Taxonomic treatment
Primulina cardaminifolia
Yan Liu & W.B. Xu, sp. nov.—TYPE: CHINA. Guangxi Zhuangzu Autonomous Region, Laibin Shi (City), Fenghuang Zhen (Township), alt. 280 m, on moist limestone rock face in a valley, 14 July 2008, Wei-Bin Xu & Yan Liu 08050 (holotype: IBK; isotypes: HAST and PE). 碎米薺葉報春苣苔 Figures 1, 2.
Diagnosis
Primulina cardaminifolia Yan Liu & W.B. Xu resembles Primulina pinnata (W.T. 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.
Description
Herbs perennial. Rhizome subterete, 3–5 mm across. Leaves 5–7, in basal rosette, imparipinnate, 10–20 cm long, papery when dry; petiole subterete, 6–12.5 cm long, densely pubescent; terminal leaflet ovate-cordate, 3–7 × 3–6.5 cm, apex obtuse, base cordate, margin repand to irregularly pinnately lobed, densely pubescent on both surfaces; lateral leaflets 1 or 2 pairs, opposite or alternate, broadly ovate to rotund, 1–3 × 1–3 cm, margin repand to irregularly pinnately lobed, densely pubescent on both surfaces, petiolules short, 2–10 mm long. Cymes 2–4, axillary, 1–3-branched, 3–10-flowered; peduncle 4–8 cm long, 1–2 mm in diam., densely pubescent; bracts 2, opposite, linear-lanceolate, 7–8 × 2–3 mm, margin entire, pubescent; pedicel 4–7 mm long, densely pubescent. Calyx 5-parted to base, lobes lanceolate-linear, 8–12 × 2–3 mm, apex acuminate, outside pubescent, inside glabrous, margins entire. Corolla white to pale purple, 3.2–3.5 cm long, outside glandular-pubescent, inside sparsely puberulent, with 2 pale-yellow stripes; corolla tube 2.1–2.3 cm long, 8–12 mm in diam. at the mouth, 2.5–3 mm in diam. at the base; limb pale purple, distinctly 2-lipped; adaxial lip 2-parted to over the middle, lobes oblong, 8–10 × 6–7 mm; abaxial lip 3-lobed to over the middle, lobes oblong, 12–13 × 4–5 mm; stamens 2, adnate to 1.5 cm above the corolla tube base; filaments linear, ca. 1.2 cm long, geniculate above the base, sparsely glandular-puberulent; anthers ca. 3 mm long, ca. 1.5 mm wide, dorsifixed, glabrous; staminodes 2, ca. 5 mm long, apex capitate, glabrous, adnate to ca. 6 mm above the base of corolla tube. Disc ring-like, ca. 1 mm in height, margin repand, glabrous. Pistil 2.5–2.8 cm long, ovary 7–8 mm long, ca. 1.5 mm across, puberulent; style 1.5–1.8 cm long, ca. 0.6 mm across, puberulent; stigma obtrapeziform, ca. 2 mm long, apex 2-lobed. Capsules not seen.
Additional specimens examined
CHINA. Guangxi Zhuangzu Autonomous Region, Laibin Shi, Fenghuang Zhen, 3 July 2008, Wei-Bin Xu & Yan Liu 08040 (IBK); same locality, 8 Sep 2008, Wei-Bin Xu & Kuo-Fang Chung 08472 (IBK), Shin-Ming Ku et al. 2035 (HAST); same locality, 28 June 2007, Hong-Jin Wei & Wei-Bin Xu 07244 (IBK).
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).
Notes
Primulina cardaminifolia resembles Primulina pinnata (W.T. Wang) YinZ. Wang (Figure 4), differing by the terminal leaflet being ovate-cordate, 3–7 × 3–6.5 cm (vs. oblong, 1.6–4.5 × 0.7–2.5 cm); the lateral leaflets 1 or 2 pairs, broadly ovate to rotund, 1–3 × 1–3 cm (vs. 3–5 pairs, oblong to oblong-lanceolate, 0.5–2.5 × 0.4–1.5 cm); cymes 1–3-branched, 3–10-flowered (vs. 1-branched, 1–3-flowered); calyx lobe with acuminate apex and entire margins (vs. acute to obtuse at apex and margins denticulate).
Chromosome cytology
Somatic chromosomes at metaphase of Primulina cardaminifolia 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.
In Primulina, chromosome numbers are uniformly diploid with 2n = 36 except for 2n = 32 in P. tamiana that was misplaced in the genus (Christie et al. 2012) and a polyploid with 2n = 72 in P. longgangensis (W.T. Wang) YinZ. Wang (Christie et al. 2012; Liu et al. 2012; Yang et al. 2012). Chromosomes of Primulina at somatic metaphase are generally small, ranging from 0.7 to 1.6 μm (Christie et al. 2012; Liu et al. 2012), to which P. cardaminifolia agrees (Figure 5). Our chromosome count of 2n = 36 in P. cardaminifolia agrees with basic chromosome number, x = 18, and supports its generic placement in the genus.
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 likelihood = −4114.9869) was recovered (Figure 6A). The MP analysis resulted in 6 equally parsimonious trees with 642 steps (CI = 0.70, RI = 0.64, RCI = 0.44). The topology of the strict consensus tree of the 6 equally parsimonious trees was congruent with the ML tree (not shown). The cpDNA matrix contained 27 accessions of 694 aligned positions, of which only 19 (1.3%) were parsimony informative. Based on the Hasegawa-Kishino-Yano (HKY) selected by the AICc implemented in MEGA5, a single ML tree (log likelihood = −1483.76) was obtained (Figure 6B). The MP analysis uncovered 23 equally parsimonious trees with 77 steps (CI = 0.97, RI = 0.97, RCI = 0.94). The topology of the strict consensus tree of the 270 equally parsimonious trees was largely consistent with the ML tree (not shown).
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.