﻿Didymocarpuspingyuanensis (Gesneriaceae), a new species endemic to Danxia landscape from Guangdong Province, China, and two new combinations in Didymocarpus

﻿Abstract Didymocarpuspingyuanensis, endemic to the Danxia landscape in Pingyuan County, Guangdong, China, is described and illustrated here. This species can be distinguished from other members of Didymocarpussect.Heteroboea by its calyx deeply 5-lobed to about three quarters of its length. The phylogenetic position of the new species within Didymocarpus was examined using nuclear ribosomal internal transcribed spacer (ITS) sequences. Based on phylogenetics analysis and morphological evidence, we propose two new combinations, elevating the two varieties to species level, namely D.yinzhengii and D.gamosepalus.


Introduction
Didymocarpus Wall.was once a large genus with approximately 200 species (Weber and Brutt 1998).Recent molecular phylogenetic studies and morphological revisions of Didymocarpus have led to a reduction in the estimated number of species from 200 down to 60-80, with some species being transferred to Henckelia Spreng., Hovanella A. Weber & B.L.Burtt, Petrocodon Hance (Weber and Brutt 1998;Weber et al. 2000Weber et al. , 2011;;Möller and Clark 2013).
During a field investigation of Danxia landscapes in Pingyuan County, Guangdong, in April 2023, we encountered a Didymocarpus species in bloom and confirmed its classification within sect.Heteroboea of this genus as it was stemless herb (Wang et al. 1990).However, it differs from all known species in this section by having a calyx that is deeply 5-lobed to about three quarters of its length.After thorough morphological comparisons using herbarium specimens, digital images, and relevant literature on other similar species, we concluded that this plant represents an undescribed species.Here, the putative species is described and illustrated based on morphological observations and compared with closely related species.Additionally, we used nuclear DNA internal transcribed spacer (ITS) to reconstruct the phylogeny to evaluate the phylogenetic position of Didymocarpus pingyuanensis.

Morphological study
We used a micrometer and a stereomicroscope to observe and measure the morphological traits of the putative species.Morphological comparisons between Didymocarpus pingyuanensis and its related species were based on dry specimens we collected, relevant literature (Wen et al. 2013;Li and Li 2014;Xu et al. 2019;Xie et al. 2020), as well as digital images on the Chinese Virtual Herbarium (https://www.cvh.ac.cn/) and the China Field Herbarium (https:// www.cfh.ac.cn/).Morphological observation was conducted in the Herbarium of Sun Yat-sen University (SYS).

Taxon sampling and molecular analysis
The ITS region was used for examining the phylogenetic position of the putative species.During May 2023, we collected 6 taxa belonging to sect.Heteroboea for this study.These taxa included Didymocarpus cortusifolius (Hance) H.Lév., D. salviiflorus Chun, D. lobulatus, D. heucherifolius var.heucherifolius Hand.-Mazz,D. heucherifolius var.yinzhengii, D. heucherifolius var.gamosepalus.Except for D. heucherifolius var.gamosepalus, all species were collected from their type localities.The population of D. heucherifolius var.gamosepalus at its type locality has gone extinct due to human disturbance caused by nearby village activities.Consequently, we were unable to find it at its type locality in Pingyuan County, Guangdong.Instead, we collected specimens from Zijin County, Guangdong.Voucher specimens were deposited in SYS.We sequenced their ITS gene sequences to verify the molecular differences; the sequences have been uploaded to GenBank.Thirty-eight species of Didymocarpus and related genera with ITS gene sequences in NCBI GenBank were downloaded for analysis.In total, we got 45 ITS gene sequences of 45 taxa, GenBank accession numbers were followed after their Latin name in the phylogenetic tree.These taxa encompass three Gyrocheilos W.T.Wang species, one Allocheilos W.T.Wang species, one Raphiocarpus Chun species, six Primulina species, three Petrocodon species, nineteen species from Didymocarpus sect.Didymocarpus and nine from Didymocarpus sect.Heteroboea.Sinningia incarnata (Aubl.)D.L.Denham and S. tubiflora Fritsch were selected as outgroups.All but one species of Didymocarpus sect.Heteroboea were included in this study.The excluded species was Didymocarpus reniformis W.T.Wang, which was not located during multiple expeditions, and only imprecise locality data were available.
Total DNA was extracted from silica-gel-dried leaves using the modified cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle 1987).Polymerase chain reaction was carried out based on the program setting as proposed by Lee et al. (2022) using the universal primers, ITS1 and ITS4 (White et al. 1990).The ITS sequence was aligned with Clustal W which is embedded in MEGA-11 (Tamura et al. 2021).The sequences in the alignment were manually adjusted.Phylogenetic trees were reconstructed using the maximum likelihood (ML) methods and Bayesian inference (BI) methods via MEGA-11 (Tamura et al. 2021) and MrBayes v3.2.1 (Ronquist et al. 2012).The optimum DNA substitution model calculated using the "Find best DNA/ Protein Models (ML) function embedded in MEGA-11 was Kimura 2-parameter model (K2) with Gamma Distributed With Invariant Sites (G+I) (=K2+G+I).All branch nodes were calculated with 1000 bootstrap (BS) replicates.BI analysis employed random starting trees and four Markov chain Monte Carlo (MCMC) simulations were run simultaneously and sampled every 1000 generations for 1 million generations.Bayesian posterior probabilities (PP) were calculated as the majority consensus of all sampled trees with the first 25% discarded as burn-in.

Morphological comparison
In Didymocarpus sect.Heteroboea, most species exhibit similar vegetative characteristics, except for D. dissectus, whose leaf margin is irregularly and distinctly 3-or 4-lobed in the distal 1/3-1/2.There are two varieties, D. heucherifolius var.gamosepalus and D. heucherifolius var.heucherifolius distributed in Guangdong Province, China.Both D. pingyuanensis and D. heucherifolius var.gamosepalus share the same distribution area, namely Pingyuan County in Guangdong, and both are distributed within the Danxia landscape.During the field investigation, we also found D. heucherifolius var.heucherifolius in Wuzhishi scenic spot, which is about 50 km from the locality of D. pingyuanensis.However, D. pingyuanensis can be distinguished from D. heucherifolius var.heucherifolius by its glabrous corolla.Furthermore, it can be distinguished from D. heucherifolius var.gamosepalus by its shorter corolla, and calyx deeply 5-lobed to about three quarters of the calyx length, lobes equal, obovate, apex rounded, rarely cuspidate, overlapping at margin.
Although Didymocarpus pingyuanensis and D. salviiflorus share calyx lobed and overlapping at margin, however, the former is deeply 5-lobed to about three quarters of the calyx length, contrasting with the latter 5-lobed to about half of the calyx length from the base.Additionally, D. pingyuanensis can also be distinguished from D. salviiflorus by bracts free, elliptic, and corolla glabrous outside.
Differences between the putative species and its morphologically related species, Didymocarpus heucherifolius var.heucherifolius, D. heucherifolius var.gamosepalus, and D. salviiflorus are shown in the following identification table (Table 1) and Fig. 2.
Phenology.The flowering of Didymocarpus pingyuanensis is from April to May; and the fruiting is in June.
Distribution and habitat.Didymocarpus pingyuanensis so far has only been found on three Danxia landscapes within Pingyuan County.It is locally abundant and endemic to a narrow area near the type locality.This species prefers moist, but sunny cliffs, at an elevation of 100-500 m a.s.l.In Pingyuan, the average temperature is 21.7 °C, and the average annual precipitation is 1637 mm.

Figure 1 .
Figure 1.Bayesian inference (BI) tree based on ITS sequences of the new species Didymocarpus pingyuanensis and related species.Bayesian posterior probabilities are shown along the branches.The new species described in this study is shown in bold.

Figure 3 .
Figure 3. Didymocarpus pingyuanensis A habitat in flowering B adaxial surface view of leaf blade C abaxial surface view of leaf blade D capsule E front view of corolla F lateral view of corolla, showing calyx deeply 5-lobed to about a quarter of the calyx length from the base G bracts H opened corolla I staminodes J pistil K stamens (Photographers: A-K by Qiang Fan).

Figure 4 .
Figure 4. Didymocarpus pingyuanensis Ling H. Yang, Q. Fan & F. Wen A habit B flower in front view C flower in lateral view D opened corolla, showing stamens and staminodes E stamens F pistil and stigma (Drawn by Rong-En Wu).

F
PhytoKeys 244: 213-224 (2024), DOI: 10.3897/phytokeys.244.126137Ling-Han Yang et al.: A new species and two new combinations in Didymocarpus var.gamosepalus have been submitted to the NCBI website.The ITS sequence data supporting the findings of this study are available in Fig. 1.