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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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... The Leucolirion clade (sensu Gao et al. 2015) of Lilium L. (Fig. 1) comprises six species: L. regale Wilson, L. leucanthum (Baker) Baker, L. sulphureum Baker ex J.D.Hooker, L. sargentiae Wilson with huge trumpetshaped flowers and L. henryi Baker and L. rosthornii Diels with recurved tepals and exserted sexual organs (Gao, Harris & He, 2015). Although highly dissimilar in floral syndrome, especially morphology, these species show rather low levels of intraspecific genetic variation, and the molecular dating indicated a rapid radiation in this clade (Gao et al., 2013(Gao et al., , 2015. ...
... The Leucolirion clade (sensu Gao et al. 2015) of Lilium L. (Fig. 1) comprises six species: L. regale Wilson, L. leucanthum (Baker) Baker, L. sulphureum Baker ex J.D.Hooker, L. sargentiae Wilson with huge trumpetshaped flowers and L. henryi Baker and L. rosthornii Diels with recurved tepals and exserted sexual organs (Gao, Harris & He, 2015). Although highly dissimilar in floral syndrome, especially morphology, these species show rather low levels of intraspecific genetic variation, and the molecular dating indicated a rapid radiation in this clade (Gao et al., 2013(Gao et al., , 2015. ...
... The Leucolirion clade (sensu Gao et al. 2015) of Lilium L. (Fig. 1) comprises six species: L. regale Wilson, L. leucanthum (Baker) Baker, L. sulphureum Baker ex J.D.Hooker, L. sargentiae Wilson with huge trumpetshaped flowers and L. henryi Baker and L. rosthornii Diels with recurved tepals and exserted sexual organs (Gao, Harris & He, 2015). Although highly dissimilar in floral syndrome, especially morphology, these species show rather low levels of intraspecific genetic variation, and the molecular dating indicated a rapid radiation in this clade (Gao et al., 2013(Gao et al., , 2015. Recent re-evaluation of the phylogenetic relationship using transcriptome sequences produced using highthroughput sequencing provided further resolution that recovered two sister groups, one consisting of the two species with trumpet-shaped and the other consisting of the species with tepal-recurved flowers (Gao et al., unpublished data). ...
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Pollination niche shifts can drive remarkable floral divergence between closely related plant species. The Leucolirion clade of Lilium contains species with either tepal-recurved or trumpet-shaped flowers. The tepal-recurved flowers are bright orange and might be pollinated by butterflies and/or birds. The trumpet-shaped flowers are mostly pale and strongly fragrant and might permit visitation by a variety of hawkmoths. Lilium leucanthum has trumpet-shaped flowers, and some populations of this species show dark coloration on the floral outer surface, suggesting pollination by mammals. We identified pollinators and examined the dependence of reproduction on pollinators by floral visitor observations, pollen load analysis and pollination experiments. We also analysed floral traits to contrast the two floral syndromes involving different lepidopteran groups. The tepal-recurved lilies are specialized on a group of Papilio butterflies for pollination with pollen predominantly attached to the hindwings. The trumpet-shaped flowers are almost exclusively pollinated by hawkmoths, including diverse species with proboscises of different lengths. No mammal visitation was found to the populations of L. leucanthum with dark outer surfaces of flowers. Self-incompatibility prevails throughout the clade, including the populations in which pollinators were scarce. The butterfly- and hawkmoth-pollinated species display contrasting floral syndromes. Our findings confirmed that the dichotomy in floral syndrome in the Leucolirion clade is associated with Papilio butterfly vs. hawkmoth pollination, whereas intraspecific variation in colour of the floral outer surface of L. leucanthum many need a non-pollinator explanation.
... 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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... Lilium Linnaeus (1753: 302) comprises about 115~130 species distributed in temperate and alpine regions of the North Hemisphere, especially in eastern Asia (Liang & Tamura 2000), where members with most remarkable and variably shaped perianths in many colours can be found. After a long-term debates about the status of Lilium and Nomocharis Franchet (1889: 113), the latter was eventually accommodated in Lilium as a result of a series of molecular, morphological and ecological studies (Gao et al. 2015, Gao & Gao 2016. ...
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... We accommodated L. xanthellum on Clade X, away from Leucolirion. According to ultrametric chronograms, L. xanthellum is closer to L. lophophorum and L. matangense 95 . Totally, the composition of clade X in our phylogeny (three species of Lophophorum including L. fargesii, L. lophophorum, and L. matangense, along with L. nanum of Sinomartagon and L. xanthellum of Leucolirion) is in agreement with Du et al. 92 based on ITS regions. ...
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... Several molecular DNA-based studies using both plastid and nuclear markers have helped resolve some of the relationships amongst these species (Nishikawa et al. 2001;Lee et al. 2011;Du et al. 2014;Gao et al. 2015;Huang et al. 2018;Givnish et al. 2020). These molecular studies have shown that the Asian species of Lilium with trumpet-shaped flowers belong in two clades: One comprises Lilium brownii, L. formosanum A.Wallace, L. longiflorum Thunb., L. neilgherrense Wight, L. philippinense Baker and L. wallichianum Schult. ...
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