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Morphological and ecological divergence of Lilium and Nomocharis within the Hengduan Mountains and Qinghai-Tibetan Plateau may result from habitat specialization and hybridization

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Several previous studies have shown that some morphologically distinctive, small genera of vascular plants that are endemic to the Qinghai-Tibetan Plateau and adjacent Hengduan Mountains appear to have unexpected and complex phylogenetic relationships with their putative sisters, which are typically more widespread and more species rich. In particular, the endemic genera may form one or more poorly resolved paraphyletic clades within the sister group despite distinctive morphology. Plausible explanations for this evolutionary and biogeographic pattern include extreme habitat specialization and hybridization. One genus consistent with this pattern is Nomocharis Franchet. Nomocharis comprises 7-15 species bearing showy-flowers that are endemic to the H-D Mountains. Nomocharis has long been treated as sister to Lilium L., which is comprised of more than 120 species distributed throughout the temperate Northern Hemisphere. Although Nomocharis appears morphologically distinctive, recent molecular studies have shown that it is nested within Lilium, from which is exhibits very little sequence divergence. In this study, we have used a dated molecular phylogenetic framework to gain insight into the timing of morphological and ecological divergence in Lilium-Nomocharis and to preliminarily explore possible hybridization events. We accomplished our objectives using dated phylogenies reconstructed from nuclear internal transcribed spacers (ITS) and six chloroplast markers. Our phylogenetic reconstruction revealed several Lilium species nested within a clade of Nomocharis, which evolved ca. 12 million years ago and is itself nested within the rest of Lilium. Flat/open and horizon oriented flowers are ancestral in Nomocharis. Species of Lilium nested within Nomocharis diverged from Nomocharis ca. 6.5 million years ago. These Lilium evolved recurved and campanifolium flowers as well as the nodding habit by at least 3.5 million years ago. Nomocharis and the nested Lilium species had relatively low elevation ancestors (<1000 m) and underwent diversification into new, higher elevational habitats 3.5 and 5.5 million years ago, respectively. Our phylogeny reveals signatures of hybridization including incongruence between the plastid and nuclear gene trees, geographic clustering of the maternal (i.e., plastid) lineages, and divergence ages of the nuclear gene trees consistent with speciation and secondary contact, respectively. The timing of speciation and ecological and morphological evolutionary events in Nomocharis are temporally consistent with uplift in the Qinghai-Tibetan Plateau and of the Hengduan Mountains 7 and 3-4 million years ago, respectively. Thus, we speculate that the mountain building may have provided new habitats that led to specialization of morphological and ecological features in Nomocharis and the nested Lilium along ecological gradients. Additionally, we suspect that the mountain building may have led to secondary contact events that enabled hybridization in Lilium-Nomocharis. Both the habitat specialization and hybridization have probably played a role in generating the striking morphological differences between Lilium and Nomocharis.
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... Sealy, distinguished primarily by the exquisite symmetry of its whorled leaves (Figure 1;Stearn 1956;Liang 1980;Liang and Tamura 2000). The genetic relationship based on internal transcribed spacer (ITS) and chloroplast genome fragments reveals that it belongs to Nomocharis clade, which exhibits the contradiction of high divergence in morphology but with low genetic distancing (Liang and Tamura 2000;Gao et al. 2012Gao et al. , 2013Gao et al. , 2015. More genetic information is needed to resolve their phylogenetic relationships. ...
... Additionally, we included 3 outgroups from closely related genera. The selection of sequences is based on our previous work (Gao et al. 2012(Gao et al. , 2013(Gao et al. , 2015. ...
... To clarify the evolutionary and position of L. paradoxum, we utilized three closely related genus species as outgroups and constructed Bayesian Inference (BI) tree based on 18 representative lilies from different clades in the genus Lilium, and the results (Figure 3) mostly consistent with previous studies (Gao et al. 2012(Gao et al. , 2013(Gao et al. , 2015. The clade Nomocharis and Lophophorum form two well-supported monophyly, and L. paradoxum is in the former, being most closely related to L. gongshanense (Y.D. Gao et X.J. He) Y.D. Gao, L. apertum Franch. ...
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Lilium paradoxum, a herb from southeastern Xizang, China, has its first complete chloroplast genome sequenced using next-generation sequencing. The genome is 151,814 bp, consisting of inverted repeats (IRs; 26,323 bp), small single-copy (SSC; 17,524 bp), and large single-copy regions (LSC; 81,644 bp). It encodes 112 unique genes: 78 protein-coding, 30 tRNA, and 4 rRNA genes. Phylogenetic analysis of 22 genomes shows L. paradoxum is closely related to L. gongshanense, L. apertum, and L. souliei. These findings enhance understanding of Lilium's phylogenetic relationships and evolution, particularly the Nomocharis clade in the Hengduan Mountains.
... Nomocharis Franchet (Franchet, 1889) was initially established as an independent genus close to Lilium and Fritillaria in the late 19th century. After much debate about the status of Lilium and Nomocharis (Balfour, 1918;Evans, 1925;Sealy, 1950Sealy, , 1983Liang, 1984), the latter was eventually accommodated and nested in Lilium as a subgenus clade based upon molecular phylogenetics (Nishikawa et al., 1999;Peruzzi et al., 2009;Gao et al., 2013;Du et al., 2014;Gao et al., 2015;Gao and Gao, 2016;Huang et al., 2018;Li et al., 2022a;Zhou et al., 2023). As one of the most complicated and unclear subgroups in Lilium, previous studies have provided us with a superficial understanding of its position within the genus and the possible processes of speciation that were involved (Gao et al., 2012(Gao et al., , 2013(Gao et al., , 2015Zhou et al., 2023), but the detailed phylogenetic framework and evolutionary mechanisms are not clear due to limited sampling. ...
... After much debate about the status of Lilium and Nomocharis (Balfour, 1918;Evans, 1925;Sealy, 1950Sealy, , 1983Liang, 1984), the latter was eventually accommodated and nested in Lilium as a subgenus clade based upon molecular phylogenetics (Nishikawa et al., 1999;Peruzzi et al., 2009;Gao et al., 2013;Du et al., 2014;Gao et al., 2015;Gao and Gao, 2016;Huang et al., 2018;Li et al., 2022a;Zhou et al., 2023). As one of the most complicated and unclear subgroups in Lilium, previous studies have provided us with a superficial understanding of its position within the genus and the possible processes of speciation that were involved (Gao et al., 2012(Gao et al., , 2013(Gao et al., , 2015Zhou et al., 2023), but the detailed phylogenetic framework and evolutionary mechanisms are not clear due to limited sampling. ...
... The group's distribution is narrow and limited to the Hengduan (H-D) Mountains of southwestern China, the eastern Qinghai-Tibetan Plateau (QTP), and adjacent Myanmar and India (Sealy, 1950(Sealy, , 1983Liang, 1984;1995;Liang and Tamura, 2000). These regions were formed relatively recently and underwent quite severe changes in geographic features, and this led previous authors to speculate upon the likelihood of a comparatively short process of speciation and evolution in Nomocharis clade taxa contemporaneously with the uplift of eastern Tibet and the H-D Mountains (Liang, 1984;1995;Gao et al., 2015) and, therefore, that the group had a recent origin. ...
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The former genus Nomocharis, which has been merged as a clade within the genus Lilium (Liliaceae), represents one of the most complicated and unclear groups included in the latter. Research on members of the Nomocharis clade has been quite limited due to the sampling difficulties caused by its selective environmental preferences. In this study, we propose a new species within this clade, Lilium liangiae, as a further bridge connecting the former genus Nomocharis with other members of the genus Lilium. We conducted morphological clustering, phylogenetic, and comparative genomics analyses of nuclear internal spacers and the newly generated complete chloroplast genome, in conjunction with previously published sequences, and performed ancestral state reconstruction to clarify the evolutionary pattern of important traits in Lilium. The clustering results of 38 morphological traits indicated that the new species is allied to Nomocharis, further increasing the morphological polymorphism in the latter. The phylogenetic results and morphological clustering both supported L. liangiae belonging to the subclade Ecristata in Nomocharis, its closest affinity being Lilium gongshanense. Inconsistencies in phylogenetic relationships were detected between nuclear and plastid datasets, possibly due to ancient hybridization and ongoing introgression. Comparative genomics revealed the conservation and similarity of their chloroplast genomes, with variations observed in the expansion and contraction of the IR regions. A/T and palindromic repeat sequences were the most abundant. Seven highly variable regions (Pi≥0.015) were identified as potential molecular markers based on the chloroplast genomes of 47 species within Lilium. Both nuclear and plastid genes exhibited very low variability within the Nomocharis clade, contrasting with their highly variable morphological appearance. The ancestral state reconstruction analysis suggests that the campanulate flower form, as in L. liangiae, arose at least three times within the genus Lilium, revealing parallel evolution in the latter. Overall, this study adds important genetic and morphological evidence for understanding the phylogenetic relationships and parallel evolution patterns of species within the genus Lilium.
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... At the top, it has numerous whorls of spectacular trumpet-shaped flowers. The colour of the stem can range from green to reddish-purple, depending on genetic differences and environmental conditions (Gao et al., 2015). ...
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... Additionally, chloroplast genomes are high conservation over nuclear and mitochondrial genomes. Therefore, partial chloroplast genome sequences are preferred and suitable to use for phylogenetic studies and species/varieties identification and discrimination with similar morphology characteristics [38][39][40][41][42][43] . DNA barcoding has been deployed in prior studies owing to its effectiveness in plant variety and cultivar identification. ...
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