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Article

Ilex danxiaensis (Aquifoliaceae), a Distinct New Tree Species Endemic to Danxia Mountain in Guangdong Province, China, Based on Molecular and Morphological Evidence

by
Chenxue Lin
1,
Wanyi Zhao
2,
Zaixiong Chen
3,
Qiang Fan
2,* and
Kewang Xu
1,*
1
Key Laboratory of National Forestry and Grassland Administration on Subtropical Forest Biodiversity Conservation, Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China
2
State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China
3
Administrative Commission of Danxiashan National Park, Shaoguan 512300, China
*
Authors to whom correspondence should be addressed.
Forests 2023, 14(3), 583; https://doi.org/10.3390/f14030583
Submission received: 17 February 2023 / Revised: 8 March 2023 / Accepted: 13 March 2023 / Published: 15 March 2023
(This article belongs to the Special Issue Advances in Plant Science Research: Celebrating Nanjing Forestry University’s 120th Anniversary)
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Abstract

:
Many holly species are dominant species in evergreen broad-leaved forests, and they play an important role in the formation and maintenance of forest ecosystems in tropical and subtropical regions. Here, a new holly species (Ilex danxiaensis K. W. Xu & Q. Fan) of Aquifoliaceae was discovered from Guangdong, China, when conducting biodiversity investigations in the Danxia landform. We inferred the phylogenetic position of the new species based on sequences from three nuclear markers, namely internal transcribed spacer (ITS), external transcribed spacer (ETS), and chloroplast glutamine synthetase gene (nepGS). The results demonstrated a close relationship among the new species and members of I. sect. Lioprinos. The inferred phylogenetic position of the new species is also supported by morphological evidence that all of these species are characterized by having axillary cymes inflorescences and abaxially 1-sulcate pyrenes. However, the new species can be distinguished from others by having puberulent branchlets, petioles, peduncles, and pedicel; (1–)2–3-flowered cymes of male inflorescences; 1-flowered cymes of female inflorescences; ciliate sepals, 4-merous flowers, pink or white female flowers; and 4 pyrenes.

1. Introduction

As the only genus of Aquifoliaceae, Ilex is characterized by unisexual flowers, axillary cymose inflorescences (sometimes reduced to one flower), and fruits containing 1–6(–23) pyrenes [1]. It comprises more than 400 species in the tropical to temperate regions of the world, with its biodiversity center in South America and Asia [2]. The total species number in the genus is still ill-defined due to insufficient taxonomic revision of the genus worldwide [3]. A total of 204 species have been recorded, and additional new species have been discovered and added to the checklist of Ilex in China [4,5,6,7,8,9,10,11]. The majority of these Ilex species are distributed in montane forests in southern areas of the Yangtze River [4]. The Ilex species are evergreen or deciduous trees and shrubs, and plants of the genus are dioecious [1]. Many species of the holly genus are dominant species in evergreen broad-leaved forests, and they play an important role in the formation and maintenance of forest ecosystems in tropical and subtropical areas [12,13]. Therefore, determining the phylogeny and taxonomy of genus Ilex has important implications for the study of forest biodiversity and the role of pivotal species in forest ecosystems.
Phylogenetically, Ilex (Aquifoliaceae) are well placed within the order Aquifoliales, at the base of the Campanulids [14]. However, the infrageneric relationships and taxonomy of the genus remain poorly understood [15,16,17,18]. Recently, Yang et al. [19] proposed a comprehensive classification of the genus based on a combination of morphological traits, distribution patterns, and phylogeny that was reconstructed using the three nuclear genes of internal transcribed spacer (ITS), external transcribed spacer (ETS), and chloroplast glutamine synthetase gene (nepGS). They made a major revision of the previous classification because the nonmonophyly taxa (subgenera, sections, and series) in the previous classification were based only on morphology data. A total of 14 sections of the genus were recognized in their study: I. sect. Cassinoides Loes.; I. sect. Glabra Yi Yang & H. Peng; I. sect. Lioprinos (Loes.) S. Y. Hu; I. sect. Ilex; I. sect. Indico-Malaicae Loes.; I. sect. Micrococca (Loes.) S. Y. Hu; I. sect. Microdontae Loes.; I. sect. Paltoria (Ruiz & Pav.); I. sect. Prinoides (DC.) A. Gray; I. sect. Prinos (L.) A. Gray; I. sect. Prinifoliae Loes.; I. sect. Pseudoprinos S. Y. Hu; I. sect. Pseudoaquifolium S. Y. Hu; and I. sect. Repandae (Loes.) Yi Yang & H. Peng. However, interspecific hybridization and introgression events make this lineage very complex in terms of phylogeny and taxonomy [16,20]. There are still some clades/taxa that have not been well resolved due to the low resolution of the phylogeny. In addition, the macromorphology (e.g., leaf shape and leaf size) of the Ilex species often shows great variability at the intraspecific level, resulting in difficulties in discriminating between different species [2].
Recently, we conducted a comprehensive botanical expedition in the Danxia Mountain National Nature Reserve, Renhua County, Guangdong Province, China (Figure 1). During this expedition, we encountered an unusual species of Ilex. In general, this species belongs to members of I. sect. Lioprinus, and, morphologically, it is most similar to I. hirsuta C. J. Tseng ex S. K. Chen et Y. X. Feng in having branchlets, petioles, peduncles, and pedicel puberulent; leaf margin sparsely serrate; inflorescences cymes; and pyrenes broadly one-sulcate abaxially. However, this species has male inflorescences monochasium, flowers four-merous, female flowers pink or white, and pyrenes four, while I. hirsuta has male inflorescences monochasium or dichasium, flowers five-merous, and four or five pyrenes. After carefully examining herbarium specimens and the relevant literature, we confirmed that it represents an undescribed new species [21,22,23]. Here, we describe it as I. danxiaensis K. W. Xu & Q. Fan with a report on its phylogenetic position based on three nuclear genes.

2. Material and Methods

2.1. Morphological Study

The investigations were carried out on pollen samples from two different origins (Agongyan and Huangshakeng of Danxia Mountain) obtained from living specimens in wild populations. Mature and well-developed pollen grains were selected for investigation. Measurements of stomate morphology were conducted using mature and intact leaves obtained from living specimens. Morphology of pollen grains and stomates was observed using an ESEM-Quanta 200 (FEI, Hillsboro, OR, USA) after being sputter-coated with gold. To determine pyrene morphology, we collected the mature red fruits and rinsed each fruit sarcocarp using clean water. We counted the pyrene number of each fruit. Then, the clean pyrene features of the new species were obtained using an Olympus SZX10 dissecting microscope (Olympus Corp., Tokyo, Japan). Quantitative values of each morphological character were measured using ImageJ software (Version 1.54b, Bethesda, MD, USA, Rasband 1997–2017) [24]. All digital images of specimens examined in this study were provided by the National Specimen Information Infrastructure (available at http://www.nsii.org.cn/ accessed 5 August 2022), Global Biodiversity Information Facility (available at https://www.gbif.org/ accessed 6 August 2022), and JSTOR (available at https://plants.jstor.org/ accessed 5 August 2022). Voucher specimens were deposited in the Herbarium of Sun Yat-Sen University (SYS) and Nanjing Forestry University (NF).

2.2. Phylogenetic Study

To identify the new species, genomic DNA was extracted from silica-gel-dried leaves using the modified 2 × CTAB procedure according to Doyle et al. [25] and used as the template for polymerase chain reaction (PCR). Two nuclear markers were selected for amplification. The primers ITSF and ITSR were used for ITS amplification [18], and the primers GScp687f and GScp994r were used for nepGS amplification [26]. Each amplification was carried out in 50 μL of a reaction mixture containing 30 μL 2 × PCR Master Mixture (containing 2 × Taq DNA polymerase, 2 × polymerase chain reaction buffer, 2 × dNTP, and Mg2+, Tiangen), 14 μL of ddH20, 1 μL of each primer, and 4 μL of DNA template. Polymerase chain reaction thermocycling conditions for ITS and nepGS involved an initial denaturation step of 95 °C for 4 min, followed by 35 cycles of 95 °C for 45 s, 58 °C for 1 min, 72 °C for 2 min, and a final extension of 72 °C for 10 min. PCR products were purified and sequenced by TSINGKE Biological Technology (Guangzhou, China).
To test the precise phylogenetic position of the new species Ilex danxiaensis, we reconducted phylogenetic analyses using a combined matrix of three nuclear (ITS including ITS1, 5.8S, ITS2, ETS, and nepGS) sequences according to results of Yang et al. [19]. We preliminarily speculated that the new species belongs to I. sect. Lioprinos (Loes.) S. Y. Hu according to its morphological characteristics in our field work. Therefore, the sampling strategy of this study tried to include as many members of I. sect. Lioprinos as possible, and representative species of each section proposed by Yang et al. [19]. Finally, our data matrix totally contained 91 accessions representing 89 species, including 3 accessions of the new species, 85 accessions of other Ilex species, and 3 accessions of outgroup species (Supplemental Data S1). The ITS and nepGS of the new species were newly sequenced in this study, and the rest were downloaded from GenBank.
The newly generated raw sequences were assembled and edited using Sequencher (Version 4.14, Ann Arbor, MI, USA). All sequences of ITS, ETS, and nepGS were initially aligned using MAFFT (Version 7.480, Tokyo, Japan) [27] and manually adjusted using BioEdit (Version 7.5.5, Wooster, OH, USA) [28]. For each nuclear marker, the maximum-likelihood (ML) tree searches and bootstrapping (BS) were conducted using RAxML-HPC2 on XSEDE (Version 8.2.10, San Diego, CA, US) on the CIPRES web server with 1000 bootstrap replicates [29], with 5000 rapid bootstrap analyses followed by a search for the best-scoring tree in a single run [30,31]. Comparisons of tree topologies from the ML analyses of each individual marker did not identify any well-supported conflicts. Thus, the three nuclear genes were concatenated and analyzed in unison. Bayesian inference (BI) was conducted using MrBayes (Version 3.2.7a, Stockholm, Sweden) [32] on the CIPRES web server [29] with the temperature parameter set to 0.2, and other priors set to their default values. Two independent runs, each with four simultaneous chains (one cold and three heated), were conducted, each beginning with a random tree and sampling one tree every 1000 generations of 10,000,000 generations. After the first 25% of trees were discarded as burn-in, the remaining trees were used to construct a majority-rule consensus tree with Bayesian posterior probabilities (PPs). ModelFinder [33] was used to select the best-fitting model for ML and BI analyses. The Akaike information criterion (AIC) [34] was used to select among models instead of the hierarchical likelihood ratio test.

3. Results

3.1. Morphological Characteristics of the New Species

Our morphological study showed that the new species has puberulent branchlets, petioles, peduncles, and pedicel; (1–)2–3-flowered cymes of male inflorescences; 1-flowered cymes of female inflorescences; ciliate sepals; 4-merous flowers; pink or white female flowers; and 4 pyrenes, longitudinally 1-sulcate abaxially (Figure 2 and Figure 3). The pollen of the new species is tricolporate, isopolar, and clavate. The polar axis (P) is ca. 45.13 μm; the equatorial axils (E) are ca. 30.98 μm. The exine is ornamented with conspicuous gemmae and clavae of variable size (Figure 4A–C). The stoma (Figure 4D,E) of this new species is amphicyclocytic. The average density of stomata is ca. 275/μm2 (Figure 4D). The average size of the normal stomata is 37.3 × 33.3 μm. The leaf blades are sparsely puberulent (Figure 4F).

3.2. Phylogenetic Position of the New Species

In this study, a total of six new sequences of the two nuclear genes ITS and nepGS were newly generated for the new species (Supplemental Data S1), while other sequences of three nuclear genes (ITS, ETS, and nepGS) were downloaded from GenBank. Our final data matrix totally contained 91 accessions representing 89 species, including 3 accessions of the new species, 85 accessions of other Ilex species, and 3 accessions of outgroup species (Supplemental Data S1). The alignment length of the nuclear data matrix was 2417 bp, of which 1654 sites were identical, 486 characters were parsimony-informative, and 277 variable characters were parsimony-uninformative. The best-fit nucleotide substitution models GTR+F+I+G4 for ML and BI analyses were selected using ModelFinder [33] based on the Akaike information criterion. The tree topologies by the ML and BI analyses were generally concordant when using the concatenated dataset. The ML phylogeny of genus Ilex and outgroups based on the concatenated data is presented in Figure 5.
Our phylogenetic tree inferred from the three nuclear genes resolved our 88 accessions of Ilex into 16 strongly or moderately supported clades (Figure 5), of which the moderately supported (MLBS: 66%; BIPP: 1) clade I. sect. Lioprinos contains the most species (including 24 species) compared with the other strongly or moderately supported clades. Each section proposed by Yang et al. [19] was also resolved as a monophyletic clade with a high or moderate support value in our analyses. In addition to the 14 sections, the strongly supported (MLBS: 83%; BIPP: 0.9) clade containing 2 species (I. mucronate (L.) M. Powell, Savol., & S. Andrews and I. amelanchier M. A. Curtis ex Chapm.) and the clade only containing 1 species (I. canariensis Poir) were also resolved as monophyletic clades (Figure 5). The phylogenetic relationships within each clade were well resolved in the previous study, while the backbone relationships of genus Ilex were not well resolved with many weakly supported nodes using the current three nuclear dataset (Figure 5).
Just as we predicted based on morphology evidence, the new species was resolved as a member of I. sect. Lioprinos in our phylogenetic tree. Together with I. chinensis Sims and I. purpurea Hassk., they formed a moderately supported clade (MLBS: 65%; BIPP: 1) that was resolved as sister to a clade containing I. ficifolia C. J. Tseng ex S. K. Chen & Y. X. Feng, I. hirsuta, I. melanophylla H. T. Chang, and I. tainingensis G. S. He. Although the new species is most similar to I. ficifolia and I. hirsuta in morphology, our phylogenetic analyses demonstrate that they are not very closely related.

4. Discussion

4.1. Morphological Comparison

Ilex danxiaensis is morphologically most similar to I. hirsuta in having branchlets, petioles, peduncles, and pedicel puberulent; leaf margin sparsely serrate; inflorescences cymes; and pyrenes broadly one-sulcate abaxially. However, the new species can be easily distinguished from I. hirsuta by having male inflorescences monochasium, flowers four-merous, female flowers pink or white, and four pyrenes (Figure 2 and Figure 3). In addition, the new species is morphologically similar to I. ficifolia in having leaf margin serrulate, inflorescences cymes, peduncles and midvein pubescent, sepals ciliate, and pyrenes broadly one-sulcate abaxially. Nevertheless, the new species has flowers 4-merous, (1–)2–3-flowered cymes of male inflorescences, calyx shallowly 4-lobed, 4 stamens, 1-flowered cymes of female inflorescences, 1-fruited infructescences, and fruit pedicels pubescent (Figure 2 and Figure 3). In contrast, I. ficifolia has flowers 4–5-merous, 7-flowered cymes of male inflorescences, calyx shallowly 5-lobed, stamens 5, 3-flowered cymes of female inflorescences, 1–3-fruited infructescences, and fruit pedicels glabrescent [4]. The species can be easily distinguished from each other by the morphological characters listed in Table 1. The pollen size of Ilex species is between 17 and 47 mm × 18 and 48 mm [1], which is also supported by that of the new species (average size ca. 45.13 × 30.98 μm). The stoma of this new species is amphicyclocytic, which is similar to the members of I. sect. Lioprinus (e.g., I. editicostata Hu et Tang, I. ficifolia, I. hirsuta, I. lancilimba Merr., etc.) [35].

4.2. Phylogenetic Analyses

To further determine the phylogenetic position of the new species, we conducted ML and BI analyses for the genus Ilex using the sequence data of ITS, ETS, and nepGS. With the genus Helwingia as the outgroup, the phylogram showed that the genus Ilex is well supported as monophyly (Figure 5), which was also supported by the previous phylogenetic studies of the family Aquifoliaceae [16,17,18,19]. By using a similar data matrix when conducting our phylogenetic analyses, we generated shallow phylogenetic relationships of the genus Ilex similar to those reported by Yang et al. [19]. All the accessions of genus Ilex sampled in this study were divided into 16 major clades based on a combination of morphological and distributional characteristics in our phylogenetic tree (Figure 5). Comparisons of our tree topologies with those of Yang et al. [19] did not identify any well-supported conflicts. However, the deep relationships of the genus were somewhat discrepant between the two analyses largely due to the low resolution of the phylogenetic tree reconstructed using the three nuclear genes. For example, in the present study, I. sect. Lioprinos was weakly resolved as sister to a clade containing three sections (I. sect. Prinifoliae, I. sect. Prinos, and I. sect. Pseudoaquifolium), while in the analyses of Yang et al. [19], I. sect. Lioprinos was weakly resolved as sister to a clade containing four sections (I. sect. Microdontae, I. sect. Prinifoliae, I. sect. Prinos, and I. sect. Pseudoaquifolium). Another example, I. sect. paltoria, was resolved as sister to I. sect. repandae in our phylogeny, while in the analyses of Yang et al. [19], I. sect. paltoria was resolved as sister to all other members of genus Ilex. These phylogenetic inconsistencies demonstrated that the current backbone phylogenetic relationships of the genus Ilex are unstable based on the data matrix of three nuclear genes. Future phylogenetic studies using more comprehensive datasets are urgently needed for the troublesome genus Ilex (Figure 5).
Though the deep relationships within the genus were not well resolved in the current analyses, three accessions of I. danxiaensis were well resolved to cluster together in our phylogenetic tree. Together with I. chinensis and I. purpurea, they formed a moderately supported clade, which revealed that they might be closely related to each other (Figure 5). In fact, I. chinensis and I. purpurea were initially assigned to I. sect. Lioprinos based on morphological evidence [4]. Members of this section are characterized by having leaf margin serrulate, inflorescences cymes, and pyrenes broadly one-sulcate abaxially [4]. Recently, Yang et al. [19] also confirmed that they belong to members of I. sect. Lioprinos based on their phylogenetic analyses. Therefore, the new species is unarguably a member of I. sect. Lioprinos based on our phylogenetic analyses (Figure 5). Morphologically, the new species is characterized by having branchlets, petioles, peduncles, and pedicel puberulent, leaf margin sparsely serrate, inflorescences cymes, flowers white or pink, fruit red, four pyrenes each fruit, and pyrenes broadly one-sulcate abaxially. These features also support the conclusion that this species is a member of I. sect. Lioprinos based on molecular phylogeny. Thus, the status of the new species was confirmed, and the relationships among the new species and its closely related species in I. sect. Lioprinus were supported by both morphology and molecular data.

4.3. Distribution Ranges of Ilex danxiaensis and the Two Morphologically Similar Species

Ilex danxiaensis is currently known only from two populations on Danxia Mountain in Renhua County, Guangdong Province of China. It might be restricted to the Danxia landform of South China (Figure 1). However, its morphologically similar species I. hirsuta is sporadically distributed in regions of central and east China and I. ficifolia is widely distributed in regions of central, east, and south China (Figure 1). The latter two species I. hirsuta and I. ficifolia mainly occur in acidic soils, rarely in the areas of the Danxia landform.

4.4. Conservation Significance

The conservation status of the new species I. danxiaensis caught our attention. The new species is suggested as an endangered species and listed as CR, which is attributed to its extremely small populations with less than 50 mature individuals. In addition, its typical locality, Danxia Mountain, is a well-known national scenic area. The activities of tourists might be a potential threat for regional biodiversity in Danxia Mountain.
Danxia Mountain, located in the northern part of Guangdong province, is one of the famous examples of Danxia landform characterized by red-colored sandstones and conglomerates of largely Cretaceous age [36]. Danxia Mountain harbors many endemic species and is a mountain with very rich endemic elements owing to its unique geological and geomorphic structure [36]. Local governments have made great efforts to protect biodiversity in the mountain and carried out numerous biodiversity surveys in recent years. Consequently, a lot of new and endemic species have been found there, e.g., Lespedeza danxiaensis Q. Fan, W. Y. Zhao & K. W. Jiang [37] and Asplenium danxiaense K. W. Xu [38]. The discovery of these species serves to reveal the formation and maintenance mechanism of regional biodiversity on Danxia Mountain. As a result, the biological conservation of primary vegetation and endangered species of this biodiversity hot spot is quite important, and conservation action is necessary.

4.5. Taxonomic Treatment

Ilex danxiaensis K. W. Xu & Q. Fan, sp. nov. (Figure 2 and Figure 3).
Type. China. Guangdong: Shaoguan City, Danxia Mountain, Agongyan, elev. 210 m, 25°01′39.08″ N, 113°45′15.13″ E, 11 October 2020, Q. Fan 18438 (holotype SYS!; isotype NF!).
Diagnosis. The new species morphologically resembles I. hirsuta in having branchlets, petioles, peduncles, and pedicel puberulent; leaf margin sparsely serrate; inflorescences cymes; and pyrenes broadly one-sulcate abaxially. It differs in having male inflorescences monochasium (vs. male inflorescences monochasium or dichasium), flowers four-merous (vs. flowers five-merous), and four pyrenes (vs. four or five pyrenes).
Description. Shrubs or trees, evergreen, up to 8 m tall. Annual branches subterete, green to greyish-green, densely puberulent, hairs yellowish-brown; perennial branches grayish white, glabrescent; lenticels sparse, indistinct. Petiole 2–5 mm, adaxially longitudinally sulcate, densely puberulent when young; leaf blade abaxially greenish, adaxially dark green, narrow elliptic or ovate-lanceolate, (1.5–)6–8 × (1.2–)2.5–3.5 cm, leathery, midvein raised and pubescent abaxially when mature, flat, or impressed and glabrescent adaxially, lateral veins 4–6(–8) pairs, slightly raised on both surfaces, anastomosing near margin, reticulate veins obvious abaxially, obscure adaxially, base cuneate or obtuse, basal margin near entire, upper serrulate, slightly recurved, apex acuminate. Inflorescences: cymes, solitary, axillary. Male inflorescences: cymes, (1–)2–3-flowered; peduncles (3–)5–12 mm, brown-yellow pilose; bracteoles 1, basal, subulate, ca. 1 mm; flowers pink, 4-merous; pedicel 3–5 mm; calyx shallowly 4-lobed, lobes obtuse, ciliate; petal rotate, ovate, ca. 1.5 × 3 mm, basally slightly connate; stamens 4, ca. 1/2 as long as petals, anthers oblong. Female inflorescences: one-flowered cymes; peduncles ca. 4 mm, brown-yellow pilose; bracteoles 2, subulate, ca. 1 mm; pedicels ca. 1 cm; calyx four-lobed, lobes ciliate; four petals, ovate, ca. 1.5 × 3 mm; staminodes ca. 1.5 mm, sterile anthers ovoid; ovary broadly globose, ca. 2 mm in diam., persistent stigma discoid. Infructescences: 1-fruited cymes; peduncles ca. 5 mm, brown-yellow pilose; fruit pedicels 9–10 mm, pubescent; bracteoles 2, subulate. Fruit yellowish-brown, shiny, subglobose, 5–7 mm in diam.; persistent calyx patelliform, explanate, ciliate, four-lobed; stigma persistent; four pyrenes, ellipsoidal, ca. 4.8 mm long, abaxially broadly and deeply one-sulcate. Fl. May, Fr. Oct–Dec.
Phenology. It starts to flower in May and bears fruits from October to December.
Distribution and habitat. Ilex danxiaensis is currently known only from the type of locality, Danxia Mountain National Nature Reserve (Agongyan and Huangshakeng), Guangdong, China (Figure 1). It usually occurs in subtropical evergreen broad-leaved forest at ca. 200 m elevation of mountains with Danxia landform.
Conservation assessment. Ilex danxiaensis is endemic to Danxia Mountain National Nature Reserve of Shaoguan City. Currently, only two large populations with no more than 50 mature individuals have been found. According to IUCN Red List criteria D [39], this species should be listed as critically endangered (CR). More extensive fieldwork in nearby mountains with Danxia landform will be needed to accurately assess its conservation status.
Etymology. Based on the mountain name, Danxia, in northern Guangdong, China, and the Latin suffix, -ensis, of origin, referring to the type of locality of the species.
Vernacular name. We propose a Chinese name, Dānxiáshān dōngqīng (丹霞山冬青) to reflect the type locality of the new species.
Additional specimens examined (paratypes). China. Guangdong: Shaoguan City, Danxia Mountain, Agongyan, elev. ca. 210 m, 25°01′39.08″ N, 113°45′15.13″ E, 17 April 2021, Q. Fan 18960 (SYS!, NF!); China. Guangdong: Shaoguan City, Danxia Mountain, Huangshakeng, elev. 211 m, 25°00′52.23″ N, 113°44′22.33″ E, 18 April 2021, Q. Fan 18966 (SYS!, NF!).

5. Conclusions

We described a new species of the holly genus Ilex (Aquifoliaceae) from Guangdong Province in China, and provided evidence for its phylogenetic position based on the sequence data of ITS, ETS, and nepGS. Ilex danxiaensis K. W. Xu & Q. Fan, sp. Nov. was only known from a narrow range of the Danxia Mountain in the northern part of Guangdong Province in China, and occurred on the hillside near mountaintop. In our fieldwork, only two populations of the new species were found, and further more detailed investigations on the species are urgently needed to ascertain its distributional range and population status. With our description, we contribute to a better knowledge of the diversity of the genus Ilex in southern China, and suggested that more comprehensive phylogenetic and taxonomic studies would highlight the evolution patterns of this group.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14030583/s1, Supplemental Data S1. List of taxa sampled, GenBank accession numbers, and voucher information (/indicates accessions with missing data).

Author Contributions

Field observation and collection, W.Z., Z.C., and Q.F.; data analysis and morphological study, K.X.; experiment and photo typesetting, C.L.; writing—original draft preparation, C.L.; writing—review and editing, K.X. and Q.F. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Guangdong Provincial Special Research Grant for the Creation of National Parks (2021GJGY034), the Natural Science Foundation of Jiangsu Province (#BK20210612), the National Natural Science Foundation of China (#32100167), and the Nanjing Forestry University project funding (#163108093).

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Distribution map of the three morphological similar species Ilex danxiaensis, I. ficifolia, and I. hirsuta in China, on the basis of the specimens examined in this study and flora of China.
Figure 1. Distribution map of the three morphological similar species Ilex danxiaensis, I. ficifolia, and I. hirsuta in China, on the basis of the specimens examined in this study and flora of China.
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Figure 2. Ilex danxiaensis K. W. Xu & Q. Fan, sp. nov. (A) Habitat; (B) tree trunk; (C) annual branches; (D) morphological variation of leaf blades; (E) female flower buds; (F) male flower buds; (G) female inflorescence with pink flowers; (H) female inflorescence with white flowers; (I) male inflorescence and flowers; (J) infructescences and fruit; (K) pyrenes.
Figure 2. Ilex danxiaensis K. W. Xu & Q. Fan, sp. nov. (A) Habitat; (B) tree trunk; (C) annual branches; (D) morphological variation of leaf blades; (E) female flower buds; (F) male flower buds; (G) female inflorescence with pink flowers; (H) female inflorescence with white flowers; (I) male inflorescence and flowers; (J) infructescences and fruit; (K) pyrenes.
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Figure 3. Ilex danxiaensis K. W. Xu & Q. Fan, sp. nov. (A) Fruiting branches; (B) female flowering branches; (C) fertile stamen; (D) staminodes; (E) petals and stamens of male flowers; (F) petals and staminodes of female flowers; (G) ciliate sepal; (H) male flower; (I) female flower; (J) dorsal view of male flower; (K) fruit. Drawn by Yun-Xiao Liu.
Figure 3. Ilex danxiaensis K. W. Xu & Q. Fan, sp. nov. (A) Fruiting branches; (B) female flowering branches; (C) fertile stamen; (D) staminodes; (E) petals and stamens of male flowers; (F) petals and staminodes of female flowers; (G) ciliate sepal; (H) male flower; (I) female flower; (J) dorsal view of male flower; (K) fruit. Drawn by Yun-Xiao Liu.
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Figure 4. SEM photomicrographs of pollen grains and stomata of Ilex danxiaensis sp. nov. (A–C) Pollen grains; (D–F) stomata.
Figure 4. SEM photomicrographs of pollen grains and stomata of Ilex danxiaensis sp. nov. (A–C) Pollen grains; (D–F) stomata.
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Figure 5. Maximum likelihood (ML) tree of genus Ilex from phylogenetic analysis of combined ITS, ETS, and nepGS sequence data. Numbers near each branch are maximum-likelihood bootstrap support (MLBS) and Bayesian inference posterior probability (BIPP). Dash means that MLBS ≤ 50% or BIPP ≤ 0.5. ”*” means that MLBS =100% or BIPP=1.0. The new species Ilex danxiaensis is indicated in bold in the ML tree.
Figure 5. Maximum likelihood (ML) tree of genus Ilex from phylogenetic analysis of combined ITS, ETS, and nepGS sequence data. Numbers near each branch are maximum-likelihood bootstrap support (MLBS) and Bayesian inference posterior probability (BIPP). Dash means that MLBS ≤ 50% or BIPP ≤ 0.5. ”*” means that MLBS =100% or BIPP=1.0. The new species Ilex danxiaensis is indicated in bold in the ML tree.
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Table
Table 1. Morphological comparisons among Ilex danxiaensis and its morphologically similar species.
Table 1. Morphological comparisons among Ilex danxiaensis and its morphologically similar species.
CharacteristicIlex danxiaensisI. ficifoliaI. hirsuta
Annual branchdensely puberulentglabrescentdensely puberulent
Petiole2–5 mm5–10 mmca. 15 mm
Leaf blade(1.5–)6–8 × (1.2–)2.5–3.5 cm; sparsely puberulent4–7 × 1.5–3 cm; glabrous6–7 × 2.5–4 cm; densely hirsute
Male inflorescencecymes, (1–)2–3-floweredcymes, 7-flowered cymes, 1-3-flowered
Male flowerflowers 4-merous; calyx shallowly 4-lobedflowers 4- or 5-merous; calyx shallowly 5-lobedflowers 5-merous; calyx shallowly 5-lobed
Female inflorescencecymes, 1-flowered cymes, 3-floweredunknown
Female flowerflowers 4-merousflowers 4- or 5-merousunknown
Infructescences1-fruited1–3-fruited1–3-fruited;
Pedunclesca. 5 mm0.7–15 mmca. 15 mm
Pyrene number per fruit454 or 5
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Lin, C.; Zhao, W.; Chen, Z.; Fan, Q.; Xu, K. Ilex danxiaensis (Aquifoliaceae), a Distinct New Tree Species Endemic to Danxia Mountain in Guangdong Province, China, Based on Molecular and Morphological Evidence. Forests 2023, 14, 583. https://doi.org/10.3390/f14030583

AMA Style

Lin C, Zhao W, Chen Z, Fan Q, Xu K. Ilex danxiaensis (Aquifoliaceae), a Distinct New Tree Species Endemic to Danxia Mountain in Guangdong Province, China, Based on Molecular and Morphological Evidence. Forests. 2023; 14(3):583. https://doi.org/10.3390/f14030583

Chicago/Turabian Style

Lin, Chenxue, Wanyi Zhao, Zaixiong Chen, Qiang Fan, and Kewang Xu. 2023. "Ilex danxiaensis (Aquifoliaceae), a Distinct New Tree Species Endemic to Danxia Mountain in Guangdong Province, China, Based on Molecular and Morphological Evidence" Forests 14, no. 3: 583. https://doi.org/10.3390/f14030583

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