﻿Actinostephanus (Gesneriaceae), a new genus and species from Guangdong, South China

﻿Abstract Actinostephanus, a new genus from southern China, is described and colorfully illustrated with a single species, A.enpingensis. This new genus is morphologically most similar to Boeica and Leptoboea, nevertheless, it can be easily distinguished from the latter two by the following characteristics, such as leaves in whorls of three, all closely clustered at the top; corolla bowl-shaped, 5-lobed, actinomorphic; capsule hard, oblong-ovoid, short, 3–4 mm long, densely appressed villous, wrapped by persistent densely pubescent calyx lobes, style persistent. The new genus and related genera were sequenced using the next-generation sequencing technique. The whole plastid genome of the new genus is 154, 315 – 154, 344 bp in length. We reconstructed phylogenetic trees using the dataset of 80 encoded protein genes of the whole plastid genome from 47 accessions based on ML and BI analyses. The result revealed that the new genus was recovering in a polytomy including Boeica, Rhynchotechum, and Leptoboea with strong support, congruent to the morphological evidence. A global conservation assessment was also performed and classifies A.enpingensis as Least Concern (LC). In addition, after a review of recently described species of Gesneriaceae, we propose that plant enthusiasts, especially Gesneriad fans, have been playing an increasingly important role in the process of new taxa-discoveries.

Apart from morphological data, recognizing and describing new taxa with molecular evidence will result in a more robust and rational taxa ). The first Chinese paper to mention the molecular method, estimated a molecular phylogeny of the previous subfamily Cyrtandroideae using two DNA regions . However, the Chinese described new taxa of Gesneriaceae in China using molecular evidence starting around 2010. The first one was a new genus, Litostigma Y.G. Wei, F.Wen & M.Möller. It was confirmed and published using classical taxonomy, palynology, and phylogenetic analysis (Wei et al. 2010). Since then, some redefined genera, such as Hemiboea Clarke (Weber et al. 2011a), Loxostigma Clarke (Möller et al. 2014), Oreocharis , Paraboea (Clarke) Ridley (Puglisi et al. 2011), Petrocodon Hance (Weber et al. 2011b), Primulina Hance (Weber et al. 2011c), etc., and newly divided or restored genera, for example, Glabrella Mich. Möller & W.H.Chen (Möller et al. 2014) and Bournea Oliv. (Chen et al. 2020), were confirmed by the molecular and morphological evidence.
In 2017, a plant enthusiast, Mr. Yi Huang, sent the authors some photos of a rare and distinct Gesneriaceae plant, and we considered it a new taxon but could not undertake further studies since no material was collected at that time. Coincidentally, in 2019, one of the authors, Mr. Hui-Feng Wang, collected this species while undertaking a field trip in southern Guangdong, China.
After a careful morphological comparison, we could not place it into any genus of Gesneriaceae despite it sharing some similarities with Boeica or Leptoboea. To better understand the generic placement of this species, molecular phylogenetic analysis was also performed. After consulting the relevant literature Li and Wang 2005;Wei et al. 2010;Wei 2018;Wen et al. 2019) and the molecular evidence, we concluded that this new species was assignable to a new genus, Actinostephanus gen. nov.

Ethics statement
The only known location where this new species was found and collected was in the Qixingkeng provincial natural reserve, Enping, Guangdong. Two authors, Ms. Xiao-Yun Chen and Mr. Jun-Jie Liang, are staff at this natural reserve. They helped us get specific permission to enter the reserve and collect specimens. Our field studies did not involve any endangered or protected species. Further, special permits to conduct this research were not required.

Material collection
This new species/genus has been monitored in the field by staff from Qixingkeng provincial natural reserve and grown by the authors at the nursery of the Gesneriad Conservation Center of China (GCCC) and National Gesneriaceae Germplasm Resources Bank of Guangxi Institute of Botany (GXIB) since the plants were collected. We also collected leaf materials of this proposed new species, using silica gel to dry them in the field for DNA extraction.

Genomic DNA extraction and sequencing
Leaf material for DNA extraction was dried using silica gel (Chase and Hills 1991). Genomic DNA was extracted using the CTAB protocol (Doyle and Doyle 1987). The total gDNA sample was sent to Majorbio (http://www.majorbio.com/, China) for library construction and next-generation sequencing. Short-insert (350 bp) paired-end read libraries preparation and 2 × 150 bp sequencing were performed on an Illumina (HiSeq4000) genome analyzer platform. Approximately 6 Gb of raw data for the new species was filtered using the FASTX-Toolkit to obtain high-quality clean data by removing adaptors and low-quality reads (http://hannonlab.cshl.edu/fastx_toolkit/download.html).

Whole plastid genome assembly and annotation
Clean reads were paired and imported in Geneious Prime (Kearse et al. 2012). For plastid genome assembly, the clean reads were mapped to published plastid genome sequence (Petrocodon jingxiensis, Genbank accession number: NC_044477.1) as reference  using the Fine Tuning option in Geneious Prime (iterating set as 10 times) to exclude nuclear and mitochondrial reads. Then, de novo assembly was performed using Geneious Prime with a medium-low sensitivity setting to assemble the plastid genome sequence. The clean reads mapped the generated contigs using the Fine Tuning option in Geneious Prime (iterating set as 10 times) to fill gaps. Contigs could be concatenated using the Repeat Finder option implemented in Geneious Prime until a ~130 kb contig (including SSC, IR, and LSC) was built. The Inverted Repeat (IR) region was determined by the Repeat Finder option in Geneious Prime and was reverse copied to obtain the complete plastid genome. The annotation approach of the plastid genome was performed using CPGAVAS2 and PGA (Qu et al. 2019;Shi et al. 2019).

Phylogenetic analyses
To confirm the placement of this new plant, we reconstructed phylogenetic trees using the dataset of 80 encoded protein genes of the whole plastid genome. The new plant is morphologically similar to Boeica or Leptoboea, both of which belong to Subtr. Leptobaeinae C.B.Clarke (Clarke 1883). Therefore, we sampled all genera within this subtribe except for Championia Gardner and representatives of other subtribes within the Gesneriaceae as in-group, and 11 species represented other families as out-group. Consequently, 11 accessions were newly generated, while 36 accessions were downloaded from NCBI. Sequences obtained from this study and their information are listed in Appendix I.
All gene sequences were extracted using the PhyloSuite v1.2.2 ) and aligned by MAFFT v7.4 (Katoh and Standley 2013). The aligned sequences were then concatenated with PhyloSuite v1.2.2 . Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian inferences (BI), respectively. For the BI tree, we employed MrBayes v3.2.6 (Ronquist et al. 2012) to obtain a maximum clade credibility (MCC) tree. The parameters set as follows: nst = 6, rates = invgamma. Bayesian inference was performed with the concatenate sequence, using two million generations, two runs, four chains, a temperature of 0.001, and 25% trees were discarded as burn-in, and trees were sampled every 1,000 generations. Then, we used ModelFinder (Kalyaanamoorthy et al. 2017) to find the best fit model for ML analysis and further conducted the ML tree using IQ-TREE v2.1.2 (Nguyen et al. 2014) with 1000 bootstrap replicates. Tree visualization was achieved in Figtree v1.4.3.

Characteristics of the complete plastid genome and ribosomal DNA
The complete plastid genome of Actinostephanus enpingensis comprised 154,315 -154,344 bp (Fig. 1). The characteristics and statistics of the plastid genome are summarized in Tables 1, 2.

Molecular phylogenetic studies
BI and ML analyses of the dataset of 80 encoded protein genes of whole plastid genome resulted in the identical tree topologies that both indicate the three accessions of the new plant formed a strongly supported clade that was recovering in a polytomy including Boeica, Rhynchotechum, and Leptoboea in the clade of Subtr. Leptobaeinae (posterior probability (PP) = 1, bootstrap support (BS) = 100%) (Fig. 2).

Ecology
Plants of the new taxon were primarily accessible in the Qixingkeng provincial natural reserve, growing on nearly vertical or steep slopes of montane yellow soil under tropical evergreen broad-leaved forest. Voucher specimens were made in the usual way (Bridson and Forman 1998) from some accessible plants that could be reached from the type locality. The conservation assessment was prepared following IUCN (2019).  Notes: Genes with one or two introns are indicated by one (*) or two asterisks (**), respectively. Genes in the IR regions are followed by the (×2) symbol. Benth. according to the molecular evidence and some morphological data, but differs from the latter two by the following distinguishing characters: leaves in whorls of three, all closely clustered at the top; corolla bowl-shaped, 5-lobed, actinomorphic; capsule hard, oblong-ovoid, short, 3-4 mm long, densely appressed villous, wrapped by persistent densely pubescent calyx lobes, style persistent. The detailed distinguishing characters of this new genus and its congeners are listed in Table 1.

Subfam. Didymocarpoideae
Type and only known species. Actinostephanus enpingensis F.Wen, Y.G. Wei & Z.B.Xin, sp. nov. Description. Herbs, perennial, acaulescent, or forming elongated rhizome slightly fleshy growing after some years, rhizomes cylindrical, surface densely brown pubescent, fibrous root filiform, forming adventitious buds and plantlets in the middle or at the end of the fibrous root. Leaves all basal, whorls of three, sometimes opposite, all closely clustered at the top, forming a rosette, or clustered forming a rosette at the top of the rhizome after years of growth. Leaf-blades obovate elliptic, asymmetric, rarely symmetric, attenuate to base and base usually oblique, rarely aequilateral. Bracts 2. Calyx actinomorphic, 5-parted to the base. Corolla actinomorphic, bowl-shaped; tube very short, shallow bowl-shaped; limb quinquelobate, lobes equal. Stamens 4, separated, anthers dorsifixed, free, dehiscing longitudinally. Disc glabrous, margin crenulate. Ovary conical, stigma punctate. Capsule oblong-ovoid, appressed villous, wrapped by persistent calyx lobes, and the abaxial surfaces of calyx lobes covered densely pubescent. The number of seeds per capsule fewer. Seeds bigger, elliptic, both ends pointed.
Preliminary conservation status. Based on the result of our joint field surveys in the type locality and adjacent regions, the EOO and AOO of Actinostephanus enpingensis are about 79.5 km 2 and 0.1 km 2 , respectively. So far, only one population of this species has been recorded along the local stream in the Qixingkeng provincial natural reserve, Enping city, Guangdong province, southern China, but we believe that more A. enpingensis populations can be found in the hills of Enping and its adjacent counties. If that is the case, the Extent of Occurrence (EOO) and Area of Occupancy (AOO) of this species will increase. Because the flowers and leaves of this species are inconspicuous, and after learning from some local people that it has no known medicinal value we feel that this species faces little risk. Moreover, almost all of these plants are growing in the protected areas of this reserve so that the species are well protected. According to the Guidelines for Using the IUCN Red List Categories and Criteria (IUCN 2019), we access this taxon as a Least Concern species (LC).

Discussion
Our phylogenetic studies revealed that the new plant fell into Subtr. Leptoboeinae C.B.Clarke (PP = 1, BS = 100%). This subtribe belongs to Tribe Trichosporeae Nees, Subfamily Didymocarpoideae. At present, six genera have been included in this subtribe (Möller et al. 2017). Although their morphologies from different genera of this Subtribe are heterogeneous, several characters, such as the absence of large flowers, the inconspicuous to capitate stigma, straight but no-twisted fruits, and commonly 4-valved and dehiscent capsules or fleshly berries were concluded as common ones (Weber at al. 2013, Weber at al. 2020. The new plant is morphologically congruent with these characters that further indicate the monophyly of Subtr. Leptoboeinae. Within Subtr. Leptoboeinae, the new plant, was recovering in a polytomy including Boeica, Rhynchotechum, and Leptoboea. Two Boeica spp. are most closely related to Rhynchotechum, and both sisters to the type species of Boeica (B. furruginea) indicated that Boeica is not monophyletic. This relationship was congruent with previous studies (Yang et al. 2020). Expanding the sampling, and exploring key characters, is needed to re-define the Boeica. Despite this, the new genus is morphologically similar to these genera based on phyllotaxis and inflorescence cyme. However, it can be easily distinguished by corolla bowl-shaped, 5-lobed, actinomorphic, capsule densely appressed villous, wrapped by persistent densely pubescent calyx lobes and style persistent ( Table 3). The most distinct characters of the new plant are its actinomorphic corolla, tiny fruit hard when mature, rarely dehiscent, occasionally split into 4-valves, style usually persistent, which are likely to be derived characters or autapomorphies. We, therefore, based on the molecular and morphological evidence, treat it as a new genus, namely, Actinostephanus.
The high levels of plant species diversity and endemism in southern and southwestern China are more and more renowned, especially in karst regions. Nevertheless, it is evident that the geographic accessibility of those mountainous areas (including townships, villages, and surrounding regions) has been hindered by terrible transport problems. It also seriously affected the understanding of plant diversity in South and Southwest China. But with the fast development of the Chinese economic and construction systems, more and more road construction projects are being carried out, forming a relatively completed road transportation system in China. Thus, there are more opportunities to discover many taxa new to science. The people have easy access to those places that were difficult to reach in past decades.
Not only do more and more taxonomists focus on the biodiversity of Gesneriaceae in China, but plant enthusiasts are also making an enormous contribution to help botanists discover rare and new Gesneriads. As previously mentioned, Mr. Yi Huang, a plant enthusiast, found the interesting Boeica-like species of Gesneriaceae in South China, and he offered this critical information to us. Thus, we will be more conscious of this uncertain species over the next few years. Therefore, we propose that plant enthusiasts, especially Gesneriad fans, are playing an increasingly important role in the process of new taxa-discoveries. Several