Molecular systematics of Chiritopsis-like Primulina (Gesneriaceae): one new species, one new name, two new combinations, and new synonyms

Background The Gesneriaceae genus Chiritopsis, confined almost exclusively to cave or cave-like microhabitats of limestone karsts of southern China, was described to distinguish it from Chirita by much smaller flowers and generally miniature plant sizes in the former genus. However, molecular phylogenetic analyses showed that Chiritopsis is polyphyletic and its species delimitation has been problematic. To understand how many times Chiritopsis-like species have evolved from within the recircumscribed Primulina and to further clarify their species identification, we sampled all but two recently described species of Chiritopsis-like Primulina and reconstructed their phylogenetic relationship based on DNA sequences of nuclear ITS and chloroplast trnL-F and trnH-psbA. Results With 182 accessions of 165 taxa of Primulina sampled, our analyses placed the 40 accessions of 25 taxa of Chiritopsis-like Primulina in 17 unrelated positions, indicating at least 17 independent origins of the traits associated with caves or cave-like microhabitats. Of the 17 clades containing Chiritopsis-like Primulina, Clade 1 is composed of P. bipinnatifida, P. cangwuensis, P. jianghuaensis, P. lingchuanensis, and P. zhoui, as well as additional samples that show variable and overlapping morphology in leaf shapes. Clade 10 includes P. cordifolia, P. huangii, and P. repanda, while Primulina repanda var. guilinensis is not placed within Clade 10. Primulina glandulosa var. yangshuoensis is not placed in the same clade of P. glandulosa. Conclusions Based on our data, P. cangwuensis, P. jianghuaensis, and P. lingchuanensis are proposed to synonymize under P. bipinnatifida, with P. zhoui treated as a variety of P. bipinnatifida. Primulina repanda var. guilinensis is transferred as P. subulata var. guilinensis comb. nov. and Primulina pseudoglandulosa nom. nov. is proposed for P. glandulosa var. yangshuoensis. One new species is named P. chingipengii to honor the late Dr. Ching-I Peng (1950–2018). Electronic supplementary material The online version of this article (10.1186/s40529-019-0266-x) contains supplementary material, which is available to authorized users.

Following the remodeling of Chirita, Chiritopsis and Chirita sect. Gibbosaccus, along with two species of Wentsaiboea D. Fang & D.H.Qin, were transferred to Primulina (Wang et al. 2011;Weber et al. 2011). With the inclusion of the ca. 130 species, Primulina is expanded drastically from a monotypic genus to one of the largest genera of the Old World Didymocarpoid Gesneriaceae and the largest genus of Chinese Gesneriaceae (Weber et al. 2013;Xu et al. 2017;Wen et al. 2019). Subsequent to its recircumscription, more than 70 new species have been added to Primulina (Fig. 1), with 10 species bearing Chiritopsis-like flowers reported from limestone karsts Cai et al. 2014;Zhang et al. 2016;Pan et al. 2017;Hong et al. 2018;Xin et al. 2018;Li et al. 2019).
With 204 described taxa, the recircumscribed Primulina is the most species-rich plant genus on the vast limestone karst terrain stretching from southern China to northern Vietnam (Möller et al. 2016;Xu et al. 2017;Hong et al. 2019;Kong et al. 2019;Li et al. 2019;Wen et al. 2019). The diverse floral morphology and habitat preference of Primulina thus makes the genus an ideal clade to study evolutionary mechanisms that generated the high species diversity in the Sino-Vietnamese limestone karsts (Chung et al. 2014;Gao et al. 2015;Kong et al. 2017). The polyphyly of Chiritopsis-like Primulina indicates the homoplasious nature of the small corollas and generally lesser plant size characterizing the former Chiritopsis, suggesting multiple and independent origins of these traits. The highly polyphyletic Chiritopsislike Primulina also suggests convergent evolution of the smaller corollas associated with their habitat preference (i.e., caves and cave-like microhabitat), reminiscent of the convergent evolution associated with pollination syndromes in the New World Gesneriaceae (e.g., Clark et al. 2012Clark et al. , 2015. Although Primulina is currently the most species-rich plant genus inhabiting the Sino-Vietnamese limestone karsts, the taxonomy and species delimitations of Primulina remain controversial (Wang et al. 2017;Yang et al. 2019), as Weber et al. (2011) has cautioned that "It is also our impression that too many species have been described with numerous pairs or small groups of species growing in adjacent areas and differing only in slight, quantitative characters." For instance, P. huangii  and P. zhoui (Fig. 3q, r) were described based on their leaf morphology; however, both species have corollas identical to P. cordifolia and P. repanda (Fig. 2) and P. bipinnatifida, P. lingchuanensis, P. jianghuaensis, and P. cangwuensis (Fig. 3), respectively, that were overlooked by the author (Xin et al. 2018). Additionally, in the type localities of abovementioned species, we also observed considerable variation in leaf shapes (Figs. 2 and 3) apparently neglected and/or ignored by previous studies (Wang 1981(Wang , 1982Liu et al. 2006;Cai et al. 2014;Hong et al. 2018;Xin et al. 2018). On the other hand, we notice two apparent cases of taxon misidentification of Chiritopsislike Primulina in the GenBank (Vilgalys 2003). Specifically, the ITS sequence of P. mollifolia collected from the type locality used in current study ; NCBI: JX506866) is very different from those of 'P. mollifolia' submitted by Li and Wang (2007;NCBI: DQ872847) and Kong et al. (2017;NCBI: KY394945). Instead, DQ872847 and KY394945 are identical to the ITS sequences of samples (JX506869 & JQ713837) collected from the type locality of P. pseudomollifolia   Kong et al. (2017;KY394922) is very different from the sequence generated from specimen collected from the type locality in current study (JX506914). Unfortunately, such GenBank misidentifications of both KY394922 (labeled as P. lingchuanensis) and KY394945 (labeled as P. mollifolia) have been transmitted in a recent publication by Ye et al. (2019). Regardless those taxon misidentifications, previous phylogenetic study by Kong et al. (2017) showed that two infraspecific taxa P. glandulosa var. yangshuoensis and P. repanda var. guilinensis are only distantly related to their respective parental binominal P. glandulosa and P. repanda, necessitating nomenclatural changes.
In our continuous efforts to investigate the diversity of the cave flora of Sino-Vietnamese karsts (Chung et al. 2014), we surveyed and collected in a considerable number of caves, including type localities of most species of former Chiritopsis, as well as species of Chiritopsis-like Primulina species described after the work of Wang et al. (2011) andWeber et al. (2011). To understand how many times the Chiritopsis-like Primulina species have evolved, we reconstructed up to date the most comprehensive phylogenetic relationship of Primulina, sampling 165 taxa (81% of existing taxa). Based on our analyses, we propose taxonomical changes to better reflect their phylogenetic relationships, including the recognition of a new species, two new name, a new combination, and three new synonyms.

Taxon sampling and DNA extraction and sequencing
A total of 117 accessions representing 103 taxa of Primulina (50.4%) were collected by the authors in southern China during 2009-2016, with additional samples provided by the Guangxi Institute of Botany in Guilin, China. Multiple accessions of Chiritopsis-like Primulina species with broader distributions (e.g., P. bipinnatifida, P. cordifolia, and P. repanda) were sampled to test species boundary. Our samples also included two unknown species of the Chiritopsis-like Primulina. Sixty-five species of Primulina with nuclear internal transcribed spacers (ITS) and trnL-F intron-spacer region sequences available in the Genbank were also included. We acknowledge the work of Kong et al. (2017) that sampled 199 populations of 159 described Primulina species using ten cpDNA regions and ten nuclear genes (including ITS). However, we did not include all DNA sequences of Kong et al. (2017) because (1) our data were sufficient to address our purpose (i.e., how many times Chiritopsis-like Primulina species have evolved) and (2) species identification of a majority of species in Kong et al. (2017) could not be confirmed because insufficient voucher information provided. A total of 182 accessions of Primulina representing 165 taxa (80.9%) were included (Additional file 1), with two species of Petrocodon chosen as outgroups based on previous studies (Wang et al. 2011;Weber et al. 2011). We sampled all but two recently described species of Chiritopsis-like Primulina [i.e., P. cerina and P. niveolanosa;Li et al. (2019)], including 40 accessions of 25 taxa (Table 1). Total genomic DNA was extracted from silicagel dried leaves using the CTAB protocol (Doyle and Doyle 1987). DNA sequences of ITS and the chloroplast trnL-F intron-spacer region and psbA-trnH intergenic spacer were amplified based on the PCR procedures outlined in Möller et al. (2009) andSmissen et al. (2004).

Molecular phylogenetic analyses
The DNA sequences were aligned using MUSCLE implemented in MEGA7 (Kumar et al. 2016) with subsequent manual adjustments. For those species with only ITS and trnL-F sequences, psbA-trnH sequences were treated as missing data. Substitution model test was selected using MrModeltest version 2.3 (Nylander 2004) for individual sequence matrix and concatenated matrix. The best-fit models under the Akaike Information Criterion (AIC) were GTR + G+I for ITS, GTR + G for trnL-F, GTR + G for psbA-trnH, and GTR + G+I for the concatenated matrix. Both sequence matrices were analyzed using maximum likelihood (ML) optimality criteria and Bayesian Inference (BI). The ML analyses with 1000 bootstrap resampling were conducted using RAxML-HPC (Stamatakis et al. 2008) via the CIPRES Portal (Miller et al. 2010), with a gamma model of rate heterogeneity and the substitution model GTR + G+I. Proportion of invariable sites was estimated by the program. The BI analyses were conducted with MrBayes version 3.2 (Ronquist et al. 2012). Four chains of Markov chain Monte Carlo were run for 5 million generations each and were sampled every 250 generations starting with a random tree. For each run, the first 25% of sampled trees were excluded as burn-in before Bayesian clade posterior probabilities and average branch lengths were calculated.

Results and discussion
The combined matrix of the three markers contained Results of the Bayesian inferences and maximum likelihood were highly congruent, differing mainly in support values. The Bayesian majority-rule consensus tree with mean branch length is depicted in Fig. 4, annotated with bootstrap support values (BS) of the ML analyses and P. lingchuanensis* (Chung 1802 (Chung 1865;Fig. 7d-f;20) P. repanda var. guilinensis* ≡ P. subulata var. guilinensis comb. nov. (Chung 1806;Fig. 10a-c;10) P. lutvittata Primulina sp. (Chung 3028; (Chung et al. 1805;Fig. 7j-l;21) P. glandulosa* (Fig. 13a,   Rooted by Petrocodon, the major clades identified in our phylogenetic analyses are basically congruent with previous studies (e.g., Guo et al. 2015;Kong et al. 2017;Ye et al. 2019), though relationships among these major clades remain poorly resolved. Nevertheless, our results are sufficient to address our main questions.
The phylogenetic analyses placed the 40 accessions of Chiritopsis-like Primulina in 17 unrelated positions (Fig. 4), indicating at least 17 independent origins of the small flowers and the habit. Because the Chiritopsis-like Primulina species are found almost exclusively in cave or cave-like environments (i.e., Chung et al. 2014), the homoplasious nature of those traits characterizing these habitat specialists suggest that strong selection and adaptation to the extreme habitat might have resulted in their convergent evolution (Pipan and Culver 2012;Trontelj et al. 2012). Further discussion on this issue will be dealt in our subsequent study.
Geographically, plants in Clade 1 are distributed in northeastern Guangxi and adjacent Hunan, with a single outlier P. zhoui in central Guangxi (Fig. 5). Although we did not observe plants with leaves similar to P. zhoui in the type locality of P. bipinnatifida, considerable variation in leaf shapes also exists in P. zhoui with leaves shallowly to deeply cleft in the margins (Fig. 3r). Phylogenetically (Fig. 4a), P. zhoui is sister to a sample from Lipu (Chung 1852;Fig. 3s;18 in Fig. 5) that is geographically the closest to P. zhoui (5 in Fig. 5) in our sampling. Interestingly, the population in Lipu possess reniform to ovate leaves with shallow pinnately cleft margins (Fig. 3s) very similar to some leaves of P. zhoui (Fig. 3r), while other leaves in the Lipu population are indistinguishable from those in P. jianghuaensis (Fig. 3j) and P. cangwuensis (Fig. 3n).
Judging from the fact that all five species discussed here were described based on individuals with extreme leaf forms and that the extent of variation in the respective type population had been neglected or ignore by previous authors (Wang 1981;Liu et al. 2006;Cai et al. 2014;Hong et al. 2018;Xin et al. 2018), our molecular data, as well as their identical floral morphology (Fig. 3) favor to recognize this clade as a single species distributed in northeastern Guangxi and southern Hunan (Fig. 5).
The extent of morphological variation in its leaf shapes is likely resulted from random genetic drift in small and isolated population in the fragmented limestone karst landscape, as shown in other widespread species of Primulina (Gao et al. 2015;Wang et al. 2017) as well as other plant groups inhabiting caves and cave-like microhabitats of the Sino-Vietnamese limestone karsts (Chung et al. 2014;Tseng et al. 2019). Indeed, the taxonomical oversplitting in Clade 1 exemplifies Weber et al. (2011)'s observation that "too many species have been described with numerous pairs or small groups of species growing in adjacent areas and differing only in slight, quantitative characters." Because C. bipinnatifida is the earliest valid name of the clade, P. lingchuanensis, P. jianghuaensis, and P. cangwuensis are proposed to be synonymized under P. bipinnatifida, with P. zhoui treated as a variety of P. bipinnatifida for its disjunct distribution and unique leaf shapes. In addition to their floral morphology, the recircumscribed P. bipinnatifida can be readily recognizable by the whitish variegation along the midveins and the secondary veins of the leaves, regardless its variable leaf shapes (Figs. 3 and 7).

Clade 10
Clade 10 is composed of nine accessions (Fig. 4b), including three accessions sampled from the type localities (P. repanda, P. cordifolia, and P. huangii), as well as three additional accessions of P. repanda (Chung 1815, 1823, andPeng 22921) and P. cordifolia (Chung 1808(Chung , 1826(Chung , and 1828 collected during our exploration in the limestone areas of Guangxi. Accessions in Clade 10 all have very similar corolla morphology (Fig. 2c, d, j, k, n, o). The holotype of Chiritopsis repanda (≡ P. repanda) is a plant possessing four petiolate leaves with slightly repand margins (Fig. 8). While one leaf is elliptic with attenuate and decurrent base extending through the petiole, three leaves are ovate with cuneate to oblique bases (Fig. 8). Our analyses (Fig. 4b) placed three additional accessions (Chung 1815 and1823, andPeng 22921) in the same clade with P. repanda with strong support values (PP: 1; BS: 93). In general, these accessions possess leaves with entire to slightly sinuate leaf margins and attenuate and decurrent base extending through the petioles (Fig. 2eg), though leaves with slightly cordate leaf bases could also be observed in those populations (e.g., Fig. 2b).
Within Clade 10 (Fig. 4b), three additional accessions of P. cordifolia (Chung 1808(Chung , 1826 and 1828) form a moderately supported clade (PP: 098; BS: 67) sister to P. cordifolia from the type locality with strong support values (PP: 0.98; BS: 99). The holotype of Chiritopsis cordifolia (≡ P. cordifolia) is a plant with five petiolate leaves, all ovate with crenate margins and cordate to slightly truncate bases (Fig. 9). Those three additional accessions all possess leaves with cordate bases and slightly sinuate to crenate margins (Fig. 2p-r) that can be identified as P. cordifolia, though other character such as texture and hairiness are quite variable among these accessions.
The recently described Primulina huangii is (Fig. 2l-o) placed sister to the P. cordifolia clade with strong support values (PP: 1; BS: 96), though Xin et al. (2018) allied this unique species to P. bipinnatifida (Figs. 3 and 7) for their 'bipinnatifid' leaf margins. In the type locality of P. huangii, we also observed many plants possessed leaves varying from shallowly sinuate to deeply cleft margins (Fig. 2m), with many plants with cordate leaf based quite similar to P. cordifolia. The sister group relationship between P. cordifolia and P. huangii revealed by our data (Fig. 4b) suggests a likely evolutionary origin of their morphological similarity.
Recently, P. niveolanosa was described from Yizhou, central Guangxi that is morphologically comparable to P. repanda (Li et al. 2019). Although we have not seen this plant in the field, the overall morphology, especially the pubescence, of P. niveolanosa appears to be quite similar to three collections of P. cordifolia, Chung 1817 (Fig. 2i), Chung 1828 (Fig. 2p), and Chung 1826 (Fig. 2r).

Clade 3 and others
Our phylogenetic analyses also necessitate the nomenclatural change for Primulina repanda var. guilinensis for its placement with P. subulata and P. subulata var. yangchunensis in Clade 3 (Fig. 4a), a relationship also evident in Kong et al. (2017). We propose to transfer it as P. subulata var. guilinensis comb. nov. (Fig. 10) to reflect its phylogenetic relationship and highlight its apparent morphological differences from P. subulata and P. subulata var. yangchunensis.
Congruent with the result of Kong et al. (2017), our analyses also indicate that P. glandulosa and P. glandulosa var. yangshuoensis are distantly related (Fig. 4a). However, raising the variety to the specific status is blocked by the existence of P. yangshuoensis Y.G.Wei & F.Wen (marked by red color in Fig. 4a). We therefore propose the new name P. pseudoglandulosa nom. nov. for this taxon. Kong et al. (2017) also showed the non-monophyly in P. xiuningensis among populations in Anhui, Zhejiang, and Hunan. Further study will be needed to clarify species circumscription in P. xiuningensis.
Our phylogenetic analyses further indicate that two unknown plants with Chiritopsis-like flowers are placed distantly from other species of the Chiritopsis-like Primulina (Fig. 4), supporting their recognition as new species. Here we provide detailed description for one species, P. chingipengii. Another species will be published by other research team shortly.

Conclusions
Based on molecular phylogenetic analyses and field observations, we propose the following taxonomical changes, including one new species, one new name, two new combination, and three new synonyms.  Fig. 2h-k) in corolla shape, differing by the leaf blade carnose, broadly ovate, cordate to suborbicular, 2-3 × 1.2-2 cm, densely pubescent on both surfaces, base broadly cuneate, round to cordate in the former species.
Distribution, habitat and ecology: Primulina chingipengii is only known from its type locality in Baxian Park, Chengjiang Town, Du'an County, Guangxi, China (Fig. 5). It grows on moist rock face at the entrance of a karst cave (Fig. 12a).
Phenology: Flowering from Sep to Oct, fruiting from Nov to Dec. Notes: Primulina chingipengii is similar to P. cordifolia in corolla shape, but it is easily distinguished from the latter by the leaf blades. Phenologically, P. chingipengii and P. cordifolia are also different. Our phylogenetic analyses revealed that P. chingipengii is placed in a strongly supported clade also including P. albicalyx B.Pan & LiH.   Fig. 13c-g).
Distribution, habitat and ecology: Primulina pseudoglandulosa grows on moist shade cliffs of limestone, and also on moist rock face at the entrance of karst cave in Yangshuo, Guangxi (Fig. 5), at alt 120-150 m.
Phenology: Flowering from Jun to Jul, fruiting from Aug to Oct. entrance of karst cave, and also on moist shade cliffs of limestone, in Guangxi (Gongcheng, Guilin, Hezhou, Pingle, Quanzhou, Zhongshan) and Hunan (Jiangyong), at alt 100-230 m. The record of Chiritopsis repanda var. guilinensis in Luzhai County, Liuzhou City in Li and Wang (2004) is incorrect based on our field observation.
Phenology: Flowering from Jul to Aug, fruiting from Sep to Oct.
Notes: Phylogenetic analyses indicate that Primulina repanda var. guilinensis is placed in a strongly supported clade that also includes P. subulata and P. subulata var. yangchunensis, distantly related to P. repanda (Fig. 4).