A new species of Bredia (Sonerileae, Melastomataceae) from Sichuan, China

Abstract Bredia hispida (Sonerileae, Melastomataceae), a species occurring in southeastern Sichuan, China, is newly described based on morphological and molecular data. The generic placement of B. hispida is well supported by phylogenetic analysis and morphological characters, including basally cordate, hairy leaf blade, cymose inflorescence, basally gibbous anthers and enlarged ovary crown enclosing an inverted frustum-shaped depression. Both molecular and morphological divergence showed that B. hispida is well separated from its close relatives, justifying its recognition as a distinct species. The new species resembles B. repens, B. changii and B. guidongensis in the prostrate habit and isomorphic stamens but differs markedly in the unequal opposed leaves, the 2–4 mm long, stout bristles on the adaxial surface of leaf blade and acuminate leaf apex. Bredia hispida co-occurs with B. esquirolii in the wild. No morphologically putative hybrids between them were observed despite their overlap in flowering season. The isolating mechanism remains unclear, pending further investigation.


Introduction
Bredia Blume was originally established based on B. hirsuta Blume, a species endemic to Taiwan and the Ryukyu islands (Blume 1849). Circumscription of this genus had long been controversial. The dispute mainly concerned whether to include Tashiroea Matsum. ex T. Itô & Matsum. and certain species of Phyllagathis Blume in Bredia (Diels 1924(Diels , 1932Merrill and Chun 1940;Li 1944;Chen 1979Chen , 1984Hansen 1992;Chen and Renner 2007). By combining molecular phylogenetic and morphological data, recent studies have provided strong evidence for a new generic limit of Bredia (Zhou et al. 2019a(Zhou et al. , 2019b(Zhou et al. , 2019c. Bredia was redefined as excluding Tashiroea while incorporating seven species previously treated in Phyllagathis (Zhou et al. 2019b). Together with two recently published species, Bredia as currently circumscribed includes 23 species distributed from central and southern mainland China, Taiwan, to the Ryukyu islands and northern Vietnam (Zhou et al. 2019b;Wen et al. 2019;He et al. in press). Species of Bredia are characterized by the leaf blade papery, usually hairy, inflorescences cymose, umbellate, or a cymose panicle, anther basally gibbose or tuberculate, and ovary crown persistent and enlarged enclosing an inverted frustum-shaped depression.
During a survey of specimens in Chinese herbaria for a project on species delimitation of Bredia, several collections from Xuyong County, southeastern Sichuan Province, caught our attention. These collections (e.g. Fig. 1A-C) were identified as Phyllagathis deltoidea C. Chen ( Fig. 1D-F). Upon closer examination, however, the plants from Xuyong are morphologically quite different from P. deltoidea in having basally cordate (vs. cuneate) lamina adaxially hispid with stout long bristles (vs. puberulous and sparsely setose) and linear-lanceolate calyx lobes (vs. broadly triangular lobes) (Fig. 1). In addition, P. deltoidea is only recorded from its type locality in Ningming County, southwestern Guangxi, which is about 800 kilometers away from Xuyong County, southeastern Sichuan. Both morphology and distribution suggest that the current identification is erroneous.
In September 2019, we made a field expedition to Xuyong County and collected flowering and fruiting specimens of the plant in question (Figs 2, 3 Fig. 4), but differs markedly from the latter species in leaf morphology (Fig. 2). Judging from morphological aspects, this plant may represent an undescribed species in Bredia.
To test the generic affiliation of the unknown plant and its closest relative in the genus, we performed phylogenetic analyses based on DNA sequence data of nuclear ribosomal internal transcribed spacer (nrITS), sampling all species so far recorded in Bredia. We also calculated pairwise genetic distances among this plant and 23 species of Bredia to evaluate its distinctness. The results confirmed our suspicion that this plant represented a species new to science, which we described as B. hispida below.

Materials and methods
To test the generic affiliation of B. hispida and its position in the genus, we selected ingroup taxa based on previous studies (Zhou et al. 2019a(Zhou et al. , 2019b(Zhou et al. , 2019c. The final nrITS dataset contained 35 accessions representing Blastus Lour., Dissochaeta Blume,     Total DNA was extracted from fresh leaves using the modified CTAB procedure (Doyle and Doyle 1987). The nrITS region of B. hispida, B. violacea and B. reniformis were amplified and sequenced using universal primers ITS4 and ITS5 (White et al. 1990), following the procedure described in Zou et al. (2017).
Sequences were aligned using SeqMan v.7.1.0 (DNASTAR Inc., Madison, WI). The Akaike information criterion in Modeltest version 3.7 (Posada and Crandall 1998) was used to select the best-fitting nucleotide substitution model (GTR+G) prior to phylogenetic analyses. Bayes ian inference (BI), maximum likelihood (ML) and maximum parsimony (MP) analyses were performed using MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001), RAxML version 8.2.10 (Stamatakis 2014) and PAUP version 4a165 (Swofford 2002) respectively. For BI analysis, two independent Markov chain Monte Carlo analyses (MCMC) were performed with four simultaneous chains of 2,000,000 generations sampling one tree every 100 generations. The first 25% of trees were discarded as burn-in and the remaining were used to construct a majority-rule consensus tree with Bayesian posterior probabilities (PP). We verified that the average deviation of split frequencies had reached a value below 0.01 at the end of MCMC analyses. We also assessed the effective sample sizes (ESS) for all parameters and statistics using Tracer version 1.7.1 (Rambaut et al. 2018). ML analyses were performed under GTR+G model as recommended by the author. Node support was estimated with 1,000 bootstrap replicates using a fast bootstrapping algorithm (Stamatakis et al. 2008). For MP analyses, a heuristic search strategy was conducted of 1000 random addition replicates, with the tree-bisection-reconnection (TBR) branch swapping algorithm and MultTrees on. Maxtree was set to 500. Node support was evaluated by 1000 bootstrap replicates of 1000 random additions. Pairwise genetic distances among B. hispida and species of Bredia were calculated using the Kimura 2-parameter method (Kimura 1980).

Results
The aligned sequence matrix contained 665 characters. Statistics of sequences sampled were summarized in Supplementary material 2. The tree resulting from ML analysis is shown in Fig. 5,

Phylogenetic position and specific status of B. hispida
The placement of B. hispida in Bredia is supported by phylogenetic and morphological data. Our phylogenetic analyses with complete taxon sampling of Bredia confirmed that B. hispida is a member of this clade. Morphologically, its basally cordate, hairy leaf blade, cymose inflorescence, two whorls of eight isomorphic stamens, basally slightly gibbous anthers, decurrent connectives, and enlarged ovary crown during the fruiting stage all fit well within Bredia.
Bredia hispida is phylogenetically most closely related to B. repens, B. tuberculata and B. yunnanensis. It is a dwarf subshrub up to 15 cm tall with its middle and lower stem prostrate, which makes it easily distinguished from most species of Bredia, including B. tuberculata and B. yunnanensis. It closely resembles B. guidongensis (Fig. 4A), B. changii (Fig. 4B) and B. repens (Fig. 4C) in the prostrate habit and isomorphic stamens, but differs in leaf morphology: opposed leaves unequal, leaf blade larger (1.5-9.9 × 0.8-4 cm), stiffly papery, ovate to ovate elliptic and apically acuminate (Fig. 2B-E). Moreover, B. hispida is unique in the genus in its leaf blade adaxially hispid with 2-4 mm long, stout bristles (Fig. 2E), a character previously never recorded in Bredia. Pairwise genetic distances between B. hispida and remaining species of the genus range from 0.011 to 0.066, which are comparable to the distances of most species pairs in Bredia (0.005-0.077). Bredia hispida is therefore well diverged from other species of Bredia from a molecular perspective. Both molecular and morphological evidence justify the recognition of B. hispida as a distinct species.

Co-occurrence of B. hispida and B. esquirolii
Bredia hispida is currently only known from Xuyong County, Sichuan Province. It cooccurs with B. esquirolii (H. Lév.) Lauener, a species widely distributed in Guizhou, Chongqing and Sichuan. Bredia hispida grows on shady red sandstone cliff of seasonal waterfall whereas B. esquirolii is found in bushes, under forests and also on shady cliff (but a little further away from the dripping water). Several cases of sympatry have been observed elsewhere in the genus, viz. B. dulanica C. L. Yeh, S. W. Chung & T. C. Hsu and B. oldhamii Hook. f. in Taiwan, B. repens and B. latisepala (C. Chen) R. Zhou & Ying Liu in Hunan and B. esquirolii and B. tuberculata in Sichuan. In the first two cases, the co-occurring species have non-overlapping flowering seasons and thus interspecific reproductive isolation is easily maintained; in the third case, the flowering periods overlapped, and some putative hybrid individuals were found (unpublished data). During our visit in September 2019, both B. hispida and B. esquirolii were flowering. But no morphologically putative hybrids were observed. Pre-zygotic isolation via different pollinators is not a plausible explanation as flowers of the two species are of similar size (ca. 2 cm in diameter) and both can be visited by medium to small size bees. According to previous analyses (Zhou et al. 2019c), the crown age of the branch comprising close relatives of B. hispida, viz. B. esquirolii, B. repens, B. tuberculata and B. yunnanensis, was only 0.66-2.61 Mya. We suspect that other intrinsic postzygotic barriers may not have enough time to fully develop among such recently diverged species. The isolation mechanism between the sympatric B. hispida and B. esquirolii remains unclear, pending further study. Diagnosis. Resembles B. changii, B. repens and B. guidongensis in the prostrate habit and isomorphic stamens but differs from these species in its unequal leaves (vs. equal), stiffly papery leaf blade (vs. papery) hispid with 2-4 mm long, spreading stout bristles (vs. pubescent or villous with trichomes ≤ 1 mm) and acuminate apex (vs. obtuse or acute).
Phenology. Flowering July-September, young fruits in September. Etymology. The specific epithet is based on the spreading stout bristles on the leaf blade of this species.
Distribution. Bredia hispida is currently known from Xuyong County, southeastern Sichuan, China (Fig. 7). It occurs on damp steep red sandstone cliff, often below a seasonal waterfall, at 1000-1400 m.