Smilax weniae, a New Species of Smilacaceae from Limestone Areas Bordering Guizhou and Guangxi, China

A new species, Smilax weniae (Smilacaceae), from Southwest China, is described and illustrated. The new species bears peltate leaves, which was previously a unique feature of S. luei. However, it differs from the latter by having a broad ovate leaf blade, longer peduncle, and sexual dimorphic flowers. Further phylogenetic analyses revealed that the new species were placed in a unique position in a subclade of Old World Smilax based on ptDNA and nrITS sequences. Combining detailed morphological comparisons and molecular evidence, we validated that S. weniae is an undescribed new species. Moreover, the plastome characteristics of S. weniae are reported.


Introduction
Smilax L., currently considered the sole genus of Smilacaceae [1], is one of the most abundant and easily recognizable climbing plants in various ecosystems of the Old World and the New World [2]. Typically, they are characterized by vines climbing or shrubs, being woody, less frequently suberect or herbs, dioecious and with tuberous or stoloniferous rhizomes, stems and branches which are usually prickly, paired petiolar tendrils, unisexual flowers with six tepals and either six fertile stamens or staminodes in the case of pistillate flowers [3]. Smilacaceae was split into four major clades by Qi et al. [1]: clade A: Smilax aspera L., clade B: mostly American species, clade C: prickless herbs, non-climbing species and Heterosmilax, mostly Asian species, and clade D: Asian-Australian-African woody species. Clade C and clade D are primarily from the Old World. Within clade C, sect. Heterosmilax is distinguished from other Smilax by their flowers with connate tepals [4]. The subclades in clade C, i.e., sect. Heterosmilax, sect. Nervo-marginatae, sect. Vaginatae, sect. Ligneoriparia and sect. Nemexia are all prickless. In addition, Smilax is a difficult genus to classify since its plants are dioecious and exhibit extensive phenotypic variations [5].
In 2020, we found a unique Smilax population with peltate leaves from Guizhou Manlan National Natural Reserve in Southwest China. The only formerly known Smilax species with peltate leaves is Smilax luei T. Koyama, which is endemic to Taiwan in Southeast China and can be easily distinguished by the morphological characteristics of the leaf blade, peduncle and male flower. In order to observe this special species, we conducted several follow-up fieldworks in 2021 and successfully collected its female & male flowers. During the subsequent herbarium work, we found two fruiting specimens (collected from Huanjiang County, Guangxi, and now deposited in the Herbarium of Guangxi Institute of Botany, IBK) representing this new species. To reveal its systematic position, this new species and 34 other representative Old World Smilax species were selected for phylogenetic studies. The unique morphology and systematic position both confirmed that it is an undescribed Smilax species, which is described and illustrated below. Additionally, the complete plastome of the novel species is reported.

Morphological Comparison
Morphological comparisons between 'Smilax weniae' and S. luei T. Koyama are summarized in Table 1. The new species is distinctive in having sexually dimorphic flowers (male flower: tepals connate ca. 1/5; female flower: tepals separated, glabrous on both surfaces), longer peduncles, and ovate leaves.

Molecular Phylogenetic Analyses
The total alignment of the five plastid (pt) regions (matK, rbcL, ndhA, ndhF, rpl16) was 7022 bp and included 97 variable sites, 49 of which were informative. The alignment of nuclear ribosomal (nr) ITS regions was 823 bp and included 95 variable sites, 46 of which were informative. Both phylogenetic trees show that S. weniae is included in clade C. The ptDNA ML cladogram ( Figure 1) showed that Smilax weniae (PP = 0.95, ML BS = 99) is sister to a clade consisting of 20 Smilax species represented by sect. Heterosmilax + sect. Nervo-marginatae + sect. Vaginatae + sect. Ligneoriparia. While the nrITS ML cladogram ( Figure 2) showed that Smilax weniae is sister to two species (S. fui and S. ligneoriparia) of sect. Ligneoriparia with weak support (PP = 0.74, ML BS = 90). The ML phylograms of ptDNA and nrITS (Figures S1 and S2) indicated that S. weniae had a deep genetic divergence with its sister clades. Occupying a unique systematic position, together with its distinctive morphological characteristics, Smilax weniae is verified to be a new species.

Etymology
The species is named in memory of Mrs. Hequn Wen, who was a researcher and vice director of Guangxi Institute of Botany from 1995 to 2000. Then she worked in the government of Liuzhou City, and tragically died in a car accident on a business trip to a remote location near the boundary of Guangxi and Guizhou. Wen was the first person to realize that Smilax weniae probably represents an undescribed species and provided the nude name 'Smilax peltatus H.Q. Wen' on an annotation label of the Dianqiangui Team 70302 (IBK00199287!) specimen, which was collected from Huanjiang County in Guangxi. The specific epithet she proposed clearly referred to the peltate leaves. Hence, we propose the Chinese name ' both surfaces; staminodes: six; ovary superior. Staminate flowers born on pedicels 2.0-3.0 mm long, yellow-greenish; tepals: six, two whorls, 4-5 mm long, elliptic to oblong, adaxially involution, outer ones 3.0-4.0 mm wide, inner ones slightly narrower, outer ones and inner ones connate for ca. 1/5; stamens: six, the yellowish anthers on a short filament, two anther sacs obvious, adaxially involuted. Berries 1.0-3.0 mm in diameter, presumably red at maturity (based on IBK00199287!, IBK00312465!), Flowering in April, Fruiting in October.

Etymology
The species is named in memory of Mrs. Hequn Wen, who was a researcher and vice director of Guangxi Institute of Botany from 1995 to 2000. Then she worked in the government of Liuzhou City, and tragically died in a car accident on a business trip to a remote location near the boundary of Guangxi and Guizhou. Wen was the first person to realize that Smilax weniae probably represents an undescribed species and provided the nude name 'Smilax peltatus H.Q. Wen' on an annotation label of the Dianqiangui Team 70302 (IBK00199287!) specimen, which was collected from Huanjiang County in Guangxi. The specific epithet she proposed clearly referred to the peltate leaves. Hence, we propose the Chinese name '盾叶菝葜 (dun ye ba qia)'.

Distribution and Habitat
This new species is known from Libo County, southern Guizhou Province and Huanjiang Maonan Autonomous County, northern Guangxi Zhuang Autonomous Region, China. Till now, it has been recorded at one locality of Libo County and two localities of Huanjiang Maonan Autonomous County ( Figure 5). It grows on the slopes of limestone mountains at 390-950 m.

Distribution and Habitat
This new species is known from Libo County, southern Guizhou Province and Huanjiang Maonan Autonomous County, northern Guangxi Zhuang Autonomous Region, China. Till now, it has been recorded at one locality of Libo County and two localities of Huanjiang Maonan Autonomous County ( Figure 5). It grows on the slopes of limestone mountains at 390-950 m.

Conservation Status
Smilax weniae was found in Guizhou Maolan National Natural Reserve and Guangxi Mulun National Natural Reserve. Both reserves are primarily dedicated to the preservation of the subtropical karst forest ecosystem and uncommon wild animal and plant resources. The two reserves are quite abundant in plant diversity. We anticipate that more populations will probably be discovered in the near future. Nevertheless, the new species is currently known from three sites and has a restricted distribution area. More in field research is certainly needed for an appropriate definition of distribution, population size, locations (sensu IUCN), threats, etc., information which is necessary for the assessment of the conservation status according to the IUCN categories and criteria [6]. In fact, species with a restricted distribution could be attributed to different categories, as Critically endangered (CR) according to IUCN criteria B and C [7,8], as Vulnerable (VU) under IUCN criterion D [9,10], or as Data deficient (DD) [11], etc.

Taxonomic and Evolutionary Relationships
Morphologically, the new species occasionally bears prickles on the stem, whereas the stems of other species in this clade are prickless [12]. In addition, the new species bears Plants 2022, 11, 1032 7 of 13 peltate leaves, which is a rare feature in the genus. Besides, in most known Smilacaceae species, the separation/connation patterns of male and female flowers tepals are consistent, i.e., separate in both genders, basally connate in both, or completely connate in both. The new species, however, shows an unusual sexual dimorphic pattern in which the tepals are basally connate in staminate flowers but separate in pistillate flowers. This pattern was first discovered in Smilax hirtellicaulis C.Y. Wu & C. Chen ex P. Li. which had closest phylogenetic relationship with sect. Heterosmilax [13]. Instead, S. weniae is sister to a clade consisting of representatives of four Smilax sections (including sect. Vaginatae, sect. Ligneoriparia, sect. Heterosmilax and sect. Nervo-marginatae) based on ptDNA loci (Figure 1, PP = 0.95, ML BS= %). Thus, this distinct characteristic (tepals are basally connate in staminate flowers but separate in pistillate flowers) is clearly a result of parallel evolution rather than a synapomorphy.

Discussion
Smilax weniae is morphologically allied to S. luei, which is endemic to central Taiwan, China [14,15]. They both have distinct peltate leaves. Individuals with patchy white spots were found in both S. weniae and S. luei. They also share the same type of leaf vein character (3 primary and 2 marginal veins). However, S. weniae can be easily distinguished from S. luei by its unique male flower (tepals connate ca. 1/5), female flower (tepals glabrous on both surfaces), longer peduncle and ovate leaf blade (Figure 4). Furthermore, we found a few S. weniae individuals having prickles on their stems on occasion, while the stems of S. luei are always prickless. In addition, the peduncle length of the new species is usually longer than 2.5 cm, and the longest is even 10 cm, while the peduncle of S. luei is significantly shorter (1.5-2.5 cm). In addition to the above several morphological characteristics that can distinguish the new species from other species, it also has an unusual sexual dimorphic pattern, which is similar to S. hirtellicaulis (male flower: tepals connate ca. 1/5; female flower: tepals separated). Smilax weniae and S. hirtellicaulis both have similar broad ovate leaves and slightly compressed peduncles, S. weniae differs from the latter by having distinctive peltate leaves and prickly stems, and the female tepals are glabrous on both surfaces, while the female tepals of S. hirtellicaulis are thickened, and adaxially verruculose.
While incongruence was detected in the phylogenetic placement of S. weniae based on nrITS and ptDNA loci, the novel species status is confirmed. The ptDNA phylogeny provided more convincing statistical support for its sister relationship to four Smilax sections (Figure 1). The different placement inferred from nrITS could be simply due to a lack of information or a different evolutionary history between nuclear and plastid DNA. More extensive molecular systematic research is needed to elucidate the evolutionary history of S. weniae in the future. Based on morphological studies and phylogenetic analyses, Smilax weniae is confirmed to be a new species. We observed all suitable root apical materials for chromosome number, the number of chromosomes in Smilax weniae seems to be 60 ( Figure S3), while the number of chromosomes in most Smilax species is 2n = 32, and a few species are 26, 28, 30, 60, 96. This means that Smilax weniae might be a polyploid. However, whether it is an autopolyploid or an allopolyploid remains unclear, and the exact number of chromosomes still needs further verification.

Morphological Observation
In 2020 and 2021, several field excursions were made to Gengdushan, Libo County, Guizhou, China to observe this species. Specimens with pistillate or staminate flowers were collected in 2021. At the same time, several main herbaria in China (HZU, IBK, NAS, PE, acronyms according to Thiers 2020) [16] were consulted to check if similar specimens could be located. Indeed, we found two additional specimens in IBK belonging to the new species, both of which have fruits (Dianqiangui Team 70302, IBK00199287!; R.C. Peng et al. ML0339, IBK00312465!). Based on our field observations, recent collections, and these historical specimens, we documented the morphology of this new species.

Molecular Methods and Phylogenetic Analyses
For phylogenetic analyses, we sampled one individual of the new species from Libo County, Guizhou Province, China, and thirty-four species of Smilacaceae including representatives of clade C and clade D ( Table 2). Smilax aspera L. was set as an outgroup. All voucher specimens were deposited at the Herbarium of Zhejiang University (HZU) ( Table 2). Table 2. Taxa included in the present study with voucher information and GenBank accession numbers for the sequences of internal transcribed spacer (ITS) of nuclear ribosomal DNA, matK, rbcL, ndhA, ndhF and rpl16. Missing sequences are indicated by a dash (-). Accession numbers in bold are newly generated sequences.

Family
Species Voucher ITS matK rbcL ndhA ndhF rpl16 Total genomic DNA was extracted from silica-dried tissue following a modified cetyltrimethylammonium bromide (CTAB) protocol [17]. The aqueous phase was extracted with 24:1 chloroform/isoamyl alcohol, and after isopropanol precipitation, the DNA was resuspended in Tris-ethylenediamine tetra-acetic acid (TE) buffer (pH 8.0). Based on their suitability to address inter-specific phylogenetic questions, five plastid DNA fragments (ndhA, ndhF, rpl16, matK and rbcL) and a nuclear ribosomal ITS were employed for phylogenetic analyses. Amplification of the ITS region and rpl16 intron followed Cameron & Fu [2] and Fu & al. [18]. According to Shaw et al. [19], the PCR cycling conditions for the ndhA intron were 35 cycles of denaturation at 94 • C for 30 s, primer annealing at 55 • C for 30 s, and primer extension at 72 • C for 2 min. The primer design and amplification of rbcL and matK refer to Qi et al. [1]. According to Qi et al. [1], the amplification of the plastid matK gene was accomplished using designed primers 'M3': GCAACAAT-ACTTCCTATATCCGCTTCT and 'M4': GAACTCTTCTAATAATCCCGAACCTAA. The PCR cycling conditions were template denaturation at 94 • C for 6 min prior to the start of PCR cycles, then amplified for 35 cycles of 1 min at 94 • C, 1.5 min at 53 • C, 2 min at 72 • C, and one final cycle of 12 min at 72 • C. Forward and reverse sequences were assembled using GENEIOUS v11.1.5 (Biomatters Ltd., Auckland, New Zealand). All sequences were deposited in GenBank (Table 2).
Phylogenetic analyses were performed using Bayesian inference (BI) and maximum likelihood (ML). BEAST v2.4.3 [20] was used to run the BI analyses. We conducted two independent runs of 100 million generations, with samples saved every 5000 generations. After assessing the results in Tracer v1.6 [21], we discarded the first 10% of the trees as burnin. The log files were checked for convergence using Tracer. In both steps of our analyses, all ESS (explained sum of squares) values were well over 200; a maximum clade credibility tree was summarized with Tree Annotator v1.8.4 (included in the BEAST package). The ML analysis was performed using IQTREE v1.6.8 [22], of which the bootstrap values were calculated using 5000 replicates with the best selected TPM3u+F+R2 model. Trees were visualized using FigTree v1.4.3 [23].

Chromosome Counts
Chromosomal Counts were obtained by the root tip squash methods as described in Kong et al. [12]. The vigorously growing root tips were treated with 0.05% colchicine solution for 4 h, fixed with Carnot's fixative solution (glacial acetic acid: absolute ethanol = 1:3) for 24 h, and stored at 4 • C with 70% ethanol for later use. During tableting, the root tips were washed with distilled water, dissociated with 1 mol/L hydrochloric acid in a constant temperature water bath at 60 • C for 50 s, rinsed with distilled water, and then drip-dyed with a modified phenolic fuchsin solution (Carbol-Fuchsin) for tableting. Chromosome counts were observed for at least 30 complete metaphases. Chromosome spreads were observed using 100× light microscopy. Informed Consent Statement: Not applicable.

Data Availability Statement:
The molecular data that support the findings of this study are openly available in GenBank (see Table 2).