Rhamnella intermedia (Rhamnaceae), a new evergreen species from southwest Guangxi

Abstract Rhamnella intermedia, a new evergreen species from southwest Guangxi, is described and illustrated in this study. This species is similar to R. brachycarpa by the size and ratio of length to width of dried fruit and seeds, by which it differs from R. rubrinervis and R. tonkinensis. However, it differs from R. brachycarpa by rarely mucronate seed apices, larger ratio of length to width of leaves, leaf apices acuminate to long acuminate, shorter leaf petioles, and longer fruiting pedicels. Principal component analysis based on phenotypic traits further recognised three separated groups. Rhamnella rubrinervis and R. tonkinensis were clustered into one group; the other two groups represented R. brachycarpa and two Guangxi populations, respectively. Furthermore, phylogenetic analysis of nuclear ITS sequence variations highly supported that the two Guangxi populations represented an independent evolutionary lineage and were closest to R. rubrinervis. Four fixed nucleotide sites were found and were different from R. rubrinervis. However, besides the differentiated traits in seeds and fruit, densely pilose young branches also separated them from R. rubrinervis. In addition, during our field investigations, none of the three closely related species were found at locations where this new species was distributed. Therefore, this new species, based on the two Guangxi populations, is named R. intermedia. The key to four closely related species is also presented.


Introduction
Rhamnella (Miquel, 1867) in the tribe Rhamneae Hook. f. of Rhamnaceae is a small genus (Hauenschild et al. 2016, Richardson et al. 2000. It is characterised by pedicellate flowers and fleshy fruits, 1-stoned drupes, pinnately veined leaves, serrate leaf margins, semi-inferior ovaries, stipules without thorns, and flowers in axillary cymes (Chen and Schirarend 2007). To date, 10 species have been accepted into this genus (Lu and Sun 2019). Two groups are separated based on the characters of deciduous broad-leaved and evergreen leaves respectively (Fan and Yang 1997;Chen and Schirarend 2007;Lu and Sun 2019). For all species in the latter group, including R. rubrinervis, R. tonkinensis, and R. brachycarpa, the length of leaf petioles, leaf apices, ratio of length to width of leaves, and size and ratio of length to width of dried fruit and seeds are their differentiated traits (Chen and Schirarend 2007;Lu and Sun 2019). However, on the basis of these traits, two Rhamnella populations from southwest Guangxi that belonged to the evergreen group could not be ascribed to any of the three evergreen Rhamnella species. We found that they had similar size and ratio of dried fruit and seeds to R. brachycarpa, similar leaf shapes to R. rubrinervis, and the same length of fruiting petioles with R. tonkinensis. Therefore, the two Guangxi populations may represent a new species. In order to clarify this hypothesis, we carried out field investigations on the distribution and habitat of this potential new species, and characterised its morphology based on these two populations from southeast Guangxi. Furthermore, we conducted the principal component analysis (PCA) based on phenotypic traits to show their morphological differences. Finally, we sequenced the nuclear internal transcribed spacer (ITS) fragment to clarify its genetic distinctness.

Material and methods
We collected the fruit-bearing specimens of this potential new species for morphological measurement and other analyses referring to Lu and Sun (2019). These newly collected specimens in this study were deposited as Z.Q. Lu 2019YG2601-Z.Q. Lu 2019YG2619 (GXMI and HITBC), Z.Q. Lu 2018LZQ108 (HITBC), and Z.Q. Lu 2018LZQ10802 (HITBC). In order to demonstrate its morphological differences, we compared them to all closely related evergreen Rhamnella species that had been shown in Lu and Sun (2019), including R. rubrinervis, R. tonkinensis, and R. brachycarpa. In addition, 21 newly collected and 239 previous specimens were further used to perform the PCA based on 10 phenotypic traits, as described by Lu and Sun (2019). Because they were attributed to evergreen taxa closely related to R. rubrinervis, R. tonkinensis, and R. brachycarpa, and were distinctly different from deciduous species of Rhamnella by larger drupe size (Chen and Schirarend 2007;Lu and Sun 2019), we excluded all deciduous species from morphological comparison and PCA analysis. Furthermore, we investigated the population consensus and explored whether other closely related species co-occurred with this potential new species. The habitat and distribution were also characterised through our field investigations.
We also collected fresh leaves from several populations for DNA extraction and sequencing (Table 1). Taking habit differentiation into consideration, we marked each of the climbing trees. In total, 21 individuals (including six climbing trees and 15 erect ones) from the potential new species based on Pingxiang and Wude populations, 21 individuals from 6 populations of R. rubrinervis, one individual from R. tonkinensis, and one individual from R. brachycarpa were used for sequencing the nuclear ITS fragment. PCR amplification was performed according to Lu et al. (2018). In total, 5 of 21 individuals failed in the ITS sequencing, but the six climbing individuals were all sequenced successfully. Finally, we obtained 37 ITS sequences, including 7 types. All were deposited in GenBank database (Accession numbers from MT764159 to MT764165). In addition, we also downloaded some ITS sequences of deciduous species of Rhamnella. The aligned sequences were 630 bp in length. A maximum likelihood (ML) tree based on ITS sequences was constructed by MEGA version 5.0 (Tamura et al. 2011) using the Tamura-Nei model, and the bootstrap was set as 1000. Berchemiella wilsonii, B. berchemifolia, Pseudoziziphus celata, and P. parryi were selected as outgroups referring to the phylogenetic backbone presented by Hauenschild et al. (2016).
In total, two Guangxi populations characterised by the ratio of length to width of leaves and length of leaf petioles from R. rubrinervis, by the length of fruiting petioles from R. tonkinensis, and by the size and ratio of length to width of dried fruit and seeds from R. brachycarpa. Other phenotypic traits, such as leaf apices acuminate to long acuminate and rarely mucronate seed apices also showed a morphological combination from other species. PCA analysis distinguished 260 specimens into three groups, based on 10 phenotypic traits ( Figure 3). One group consisted of R. rubrinervis and R. tonkinensis, while the other two groups corresponded to R. brachycarpa and two Guangxi populations respectively. The first principal component axis (PC1; accounting for 34.40% of the variation) significantly separated these R. brachycarpa specimens and those of R. tonkinensis and R. rubrinervis into two clusters, and the two Guangxi populations showed the overlap with both clusters; the second principal component axis (PC2; accounting for 24.40% of the variation) significantly separated the two Guangxi populations from the other two clusters, and failed in the separation of others ( Figure 3). In addition, during our field investigations, none of the three closely related species R. brachycarpa, R. tonkinensis, and R. rubrinervis were found at locations where these two Guangxi populations distributed. Furthermore, phylogenetic analysis highly supported that the evergreen group was monophletic, and these two Guangxi populations represented an independent evolutionary lineage distinctly different from other species (Figure 4). They were closest to R. rubrinervis based on ITS sequence variations; however, four fixed nucleotide sites were found (Table 3). Sequence alignments showed that six climbing trees shared the same ITS sequence types with those of erect ones.
Etymology. Owing to its intermediate morphology compared to the other three closely related species, we provide the epithet intermedia.
Phenology. Flowering from June to September and fruiting from August to October. Habitat and distribution. To date, only two R. intermedia populations have been collected from southwest Guangxi. For its population census, more than 20 mature trees (3-6 m in height) and a large number of seedlings grow on the karst limestone hill. We also found that six individuals present a climbing habit at locations where there is a relative high canopy; however, all others are erect. Interestingly, they shared the same ITS types between erect and climbing trees, suggesting no genetic differentiation. In addition, this new species may be also distributed in Vietnam, because it is  Leaf blade elliptic-ovate, 6.5-11.2 × 3.1-4.5 cm, length-width ratio 2.1-2.8; base cuneate or nearly rounded, margin inconspicuously remotely serrate or subentire; apex short acuminate to long acuminate or acute; bracteole leaf similar to leaves in vegetative branches, but smaller Leaf blade elliptic-ovate, 5.8-10.3 × 3.1-4.8 cm, length-width ratio 1.9-2.4; base cuneate or nearly rounded, margin inconspicuously remotely serrate or subentire; apex short acuminate or acute; bracteole leaf similar to leaves in vegetative branches, but smaller Length of petiole 3-8 mm 3-9 mm 7-11 mm 7-12 mm Lateral veins on each side of midvein 6-7 5-8 5-6 5-7 Average distance between lateral veins located in the middle of leaf Notes. R. intermedia is morphologically similar to R. rubrinervis based on leaf characters, but they can be easily distinguished between each other by fruit and seed characters. However, it also can be significantly distinguished from R. rubrinervis by the densely pilose young branches, if the specimen has no fruit and seeds. In addition, it also needs to be mentioned that the flower of R. brachycarpa is 5-merous.

Discussion
Many differentiated phenotypic traits between the evergreen Rhamnella species have been demonstrated (Lu and Sun 2019), such as length of leaf petioles, ratio of length to width of leaves, leaf apices and size and ratio of length to width of dried fruit and seeds. In the present study, we proposed two Guangxi populations as a new evergreen species, based on the following evidence. First, they are ascribed to the evergreen group that are significantly different from those deciduous broad-leaved species within Rhamnella, at the species level (Fan and Yang 1997; Lu and Sun 2019). Other characters such as drupe and seed sizes can also separate these two groups (  (Table 2). Third, PCA analysis further sup- ported that they represent a distinct phenotypic cluster different from all published relatives (Figure 3; Lu and Sun 2019). However, the intermediate morphology of these two Guangxi populations also conforms to the characteristic of hybrids (Mallet 2005), which usually co-occur with parental species (Zha et al. 2010). Nevertheless, our field investigations show that none of the three closely related species co-occurred with this assumed new species. Furthermore, phylogenetic analysis of nuclear ITS sequence variations suggested they represented a distinct genetic lineage and closest to R. rubrinervis ( Figure 4). Four fixed nucleotide sites were found between these two Guangxi populations and R. rubrinervis in the present study. Therefore, they are not hybrids, but represent an independent evolutionary lineage sister to R. rubrinervis. This new evolutionary lineage is distinguished from R. rubrinervis by densely pilose young branches, smaller size of dried fruit and seeds, and smaller ratio of length to width of dried fruit and seeds (Figure 1; Chen and Schirarend 2007). In total, these two Guangxi populations are distinctly different from all the published relatives. Hence, the two Guangxi populations are proposed to be erected as a new species, named as R. intermedia.