Morphology and molecules support the new monotypic genus Fenghwaia (Rhamnaceae) from south China

Abstract Fenghwaia, a new monotypic genus, along with the new species Fenghwaia gardeniicarpa, is described from Guangdong Province, China. The combined features of inferior ovary, cylindrical drupaceous fruits and orbicular and dorsiventrally-compressed seeds with an elongate and pronounced basal appendage make the new genus significantly different from other genera of the family. In addition, its pollen morphology also showed great similarity to other species of this stenopalynous family. The molecular phylogenetic analysis, based on nuclear ribosomal internal transcribed spacer (ITS) and plastid trnL-F intron spacer (trnL-F) DNA sequence data from the new genus and the other 375 species representing 58 genera of Rhamnaceae, indicates that Fenghwaia is nested within the ‘rhamnoid’ group and sister to the tribe Rhamneae and then both sister to the tribe Maesopsideae. A taxonomic classification key to the ‘rhamnoid’ group is provided, based on morphological characters. A global conservation assessment is also performed and classifies Fenghwaia gardeniicarpa as Near Threatened (NT).


introduction
The buckthorns (Rhamnaceae Juss.) are a cosmopolitan family of small trees, shrubs, climbers and, occasionally, herbs and are well represented both in tropical and temperate regions (Raven and Axelrod 1974). This family includes approximately 900 species belonging to about 60 genera and 11 tribes. China hosts 13 genera and 137 species, distributed throughout the whole country, but mostly in south and southwest China (Chen and Schirarend 2007). The species of the family are mainly characterised by having basically cymose inflorescence mostly in the axillary position, usually 4-5-merous flowers, superior to inferior 2-4-loculed ovary with one ovule in each locule and indehiscent schizocarpic or capsular fruits (Medan and Schirarend 2004).
Rhamnaceae was recently revealed to be a monophyletic family and a member of the order Rosales with other eight families, viz. Rosaceae, Barbeyaceae, Dirachmaceae, Elaeagnaceae, Ulmaceae, Cannabaceae, Moraceae and Urticaceae on the basis of molecular evidence (APG IV 2016). The systematic treatment of Rhamnaceae, based on molecular data, morphological, anatomical and geographical information, indicated that Rhamnaceae should divided into three groups, viz. the 'rhamnoid' group, 'ziziphoid' group and the 'ampelozizyphoid' group (Richardson et al. 2000a(Richardson et al. , 2004Hauenschild et al. 2016b).
During our field investigation in 2018, a treelet of Rhamnaceae, morphologically similar to Sageretia species, but bearing cylindrical and more or less fleshy drupaceous fruits with an inferior ovary and persistent calyxes, somewhat similar to the capsules of Gardenia, was found along the riverside of a secondary broad-leaf forest adjacent to a reservoir in Taishan, Jiangmen City, Guangdong Province. This plant is very different from any known species and evoked great interest for clarifying the taxonomic name and its phylogenetic relationship. Several field expeditions from March to August 2019 were subsequently undertaken to clarify its inflorescence and flower characters. Phylogenetic analysis, based on ITS and trnL-F, along with morphological comparisons, suggested that this species is best treated as a new taxon belonging to a new genus of Rhamnaceae.

Material and methods
All morphological data of the new species were collected by Light Microscope and Stereomicroscope. Palynological observations followed Guo and Wang (2011). The voucher specimens have been deposited at South China Botanical Garden, Chinese Academy of Sciences (IBSC).
Since nuclear ribosomal internal transcribed spacer region (ITS) and plastid trnL-F intron spacer region (trnL-F) were already shown to include sufficient information to reconstruct well-supported topologies in Rhamnaceae (Hauenschild et al. 2016a, b), hence a total of five genomic DNAs were extracted from five different individuals, using a modified cetyltrimethylammonium bromide (CTAB) method (Allen et al. 2006).
The primers and PCR protocols were outlined by Hauenschild et al. (2016a, b). The PCR products were sent to Sangon Biotech (Shanghai, China) and sequencing was conducted using an ABI 3730xl DNA Analyzer (Applied Biosystems, Invitrogen, Foster City, CA, USA).
Multiple locus alignment of 590 operational taxonomic units (OTUs) was performed by Mafft v 7.453 (Katoh et al. 2002) with default parameters and ambiguous positions in the alignment were removed by GBlocks v 0.91b with the parameters (-b4 = 5, -b5 = h) (Castresana 2000). After filtering, the best-fit model (TIM+F+R4) was selected on the basis of the Bayesian Information Criterion (BIC) using Modelfinder (Kalyaanamoorthy et al. 2017) and the phylogenetic tree with the Maximum Likelihood (ML) method was performed by IQ-TREE v1.6.12 (Nguyen et al. 2015). Ultrafast bootstrap values were calculated with 1000 random replicates (Hoang et al. 2018). For Bayesian Inference (BI), we used the GTR+G+I model and performed four independent Markov Chain Monte Carlo (MCMC) reactions in MrBayes v3.2.6 (Ronquist and Huelsenbeck 2003), running five million generations every Markov Chain, sampling one tree every 1,000 generations, rejecting 25% of the trees as burn-in after the value of average standard deviation of split frequencies was lower than 0.01.

Palynology of Fenghwaia gardeniicarpa
Rhamnaceae is a stenopalynous family (Erdtman 1952). The pollen morphology of the Rhamnaceae usually has suboblate to oblate spheroidal or subprolate, a distinct triangular shape in polar view, oblate shape in equatorial view and 3-zonocolporate features. It does not show any special features and the descriptions usually concur with each other (Punt et al. 2003). The tectum of Rhamnaceae can be microreticulate, striate or rugulate to reticulate, baculate, verrucate, psilate, with more or less densely-spaced perforations and this variation of exine ornamentation has usually been used for classifying the pollen type (Schirarend and Köhler 1993;Medan and Schirarend 2004;Perveen and Qaiser 2005). In general, the main features of pollen grains of Fenghwaia gardeniicarpa are consistent with those of most other Rhamnaceae species with respect to the shape, polarity, symmetry, aperture number and position, size and tectum ornamentation (Medan and Schirarend 2004). However, the exine ornamentation of Fenghwaia gardeniicarpa is more similar to that of the tribe Rhamneae, as observed in Berchemia (reticulate), Frangula, Rhamnus (suprareticulate-rugulate, psilate), Sageratia (fossulate-perforate) than to that of the Maesopsistype (baculate) (Punt et al. 2003;Perveen and Qaiser 2005;Naimat et al. 2012).

Phylogenetic relationship of Fenghwaia
Based on a phylogenetic analysis of rbcL and trnL-F sequences of the plastid genome, Richardson et al. (2000b) outlined a new tribal classification of Rhamnaceae, recognising 11 tribes encompassing the informally-named groups of 'rhamnoid', 'ampeloziziphoid' and 'ziziphoid'. Our phylogenetic analysis showed that Fenghwaia is nested within the 'rhamnoid' group. Thus, the 'rhamnoid' group consists of the tribes of Rhamneae, Maesopsideae and Ventilagineae and the genus Fenghwaia. The weak support (ML = 75/BI = 0.55) of the clade Rhamneae/Fenghwaia was probably caused by lack of adequate informative sites in the applied fragments. For example, only trnL-F sequences of Maesopsis eminii were applied in the present analysis. Morphologically, Rhamneae can be easily recognised by its fleshy fruit and 2-or 4-locular ovary. Maesopsideae and Ventilagineae have superior or half inferior ovaries, 1-2-locular, drupe (tribe Maesopsideae), samara or rostrate capsules (tribe Ventilagineae). Moreover, Maesopsis differs from all other genera in Rhamnaceae in its single-celled ovary and a style laterally attached to the fruit, rather than apically. Ventilagineae is unique in its fruits with a pronounced apical appendage (Richardson et al. 2000b).
Fenghwaia has an inferior and 3-loculed ovary, orbicular and dorsiventrally compressed seeds with an elongate and pronounced basal appendage, but its morphological characters are obviously different from those of other taxa in the 'rhamnoid' group. In addition, the genera that have an inferior ovary and 3-locular in Rhamnaceae are all in the ziziphoid group, viz. Phylica, Trichocephalus and Nesiota of the tribe Phyliceae, all genera of the tribe Gouanieae, Siegfriedia, Spyridium and Cryptandra of the tribe Pomaderreae, and Alphitonia and Granitites of the undefined tribe.
A taxonomic classification key of the 'rhamnoid' group