Primulina malipoensis (Gesneriaceae), a new species from Sino-Vietnamese border area

Abstract Primulina malipoensis, a new species from limestone areas around the Sino-Vietnamese border, is described and illustrated. This new species is morphologically similar to P. maguanensis and P. lungzhouensis, but obviously differs from the latter two species by its pale greenish-yellow flowers (vs. purple, with different colour patterns). The phylogenetic affinity, illustration and photographs of this new species are provided in this paper.


Introduction
The recently redefined Primulina Hance has become a species-rich genus within the subfamily Didymocarpoideae of Gesneriaceae (Wang et al. 2011, Weber et al. 2011 and its species diversity is still growing due to numerous new species being constantly discovered (e.g. Pan et al. 2013, Guo et al. 2015, Lai and Wen 2015. This group shows high levels of endemism and ecological (edaphic) specialisation (Hao et al. 2015). The majority of its species occur in karst areas of southern and south-western China and northern Vietnam, with narrow, island distributions, often limited to a single cave or karst limestone hill system (Wang et al. 1998, Li and Wang 2004, Wei et al. 2010. Local-scale mosaics of soil type are ubiquitous features in the karst landscapes and thus, soil nutrient availability may influence diversification and speciation of Primulina via local adaptation to specific edaphic microhabitats (Hao et al. 2015). However, in the P. eburnea complex, geographical isolation has been shown to be a major driver of its diversification and speciation in Primulina (Gao et al. 2015, Wang et al. 2017. During field explorations in 2013, one of the authors (JC) found an unknown species of Primulina near the Sino-Vietnamese border at Malipo County, southeastern Yunnan, China. Several living individuals from the population found in the field were brought to the South China Botanical Garden (SCBG) and cultivated there. These plants showed leaf blade characteristics very common in Primulina. However, when flowering, they displayed uncommon yellow flowers. Flower colour has been used as an important character for the description of new Primulina species (Pan et al. 2016, Yang et al. 2017. Therefore, these plants soon caught the authors' attention. Checking of specimens and literature studies were undertaken immediately. When specimens were checked in KUN (by its online service), an interesting specimen was found (numbered KUN 1275938), which possesses a similar leaf to these plants and had been collected from nearly the same locality as the findings. This specimen was identified as Chirita eburnea (a synonym to P. eburnea). However, this specimen was represented by only a piece of leaf and without flowers, thus, its identification is doubtful. To further reveal the true taxonomic identity of both of these plants and the specimen, other field works were carried out by one of the authors (FW) in 2017. Fortunately, he found this species at the recoded site of the specimen (KUN 1275938) and also found other populations at a nearby location in Vietnam. At the same time, additional investigations, i.e. phylogenetic analysis and morphological comparison, were undertaken. Based on these results, all of these plants from the three populations are considered as the same new species, which is described and illustrated here.

Methods
Morphological observations were carried out using living cultivated plants (ten individuals) as well as dried specimens. All morphological characters were measured using dissecting microscopes and descriptions were made following the terminology presented by Wang et al. (1998). Literature studies included all relevant monographs (Wang et al. 1998, Li and Wang 2004, Wei et al. 2010) and recently published literature (Xu et al. 2008, 2012, Li and Möller 2009, Pan et al. 2013, 2016, Li et al. 2014, Lai and Wen 2015, Guo et al. 2015. Checking of specimens was undertaken at IBSC and IBK and with the help of web databases (Chinese Virtual Herbarium: http://www.cvh.ac.cn/; Herbarium, Kunming Institute of Botany, CAS: http://www.kun.ac.cn/; Global Plants: http://plants.jstor.org/). A map of the species' geographical distribution was prepared based on field records. The molecular phylogenetic analyses of the species were included in a broader study in which the most comprehensive species-level phylogeny of Primulina was reconstructed based on 20 plastid and nuclear regions  Diagnosis. Primulina malipoensis mainly differs from P. maguanensis and P. lungzhouensis by its pale greenish-yellow flowers (vs. purple, with different colour patterns). This new species can further be distinguished from P. maguanensis by its greenish bracts (vs. white) and from P. lungzhouensis by its entire bracts margin (vs. denticulate).

Distribution and habitat.
Primulina malipoensis is a narrowly endemic species restricted to a small area at both sides of the Sino-Vietnamese border (Xiajinchang Town, Malipo County, Yunnan Province, China. Khau La Village, Quyet Tien Community, Quan Ba District, Ha Qiang province, Vietnam.) (Figure 4). It grows on moist and shady limestone rocks, at ca. 1000-1500 m altitude.
Conservation status. Based on the field investigations, Primulina malipoensis is currently only known from three sites around the Sino-Vietnamese boundary. Each population possesses no more than 150 mature individuals. However, the type population, which grew close to a road, had disappeared in 2017 and thus, the primary reason why it disappeared is probably due to its destruction by human activities. Based on currently available information, P. malipoensis should be considered as Endangered (EN): B1b(iii,v)c(iv)+2b(iii,v)c(iv); C2b, following the IUCN Categories and Criteria (IUCN 2016).
Phenology. This new species was observed flowering from June to July and fruiting from August to September.
Etymology. The specific epithet is derived from the place, Malipo County in Yunnan province, China, where the new species was first found.
Note. Primulina malipoensis (Figures 1 and 2) can be morphologically connected to P. maguanensis  Figure 3D-E) by its ovate or broadly elliptic leaf blade, with inconspicuously (or conspicuously) serrate margin, obvious bracts, white calyx lobes and infundibuliform corolla tube. However, it can easily be distinguished from the latter two species by the characters summarised in the diagnosis.
The authors' molecular phylogenetic analyses illustrate that P. malipoensis, P. lungzhouensis, P. beiliuensis B. Pan & S.X. Huang (Pan et al. 2013) and P. beiliuensis B. Pan & S.X. Huang var. fimbribracteata. F. Wen & B.D. Lai (Lai and Wen 2015) form a monophyletic clade ). However, their morphology and geographical distribution allow the assumption that P. maguanensis and P. maculata W.B. Xu & J. Guo (Guo et al. 2015) are also closely related to this group. Both P. maguanensis and P. maculata were compared to P. eburnea in the original protologue (Xu et al. 2008, Guo et al. 2015. Nevertheless, based on the observation of living plants, P. maguanensis seems most similar to P. lungzhouensis and P. malipoensis; P. maculata ( Figure 3I-J) seems most similar to P. beiliuensis var. beiliuensis ( Figure 3G-H) and P. beiliuensis var. fimbribracteata ( Figure 3C-F). Further, the geographical distribution of P. maguanensis is adjacent to P. lungzhouensis and P. malipoensis (Figure 4) and the geographical distribution of P. maculata is adjacent to P. beiliuensis (Figure 4). Moreover, the results of the phylogenetical analysis in Guo et al. (2015) show that P. maculata is more closely related to P. lungzhouensis than P. eburnea. All of the above five species occur in nearly the same latitude zone of karst limestone areas from Southern China (from S-Yunnan to S-Guangdong), but with a disjunctive distribution (Figure 4). Therefore, these species perhaps represents a complex of longitudinal speciation, which may be caused by geographical isolation. Further studies are needed to confirm the  phylogenetic relationship of this species complex and to determine its evolutionary mechanism of speciation.
Primulina malipoensis could also be related to other species by its yellow flowers. However, the phylogenetic results illustrate that P. malipoensis has a distant relationship with all yellow flowering species, such as P. lutea