Cryptic Species Diversification of the Pedicularis siphonantha Complex (Orobanchaceae) in the Mountains of Southwest China Since the Pliocene

Abstract

Morphological approaches often fail to delimit species in recently derived species complexes. This can be exacerbated in historical collections which may have lost key features in specimen preparation and preservation. Here, we examine the Pedicularis siphonantha complex, endemic to the Mountains of Southwest China. This complex is characterized by its red/purple/pink and long-tubular corolla, and twisted, beaked galea. However, herbarium specimens are often difficult to identify to species. Molecular approaches using nrITS or nuclear ribosomal internal transcribed spacer (nrITS) + plastid DNA (ptDNA) have been successfully used for species identification in Pedicularis. To resolve taxonomic confusion in the Pedicularis siphonantha complex, we reconstructed phylogenies of the complex using nrITS and four plastid DNA loci (matK, rbcL, trnH-psbA, and trnL-F). To recover as much of the phylogenetic history as possible, we sampled individuals at the population level. Topological incongruence between the nrITS and ptDNA datasets was recovered in clades including two widely distributed species, Pedicularis milliana and Pedicularis tenuituba. Based on morphological, geographical, and genetic evidence, we suggest that hybridization/introgression has occurred between P. milliana and Pedicularis sigmoidea/Pedicularis sp. 1 in the Yulong Snow Mountain of Lijiang, northwest Yunnan, and between P. tenuituba and Pedicularis leptosiphon in Ninglang, northwest Yunnan. After removing conflicting DNA regions in Pedicularis dolichosiphon (nrITS) and P. milliana (ptDNA), the concatenated nrITS and ptDNA phylogenies distinguish 11 species in the P. siphonantha complex, including two undescribed species, from the Jiaozi and Yulong Snow Mountains, respectively. Phylogeographical analyses indicate that the P. siponantha complex originated from south of the Hengduan Mountains, expanding north to the Himalayas and the Yunnan-Guizhou Plateau. Moreover, the uplift of the Qinghai-Tibet Plateau and climate oscillations may have driven further diversification in the complex.

Keywords: Pedicularis siphonantha complex; mountains of Southwest China; phylogenetic delimitation; speciation; the Hengduan Mountains.

FIGURE 1

Field photos of 11 species of the Pedicularis siphonantha complex and Pedicularis delavayi, (A) Pedicularis tenuituba H. L. Li; (B) Pedicularis leptosiphon H. L. Li; (C) Pedicularis dolichosiphon (Hand.-Mazz.) H. L. Li; (D) P. siphonantha D. Don; (E) Pedicularis sp. 2 from Jiaozi snow mountain; (F) Pedicularis milliana W. B. Yu, D. Z. Li, and H. Wang; (G) Pedicularis sp. 1 from Ganheba, Lijiang; (H) Pedicularis sigmoidea Franch. ex Maxim; (I) Pedicularis variegata H. L. Li; (J) Pedicularis dolichantha Bonati; (K) Pedicularis humilis Bonati. (L) Pedicularis delavayi Franch. ex Maxim.

FIGURE 2

A geographical sampling of the Pedicularis siphonantha complex from the Hengduan Mountains, southwestern China. The colors of circles correspond to different taxa. Numbers and letters indicate populations; these abbreviations are also used in Figures 3, 4, 6. More information regarding collection vouchers can be found in Supplementary Table 1.

FIGURE 3

K-S test for intraspecific and interspecific genetic distance. The intraspecific genetic distance of Pedicularis milliana, Pedicularis tenuituba, and Pedicularis siphonantha are estimated. Statistical significance was shown on every group (**P < 0.01, *P < 0.05). -A: to Pedicularis tenuituba; -B: to Pedicularis leptosiphon; -C: to Pedicularis dolichosiphon; -D: to Pedicularis siphonantha; -E: to Pedicularis sp. 2 from Jiaozi Snow Mountain; -F: to Pedicularis milliana; -G: to Pedicularis sp. 1 from Ganheba; -H: to Pedicularis sigmoidea; -I: to Pedicularis variegata; -J: to Pedicularis dolichantha; -M: to Pedicularis amplituba (outgroup); -N: to Pedicularis tahaiensis (outgroup).

FIGURE 4

Bayesian Inference (BI) tree comparisons of Pedicularis siphonantha complex inferred from nrITS (A) and the concatenated chloroplast (B) datasets. Phylogenetic incongruence is marked by shadow, Numbers associated with the branches are bootstrap value (BS) and BI posterior probabilities (PP), and thicker lines are indicated as BS ≥ 70 and PP ≥ 0.95. Node with BS < 50 was collapsed.

FIGURE 5

Bayesian inference (BI) tree of the Pedicularis siphonantha complex inferred from the modified nrITS+ptDNA dataset by removing the conflicting sequence nrITS of Pedicularis dolichosiphon (nrITS) and the four ptDNA regions (matK, rbcL, trnH-psbA, and trnL-F) of the samples F11-F14 of Pedicularis milliana in accordance with the topological incongruence between the nrITS and ptDNA phylogenies (see Figure 4 and Table 3). Numbers associated with the branches are ML BS value and BI PP, and thicker lines indicate BS ≥ 70 and PP ≥ 0.95. Node with BS < 50 was collapsed. The topology of the P. milliana clade with short branch lengths appear on the right.

FIGURE 6

Neighbor-net analysis of Pedicularis siphonantha complex using complete nrITS + ptDNA datasets. Bootstrap support values for clusters are indicated next to the respective cluster delimitation (dashed lines); blue dashed lines indicate the split of Pedicularis milliana.

FIGURE 7

Bayesian phylogeographical reconstruction of ancestral area of the Pedicularis siphonantha complex in the Sino-Himalayan region using the modified nrITS + ptDNA dataset (see above). Pie charts and thickness of branch represent the marginal probabilities for potential ancestral areas with each subregion (Liu et al., 2021), represented by a different color. BI PPs are above branches (* = 1.00).