Two new species of Primulina (Gesneriaceae) from limestone karsts of China

The limestone karst area of South China is a major biodiversity hotspot of global terrestrial biomes. During extensive field work on the Guangxi limestone formations, two unknown species of Gesneriaceae were collected. After conducting a comprehensive study of the literature and herbarium specimens, Primulina davidioides and P. hiemalis are recognized as two species new to science, and described and illustrated here. P. davidioides is morphologically close to P. lunglinensis based on the shape of the leaf and flower, but it can be easily distinguished by the shape of the bracts, corolla and stigma, indumentum of peduncles, pedicels and pistil and number of staminodes. P. hiemalis is closely relate to P. luzhaiensis in vegetative appearance, but differs in the shape of the calyx and stigma, number of bracts and staminodes, indumentum of the leaf blade and peduncle, and position of stamens in the corolla tube. Considering that not enough is known about their populations, it is proposed that their conservation statuses should currently be classed as data deficient (DD) according to the IUCN Red List Category and Criteria.


INTRODUCTION
The tropical and subtropical karst landforms of southern and southwestern China are renowned because of their unrivalled biodiversity and high endemism among the tropical and subtropical floras of the world (Myers et al., 2000;Clements et al., 2006;Hou et al., 2010). Karst areas in South China offer a multitude of ecological niches for plant diversification and speciation , with about half of all the endemic genera of flowering plants in China (Ying & Zhang, 1994). Among these, the Gesneriaceae form the most abundant with 28 genera amounting to 90% of all endemic genera of the family in China (Wang, Pan & Li, 1990;Wang et al., 1998;Li & Wang, 2004;Möller et al., 2016). At the same time, China is a significant centre of diversity of Old World Gesneriaceae, which consists of 52 genera (Möller et al., 2016), with 75% of all species endemic to this region .
A great number of new species were described in the genus Primulina (Gesneriaceae) in recent years (Yang et al., 2018), and it is becoming one of the most interesting genera of the Old World Gesneriaceae, comprising more than 170 species (Wang et al., 2011;Möller et al., 2011;Möller et al., 2016). This group shows high levels of endemism and ecological specialisation , with narrow island distributions (Wang et al., 1998;Li & Wang, 2004;Wei et al., 2010), i.e., only in karst towers and caves . The limestone regions of southern and southwestern China and northern Vietnam possess the highest biodiversity of Primulina with about 80% of species endemic here (Wei et al., 2010). Many Primulina species pairs can successfully interbreed through artificial experiments (Wen, 2008;Zhang, Yang & Kang, 2017), suggesting that Primulina is probably a genus under recent or ongoing speciation and differentiation .
During our continuous floristic surveys of limestone karsts flora in 2009 and 2010, we revealed an additional two species of Primulina not previously known. Further fieldwork was conducted at the same locality, and flowering specimens were collected. Available information suggested that these two species are rare and usually occur only at one or two localities. After a comprehensive analysis of the literature (Wang, Pan & Li, 1990;Wang et al., 1998;Li & Wang, 2004;Wei et al., 2010), as well as herbarium specimens of E, GH, HITBC, HN, IBK, IBSC, K, MO, KUN, PE and US (herbarium acronyms according to Index Herbariorum; Thiers, 2017), the specimens were identified as two new taxa of Primulina based on results of detailed examination of morphological anatomical features, which we hereby describe and illustrate.

Ethics statement
All the collecting locations of the new species reported in this study are not in any natural conservation area and no specific permissions were required for these locations. Since the species are currently undescribed, inevitably, they are not currently included in the China Species Red List (Wang & Xie, 2004). Our field studies did not involve any endangered or protected species. No specific permits were required for the present study.

Nomenclature
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants (ICN), hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. In addition, new names contained in this work which have been issued with identifiers by IPNI will eventually be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix ''http://ipni.org/''. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.

Material collection
These two species were collected and examined during the floristic field surveys. In addition, the plants were monitored in the field and nursery in the Gesneriad Conservation Center of China (GCCC) by the authors over the past eight years. We collected leaf materials of these possible new species, using silica gel to dry them in the field for DNA extraction. We also cultivated about ten young plants from leaf cuttings of each new species for ex situ conservation in the Gesneriad Conservation Center of China (GCCC) so that we can preserve the germ plasma resource of this rare species.

DISCUSSION
In contrast to its high species diversity, the morphological variation of Primulina is relatively limited compared to other genera (Möller et al., 2016). The corolla morphology, especially, is relatively uniform, and most species possess straightly infundibuliform corollas, with only the salverform (e.g., Primulina tabacum), campanulate (e.g., P. dichroantha, P. mollifolia, P. hezhouensis, and P. renifolia), and P. curvituba having a strongly curved tube shape. In addition, Primulina displays a wide range of diversity of involucral bracts. The two opposite bracts are brightly white when flowering and serve the function of attracting pollinators as in Primulina eburnea, P. lutea, P. xiziae, P. lungzhouensis, P. lunglinensis, P. beiliuensis var. beiliuensis, P. beiliuensis var. fimbribracteata and the new species, P. davidioides. The development of cymes in these species shows that the two lateral paraclades of the pair-flowered cyme are reduced, so all flowers are clustered together. While flowers of these species always blossom for a short duration (Hong, 2016), the large white bracts and central flower cluster make the inflorescence look superficially like a single flower, perhaps to increase attraction for pollinators.

CONSERVATION ASPECTS
These two new species belong to a group of stenochoric plants which only grow in limestone areas. Currently there is insufficient information concerning the distribution and population status of these new species. Obviously, further field study is needed in northwestern Guangxi as their geographic range may well be more extensive than presently known. Considering that not enough is known about their populations, it is proposed that their conservation statuses should currently be classed as data deficient (DD) (IUCN, 2016).

CONCLUSIONS
During our continuous floristic surveys of limestone karsts in recent years, two unknown species of Gesneriaceae were collected. After conducting a comprehensive study of the literature and herbarium specimens, Primulina davidioides and P. hiemalis are recognized as two species new to science based on results of detailed examination of morphological anatomical features. Current information for these species is only known from very few collections, they appears to be narrowly endemic and locally abundant based on our careful field investigations in the past eight years.