. Multiple sequence alignment employed MAFFT software version 7.505 [17] with automatic parameter selection and standard alignment protocols. Regions of questionable alignment quality were identified and excluded using Gblocks version 0.91b [18] with the following parameter settings: minimum number of sequences for a conserved/flank position (23/23), maximum number of contiguous non-conserved positions (8), minimum length of a block (10), and allowed gap positions (with half). Substitution model selection was performed using ModelFinder version 2.2.0 [19], utilizing the Bayesian Information Criterion for evaluation. The TIM3e+G4 model emerged as optimal based on BIC scores. Phylogenetic reconstruction through maximum likelihood methodology was executed in IQ-TREE version 2.2.0 [20] under the TIM3e+G4 model for 50,000 ultrafast bootstraps (UFB) [21], using the approximate Bayes test (aBayes) [22] as well as the Shimodaira–Hasegawa-like approximate likelihood-ratio test (SH-aLRT) [23].

3. Results

3.1. Morphological Analysis

This novel species, designated as N. simingshanensis, exhibits a distinctive suite of morphological features that separate it from related taxa. Table 1 summarizes the principal diagnostic characteristics distinguishing this new species from the four previously recognized taxa, while Figure 1 illustrates the morphological features of N. simingshanensis. Most notably, it differs from N. pilosa in the following: erect stems (vs. diffuse); long straight hirsute perpendicular to stems, petioles, and peduncles (vs. retrorse hirsute); petioles 1.5–5.5 cm long (vs. 0.8–1.8 cm); leaf blades 2.5–5 × 2.5–5 cm (vs. 1.5–2 × 1.3–1.8 cm); margins unequally 5–9-crenate per side (vs. 4–5(–7)-crenate per side); female inflorescences in distal axils or branchlets (vs. distal axils or in proximal leafless nodes). It differs from N. lobata in the following: erect stems (vs. soft, decumbent or ascending); long straight hirsute perpendicular to stems, petioles, and peduncles (vs. retrorse hirsute); petioles 1.5–5.5 cm long (vs. 0.3–1 cm); leaf blades 2.5–5 × 2.5–5 cm (vs. 0.6–1.5 × 0.6–1.5 cm); margins unequally 5–9-crenate per side (vs. margins1–2-lobes per side, rarely unlobed); female inflorescences in distal axils or branchlets (vs. uppermost axils); male flowers 5-merous (vs. 4(5)-merous). It differs from N. japonica in the following: long straight hirsute perpendicular to stems (vs. antrorse hirsute); glomerulate male inflorescences shorter than leaves (vs. cymose, longer than leaves). It differs from N. zhejiangensis in the following: long straight hirsute perpendicular to stems (vs. glabrous); glomerulate male inflorescences shorter than leaves (vs. cymose, longer than leaves).

3.2. Phylogenetic Analysis

Our ITS phylogenetic tree presents a topology based on those of Aoki et al. (2023) [11] (Figure 2). The phylogenetic analysis reveals that all Nanocnide species form a well-supported monophyletic group (100/1.00/100 for UFB/aBayes/SH-aLRT). Within this genus, N. simingshanensis forms a clade with N. lobata and N. pilosa (99.8/1/100), with N. simingshanensis being sister to N. lobata (90.7/0.973/91). This three-species clade is sister to the clade containing N. japonica and N. zhejiangensis (100/1/100). In addition, the support value for the monophyly of the clade of N. simingshanensis is (78.2/0.962/82). These results indicate strong support for the placement of N. simingshanensis as a distinct species within the genus.

4. Discussion

The morphological analysis reveals that while N. simingshanensis, N. pilosa, N. lobata, N. japonica, and N. zhejiangensis share some similar traits as members of the genus Nanocnide, they exhibit distinct characteristics that warrant the recognition of N. simingshanensis as a new species. Nanocnide simingshanensis is mainly distinguished from its closest relatives by several key morphological features such as erect stems with long straight hirsute perpendicular to the surface. The most diagnostic characteristic is the unique indumentum type—long straight hirsute hairs perpendicular to the surface, which differ from the depressed hairs of N. pilosa and N. lobata and the appressed or glabrous condition exhibited in N. japonica and N. zhejiangensis. Additionally, N. simingshanensis has significantly longer petioles (1.5–5.5 cm) and larger leaf blades (2.5–5 × 2.5–5 cm) compared to N. pilosa (petioles up to 1 cm, blades up to 2.3 × 2.3 cm) and N. lobata (petioles up to 0.5 cm, blades up to 1.5 × 1.5 cm). Interestingly, our morphological comparison shows that N. simingshanensis shares certain features with N. japonica and N. zhejiangensis, particularly the erect stem habit and large male flowers. However, the indumentum type, inflorescence morphology, and phylogenetic results clearly separate the new species from these two taxa.

Our morphological measurements were taken exclusively from fresh material, ensuring the accuracy of our observations. Comparison between fresh and pressed specimens revealed no obvious differences in the key diagnostic characteristics. This consistency suggests that the diagnostic features of N. simingshanensis are stable and reliable for taxonomic identification, regardless of preservation method.

Furthermore, the molecular findings unambiguously support the distinct phylogenetic position of N. simingshanensis. Our ITS phylogenetic analysis, following the sampling framework established by Aoki et al. (2023) [11], demonstrates that N. simingshanensis forms a well-supported clade as sister to N. lobata (90.7/0.973/91 for UFB/aBayes/SH-aLRT values), and the support value for the monophyly of the clade of N. simingshanensis is (78.2/0.962/82). Comparison with the phylogenetic results of Aoki et al. (2023) [11] reveals that our inclusion of N. simingshanensis does not significantly alter the overall topology of the Nanocnide phylogeny. The relationships among the previously recognized species remain consistent, with N. japonica and N. zhejiangensis forming one clade, and N. lobata and N. pilosa forming another. However, our results provide improved resolution within the N. lobata–N. pilosa clade with the addition of N. simingshanensis. The clade containing these three species (N. simingshanensis, N. lobata, and N. pilosa) appears to share the synapomorphy of non-cymose male inflorescences and peduncles that do not exceed the uppermost leaves, distinguishing them from the N. japonica–N. zhejiangensis clade.

As Aoki et al. (2023) [11] demonstrated, N. lobata and N. pilosa are sister taxa sharing depressed hairs as a synapomorphy, but our new species shows a different hair type despite being sister to this clade. This suggests that hair type evolution in Nanocnide may be more labile than previously thought.

Our phylogenetic results expand the known diversity of Nanocnide to five species and provide new insights into the genus’s evolutionary history. The sister relationship between N. simingshanensis and N. lobata is particularly noteworthy given their contrasting morphologies—while phylogenetically close, they have diverged substantially in stem architecture, indumentum type, leaf dimensions, and reproductive structures.

In conclusion, the integration of morphological and molecular evidence strongly supports the recognition of N. simingshanensis as the fifth species in the genus Nanocnide.

Taxonomic Treatment

Nanocnide simingshanensis Y.L. Xu & B.Y. Ding, sp. nov. (Figure 1, Figure 3 and Figure 4).

Type: CHINA. Zhejiang Province, Yuyao County, Siming Mountains, Siming Mountain Geopark, in shrubs under the deciduous broad-leaved forests, 29°45′47″ N, 121°2′1″ E, elevation 649.4 m, 12 May 2020, Yue-Liang Xu, Xu1990 (holotype: ZM barcode ZM0067388, Figure 3).

Description: Herbs perennial. Stems erect, basally branched, 10–49 cm tall, straight hirsute perpendicular to stem. Branchlets in axils, with rudimentary bract-like small leaves or none. Leaves alternate, membranous; distal blades flabellate to broadly ovate, 2.5–5 cm long and wide, obtuse or abruptly acuminate at apex, adaxially deep green, sparsely hirsute, abaxially light green, slightly lustrous, densely hirsute, 5-veined from blade base, cystoliths conspicuous on both surface, botuliform, margins unequally 5–9-crenate per side, teeth triangular-ovate, with obtuse or acute tip, 2–6 mm long, base truncate or shallowly cordate, proximal leave small, flabellate, obtuse or rounded at apex; petioles 1.5–5.5 cm long, straight hirsute perpendicular to surface; stipules ovate to elliptic, 2–5 mm long, 1.5–2 mm wide, ciliate. Male inflorescences in distal axils, glomerules, peduncles 0.5–4 cm long, extensively hirsute; Female inflorescences glomerules, born in distal axils or branchlets, 3–8 mm in diameter, peduncles 0.1–2.2 cm long. Staminate flowers greenish, 2–3 mm in diameter., perianth lobes 5, ovate, ca. 1.5 mm long, abaxially distal protrusion, setulose in margin; stamens 5, ca. 2 mm long; rudimentary ovary broadly obovoid, ca. 0.5 mm long, transparent. Pistillate flowers green, perianth lobes 4, unequal, outer 2 larger, 2–2.3 mm long, boat-shaped, inner 2 smaller, ovate-lanceolate, 1.5–2 mm long, densely covered with puberulent and short stinging hair along costa and margin; stigma penicillate. Achene ovoid, compressed, pale yellow, ca. 1.3–1.5 mm long, almost smooth.

Distribution and habitat: The new species is only known from the Siming Mountain region of Yuyao County and the Yaolin National Forest Park of Tonglu County, both in Zhejiang Province (Figure 5). It grows in shrubs under deciduous broad-leaved forests at an elevation of about 650 m, and at a limestone cave entrance. Based on our field surveys, the Siming Mountain population covers approximately 1000 square meters, with about 450 individuals, while the Tonglu population covers about 100 square meters, with approximately 75 individuals.

Phenology: Flowering from April to May, fruiting in June.

Etymology: The specific epithet ‘simingshanensis’ refers to the type locality of the new species.

Similar species: The new species is closely related to N. pilosa and N. lobata. It is mainly distinguished from them by the morphology of long and straight hirsute perpendicular to stem, petiole and peduncle, longer petioles, larger leaf blades, and more serrated margins. The distinguishing characteristics of the new species and the four relative species are listed in detail in Table 1.

Additional specimen examined (Paratypes): CHINA. Zhejiang Province, Yuyao County, Siming mountain, Siming Mountain Geopark, beside the ditch, 29°45′47″ N, 121°2′1″ E, elevation 649.4 m, 28 May 2019, Yue-Liang Xu & Bing-Yang Ding, Xu1315 (ZM0067433); ibidem, 9 June 2023, Yue-Liang Xu, Xu2870 (ZM0067434); Tonglu County, Yaolin National Forest Park, limestone cave entrance, 29°56′49.6″ N, 119°32′39.3″ E, 13 May 2020, Yue-Liang Xu, Xu1994 (ZM0067435).