Primulina cardaminifolia (Gesneriaceae), a rare new species from limestone areas in Guangxi, China

Primulina cardaminifolia Yan Liu & W.B. Xu (Gesneriaceae), a distinct new species with imparipinnate leaves, is described and illustrated from a limestone valley in Guangxi Zhuangzu Autonomous Region, China. To assure its generic placement and phylogenetic affinity, phylogenetic analyses were performed using DNA sequences of nuclear ITS and chloroplast trnL-F intron spacer region. Additionally, somatic chromosome number was counted and pollen stainability was tested. Phylogenetic analyses support its placement in Primulina; however, two phylogenetically distinct ITS sequence types were detected, suggesting a probable hybrid origin. Its pollen stainability is 100% and its chromosome number, 2n = 36, is congruent with all known counts of diploid species of the genus. All available data support the recognition of the new species Primulina cardaminifolia and suggest that it could have derived from homoploid hybrid speciation. Color plates, line drawings and a distribution map are provided to aid in identification.


Background
Recent progress in molecular phylogenetic studies of the Old World Gesneriaceae has foreseen the restructuring of the highly heterogeneous Chirita D. Don (Li and Wang 2007;Möller et al. 2009;Möller et al. 2011). However it was unexpected that Chirita has been forsaken (Wang et al. 2011;Weber et al. 2011) and the formerly monotypic Primulina Hance recircumscribed and expanded to include Chirita sect. Gibbosaccus C.B. Clarke, Chiritopsis W.T. Wang, and two species of Wentsaiboea D. Fang & D.H. Qin (Wang et al. 2011;Weber et al. 2011). With over 130 species transferred to (Wang et al. 2011;Weber et al. 2011;Xu et al. 2012b) and more than ten species newly described Hong et al. 2012;Huang et al. 2012;Li et al. 2012;Wen et al. 2012a, b, c;Wu et al. 2012aWu et al. , 2012bXu et al. 2012a;Chung et al. 2013), Primulina has now become one of the largest genera of the Old World Didymocarpoid Gesneriaceae. Under this new delimitation, Primulina is essentially a calciphilous genus distributed in southern China and adjacent northern Vietnam, with the major center of diversity in the limestone karsts of Guangxi Zhuangzu Autonomous Region (Wang et al. 1998;Li and Wang 2004;Hou et al. 2010;Wei 2010;Weber et al. 2011;Xu et al. 2012a, b).
Although the new generic circumscription reflects better the evolutionary relationships and ecological preferences of the genus, diagnostic characters of Primulina, such as perennial habit and acaulescent rosette ) and the basic chromosome number, x = 18 (Christie et al. 2012;Liu et al. 2012), are not exclusive to the genus. The lack of strong morphological synapomorphies to distinguish it from related genera in the region necessitates molecular data in ascertaining generic placements . For example, the transfer of Chirita tamiana B.L. Burtt to Primulina [i.e., P. tamiana (B.L. Burtt) Mich. Möller & A. Weber] made without support from molecular data by Weber et al. (2011) was recently challenged by chromosomal cytology and molecular data (Christie et al. 2012). Similarly, our ongoing molecular phylogenetic studies have also identified a number of misplacments in Primulina (Chung, unpubl. data).
In the course of a floristic survey in central Guangxi in 2007, a distinct species of Gesneriaceae with imparipinnate leaves was collected by the authors. After consulting national and local floras and the relevant literature (Wang et al. 1998;Li and Wang 2004;Wei 2010;Liu et al. 2011;Wang et al. 2011;Wu et al. 2012a;Xu et al. 2012a, b) as well as herbarium specimens, we conclude that it represents a new species of Primulina, which is described and illustrated here. Chromosome count of the new species is in agreement with those reported for Primulina in Christie et al. (2012). Its generic placement is further confirmed by molecular and chromosome data.

Chromosome preparations
The plant for chromosome studies was collected from the type locality and cultivated in the experimental greenhouse of Academia Sinica, Taipei. A voucher specimen (Ku et al. 2035) has been deposited in HAST. Root tips were gathered and pretreated in 2 mM 8-hydroxyquinoline at 15-18°C for about 6 h and fixed overnight in an ethanol-acetic acid solution (3:1) below 4°C. The chromosomes were stained and macerated in 2% acetic orcein with 1 N hydrochloric acid (10:1). Classification of chromosome complement based on centromere position at mitotic metaphase followed Levan et al. (1964).

Molecular methods
DNA sequences of the nuclear ribosomal internal transcribed spacers (ITS) and the chloroplast trnL-F intron spacer region were gathered using protocols outlined in Xu et al. (2012a). Because direct sequencing of the ITS PCR products of the new species resulted in DNA sequences with overlapping signals, molecular cloning was performed. Following the manufacturer's protocol, the purified ITS templates were ligated to the pGEMT-T vector system (Promega, Madison, Wisconsin, USA) and subsequently transformed into competent cells (DH5α) to perform molecular cloning. After overnight culture at 37°C on the LB ampicillin/IPTG/X-gal selective plate, colonies carrying the ITS insert were identified by color (white) and further verified by PCR using the T7 and SP6 promoter primer pairs (Promega, Madison, Wisconsin, USA). Ten colonies with positive ITS insert were then transferred and grown in 2 μl LB medium at 37°C for 15 h. Plasmids were extracted using the Mini Plus™ Plasmid DNA Extraction System (Viogene, Taipei, Taiwan) and cycle-sequenced using the T7 and SP6 promoter primer pairs.
For phylogenetic analyses, matrices of Xu et al. (2012a) were adopted, keeping one accession for each species (Appendix 1) and ITS and trnL-F regions as separated matrices. Primulina pinnata (W.T. Wang) YinZ. Wang, which was not included in Xu et al. (2012a) because of the presence of an access of ambiguous sites in its ITS sequence in the GenBank (i.e., FJ501349), were added in current analyses to test for its putative relationships with the new species. The final matrix contained 24 species of Primulina with Petrocodon dealbatus Hance, Pet. scopulorum (Chun) YinZ. Wang, and Didymocarpus podocarpus C.B. Clarke chosen as outgroups based on recent phylogenetic analyses Weber et al. 2011). The DNA sequences were aligned using the program MUSCLE implemented in the software MEGA5 (Tamura et al. 2011) with minor manual adjustments. Phylogenetic trees were reconstructed separately for ITS and trnL-F based on maximum parsimony (MP) and maximum likelihood (ML) criteria implemented in MEGA5. MP trees were searched using the Tree-Bisection-Reconnection (TBR) search option with the initial trees setting at 50, MP search level setting at 5, and maximum number of trees setting at 2000. Clade supports were calculated based on 100 bootstrap resamplings (parsimony bootstrap; PB). ML trees were reconstructed using the nearestneighbor-interchange (NNI) method with all site used and the initial tree automatically selected under the model(s) selected by MEGA5. Clade supports of ML analysis were evaluated based on 100 bootstrap resamplings (likelihood bootstrap; LB).  Wang) YinZ. Wang in having imparipinnate leaves, but is clearly distinct from this species by the ovate-cordate terminal leaflet of 3-7 × 3-6.5 cm, 1 or 2 pairs of broadly ovate to sub-ovate lateral leaflets, 1-3-branched cymes with 3 to 10-flowers, and the entire-margined calyx lobes with acuminate apex.

Ecology and distribution
Primulina cardaminifolia is extremely rare, currently known only from the type locality in Laibin Shi, Guangxi Zhuangzu Autonomous Region, China (Figure 3). It grows on a moist limestone rock face in a valley.

Phenology
Flowering from June to July; fruits not observed.

Etymology
The specific epithet is derived from the leaves resembling those of the genus Cardamine L. (Brassicaceae).

Chromosome cytology
Somatic chromosomes at metaphase of Primulina carda minifolia were determined to be 2n = 36 ( Figure 5). The 36 chromosomes were small and gradually varied from ca. 0.6 μm to 1.3 μm in length. Most chromosomes had centromeres at median positions, while those of some of the shorter chromosomes could not be determined. Satellites were not observed.

Phylogenetic analyses
Results of molecular cloning of the ITS PCR product revealed two phylogenetically distinct ITS sequences ( Figure 6A) with the length of 643 (P. cardaminifolia-A) and 634 (P. cardaminifolia-B) bp, respectively, while only one cpDNA sequence type was detected in the species. With the addition of these two ITS sequences, the ITS matrix contained 28 accessions of 675 aligned positions, of which 182 (26.96%) were parsimoniously informative. Based on the Kimura 2-parameter model using a discrete Gamma distribution (K2 + G) with 5 rate categories selected by the corrected Akaike Information Criterion (AICc) implemented in MEGA5, a single ML tree (log Both ITS and cpDNA dataset placed P. cardaminifolia in Primulina with high (LB = 98, PB = 97) and low (LB = 64, PB = 70) supports in ITS and cpDNA datasets, respectively ( Figure 6), confirming its generic placement. Although the phylogenetic trees of the two datasets were not perfectly congruent with each other, especially in the deeper nodes, several well supported subclades were highly consistent between the two trees ( Figure 6). Specifically, both datasets identified the clade (Clade A) composed of P. glandulosa, P. repanda var. guilinensis, P. bipinnatifida, P. dryas, P. pinnatifida, and P. multifida. In the ITS tree, the P. cardaminifolia-A sequence was also included in Clade A ( Figure 6A). Other congruent clades included the clade consisting of P. spinulosa, P. wentsaii, P. ophiopogoides, and P. minutimaculata, the clade of P. longgangensis and P. linearifolia, and the clade of P. heterotricha and P. pteropoda.
The occurrence of intraindividual ITS polymorphism, or failure of concerted evolution among reiterated loci of ribosomal DNA arrays to nullify various rDNA repeats, could have resulted from hybridization, polyploidization, multiple nucelolar organizing regions on non-homologous chromosomes, rDNA pseudogenization, long generation time, loss of sexual recombination, or extensive introgression during domestication (Denduangboripant and Cronk 2000). The diploid chromosome number 2n = 36 and the presence of two phylogenetically distinct ITS sequence types raised the concern that the distinctive P. cardaminifolia might have been a hybrid (e.g., Peng and Chiang 2000), which has frequently been reported in Gesneriaceae (e.g., Puglisi et al. 2011). Assuming a maternal inheritance of its chloroplast genome, species in Clade B of the cpDNA tree would be the maternal parent, while Clade A could have been its paternal parent ( Figure 6). Interestingly, most species in Clade A, including P. glandulosa, P. bipinnatifida, P. pinnatifidai, and P. multifida, are characterized by deeply lobed or pinnatified leaves that are otherwise unknown in Primulina and may have contributed to the unique imparipinnate leaves of P. cardaminifolia (Figures 1 & 2). Nevertheless, the species status of P. cardaminifolia was supported by a 100% pollen fertility suggested by the high level of stainable pollen (Figure 7) using the malachite green-acid fuchsin-orange G stain (Alexander 1969). Given its perfectly developed pollen, the cytological and molecular data instead could have suggested that P. cardaminifolia might be of homoploid hybrid origin, which was formed without changes in chromosome number (cf. Howarth and Baum 2005;Rieseberg and Willis 2007;Abbott et al. 2010). Further studies will be needed to test the probable hybrid and homoploid origin of the new species.

Conclusion
All available data support the recognition of the new species Primulina cardaminifolia, which is described herein. The new species may have arisen via homoploid hybrid speciation. Its generic placement is confirmed by morphological, chromosomal, and molecular analyses.