Plastome phylogenomics, biogeography, and evolutionary diversification of Lilium (Liliaceae)

Plastome phylogenomics, biogeography, and evolutionary diversification of Lilium (Liliaceae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plastome phylogenomics, biogeography, and evolutionary diversification of Lilium (Liliaceae) Nian Zhou, Ke Miao, Changkun Liu, Linbo Jia, Jinjin Hu, Yongjiang Huang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2303338/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Lilium (Liliaceae) is an economically important genus with great medicinal, ornamental, and edible values, however evolutionary history of the genus remains poorly understood due to the lack of robust phylogeny. Based on a large plastome data set, this study aims to recover a robust backbone phylogeny of the genus to infer its historical biogeography and evolutionary diversification. Results Complete plastomes representing 50 currently accepted species in the genus Lilium were sampled for phylogenetic analysis, among which, plastomes representing 14 species were newly sequenced in this study. Under time-calibrated phylogenetic framework, biogeographic scenarios and evolutionary diversification of Lilium were explored. Phylogenetic analysis recovered a backbone phylogeny of Lilium , in which most nodes were fully supported; however, failed to resolve all intrageneric sections as monophyletic. Ancestral area reconstruction proposed that the ancestor of Lilium might widely distribute throughout the temperate regions of the Northern Hemisphere, and has experienced multiple dispersal, extinction, and vicariance events during the evolutionary course. The rate of species diversification has sharply accelerated since the late Miocene ( ca. 9 Ma) and kept increasing in the Pliocene and Pleistocene. Conclusions The results suggest that ancient climatic changes and geological tectonic activities, such as the Middle Miocene Climate Optimum (MMCO), the late Miocene global cooling, as well as the successive uplift of the Qinghai-Tibetan Plateau (QTP) and the enhancement of monsoonal climate in East Asia during the late Miocene and the Pliocene, jointly shaped the distribution range and drove evolutionary radiation of Lilium . Resulted from radiative divergence and distant dispersal events, the genus may have experienced incomplete lineage sorting (ILS) and morphological convergence. The uplift of QTP and enhancement of monsoonal climate dramatically triggered radiative divergence of species, accounting for the higher Lilium species diversity in East Asia than in Central Asia, Europe, and North America. The findings shed light on the crucial role of the unique Neogene geological and climatic events in East Asia, such as the uplift of QTP and the establishment of monsoonal climate, in shaping the uneven distribution of plant diversity in the Northern Hemisphere. Asian monsoon climatic changes distribution range evolutionary convergence radiative diversification species diversity Qinghai-Tibetan Plateau (QTP) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Lilium L. is a large genus of the monocotyledonous family Liliaceae, which encompasses approximately 115 species of bulbous herbaceous perennials [ 1 ] spreading throughout the cold and temperate regions of the Northern Hemisphere [ 2 ]. East Asia is the species diversity center of the genus [ 2 ], as nearly half of currently recognized Lilium species occur in China, chiefly in Southwest China and the Himalayas [ 3 , 4 ]. Lilium species are commercially valued ornamental plants and cultivated widely across the world for the esthetic property of their showy and colorful flowers [ 3 – 8 ]. Additionally, the flowers and bulbs of numerous Lilium species are traditionally used as herbal medicines and health food in Eurasia and North America [ 2 , 4 , 5 ]; currently, their great commercial value of “medicine food homology” is attracting universal attentions [ 9 ]. The floral morphologies of Lilium which are used to create subgeneric classification exhibit high levels of interspecific variations, resulting in the constant changes in the taxonomic delimitation of sectional boundaries according to different classification systems [ 10 – 13 ]. Among them, the classification proposed by Comber [ 12 ], which divided the genus into seven sections (i.e., sect. Archelirion , sect. Daurolirion , sect. Leucolirion , sect. Liriotypus , sect. Martagon , sect. Pseudolirium , and sect. Sinomartagon ), has been universally adopted. Nevertheless, previous phylogenetic studies identically showed that most of the section-level taxonomic units recognized by Comber [ 12 ] are non-monophyletic, and there are still massive controversies over the intrageneric relationships of Lilium [ 14 – 20 ]. Additionally, Nomocharis Franch., which was once a segregated genus, was regarded as congeneric with Lilium [ 21 ], and then reduced as a section-level taxonomic unit of Lilium [ 22 ]. Although the taxonomic proposal that synonymized Nomocharis to Lilium has been well evidenced by molecular phylogeny [ 18 , 20 ], the monophyletic nature of the sect. Nomocharis sensu Gao and Gao [ 22 ] remains uncertain. Such phylogenetic ambiguities impede to better understand the biogeographic scenario and evolutionary diversification of this economically important genus. To reconstruct robust phylogenetic relationships, extending taxon sampling and employing alternative data sets with more phylogenetically informative variations are both preferred [ 23 – 26 ]. Recently, high-throughput sequencing technologies have been increasingly used to generate genome-scale data for phylogenetic studies. Phylogenetical analysis of sequence data generated by high-throughput sequencing, such as complete plastid genomes (plastome) and genome-wide nuclear sequences, which possess orders of magnitude more sequence variations than single or multiple sequences regions produced by Sanger sequencing, have exhibited greater potential for resolving recalcitrant relationships in a wide spectrum of plant lineages [ 24 , 27 – 36 ]. Theoretically, phylogenetic reconstruction based on the uniparentally inherited plastomes recovers only the maternal (or in some cases the paternal) relationships of a certain plant lineage, compared with the relatively integrated evolutionary schemes recovered by analysis of the biparentally inherited nuclear genomes. Even so, with the wide application of plastomes in phylogenetic studies, numerous historically difficult issues in plant phylogenetics have been satisfactorily addressed [ 27 – 30 , 35 , 37 – 40 ], suggesting that plastomes are as important as nuclear genome data sets and will continue to play an indispensable role in plant phylogenetics. Previously, Du et al. [ 41 ], Kim et al. [ 42 , 43 ], and Li et al. [ 20 ] attempted to apply completed plastome DNA sequences to clarify phylogenetic ambiguities in Lilium . Although analysis of plastome data set largely improve phylogenetic resolution and support, sampling size at the species level of these studies was too low to recover high-resolution and well-supported phylogenetic backbone of the genus. Here, the taxonomic sampling of Lilium is extended to include 50 currently accepted species representing these seven sections recognized by Comber [ 12 ] plus the section Nomocharis defined by Gao and Gao [ 22 ]. Based on phylogenomic analyses of a large plastome data set, the primary objectives of the present study are: (1) to recover a robust backbone phylogeny of the economically important plant genus; and (2) to explore biogeographic scenarios and historical diversification for the genus inferred from phylogenetic framework and molecular dating. Results Phylogenetic relationships Plastomes of Lilium species sampled in this study varied from 151,655 bp ( L. bakerianum ) to 153,235 bp ( L. fargesii ) in size, which are conserved in gene content and genome rearrangement. These plastomes contained 114 unique genes, including 80 protein-coding genes, 4 rRNA genes, and 30 tRNA genes. As shown by multiple genomes mauve alignment ( Additional file 1 : Fig. S1), Lilium plastomes were consistent in genes order and no structural rearrangement was detected. Based the alignment of complete plastome DNA sequences, the ML and BI analyses (Fig. 1 ) generated almost identical tree topologies (the only difference is the placement of L. lancifolium ), with full support for most nodes (BS = 100%; PP = 1.00). Both ML and BI phylogenies recovered two major clades (BS = 100%; PP = 1.00), consisting of East Asia + North America species (Clade I) and Eurasia species (Clade II). Additionally, the seven sections recognized by Comber [ 12 ] and the section Nomocharis sensu Gao and Gao [ 22 ] were recovered as monophyletic by neither ML nor BI phylogeny. Molecular dating and historical diversification The BEAST analysis (Fig. 2 ) showed that the early divergence of Lilium , corresponding to the splitting of two major clades (Clade I and Clade II), occurred at 16.82 Ma (95% HPD:19.55–13.60 Ma), around the middle Miocene. The crown ages of Clade I and Clade II were dated at 13.03 Ma (95% HPD: 16.43–9.51 Ma) and 12.18 Ma (95% HPD:16.26–7.71 Ma), respectively. Within each clade, the divergence of shallow branches, which resulted in the formation of most extant species, intensively occurred in the late Miocene, Pliocene, and Pleistocene. The lineage through time (LTT) plots analysis (Fig. 3 ) proposed that the diversification rate was relatively stable during the early evolution Lilium , but sharply accelerated in the late Miocene ( ca. 9 Ma) and kept increasing during the Pliocene and Pleistocene. Consistent with this, the Bayesian Analysis of Macroevolutionary Mixtures (BAMM) identified a sharp shift in net species diversification rate in Lilium ca . 9 Ma, and the trend of continuous growth of net species diversification rate had been maintained in the Pliocene and Pleistocene (Fig. 4 ). Ancestral area reconstruction The Statistical-Dispersal Vicariance Analysis (S-DIVA) proposed that the most recent common ancestor of (MRCA) of Lilium might have widely occurred in Eurasia and North America, and experienced a distant dispersal and an extinction event to evolve the ancestral populations of clade I and clade II (Fig. 5 ). Although S-DIVA analysis failed to reconstruct the ancestral area of clade I, the preliminary formation of the disjunction distribution between North American and Southwest China and the Himalayas was inferred to be jointly driven by a dispersal, an extinction, and a vicariance event. Next, five dispersal, one extinction, and four vicariance events were inferred as the evolutionary driving force for the formation of the current distribution range of Clade I. Additionally, S-DIVA analysis showed that the ancestral populations of the clade II mostly likely spread throughout Eurasia from northern Indochina to Europe, and three vicariances and two dispersals were inferred during the evolutionary process of this clade. Discussion Phylogenetic inferences In this study, well-supported and highly resolved intrageneric relationship of Lilium was recovered based on a large plastome data set, in which most nodes received full branch support, providing a robust backbone phylogeny for critically exploring the classification of the genus. Similar to previous molecular phylogenetic investigations of Lilium [ 14 – 20 ], the plastome-based phylogeny failed to resolve the seven sections recognized by Comber [ 12 ] and sect. Nomocharis defined by Gao and Gao [ 22 ] as monophyletic. Remarkably, as revealed by previous studies [ 15 , 17 , 18 ], there were significant conflicts between the nuclear and plastid phylogenies (cytonuclear discordance). As frequent gene flows were observed among Lilium sections [ 17 ], the cytonuclear discordance in Lilium phylogeny, as well as the non-monophyly of these section-level taxonomic units within the genus, were proposed to have been caused by inter-sectional hybridization rather than by ILS [ 15 , 17 ]. Nevertheless, our data imply that the ILS caused by evolutionary radiation cannot be ruled out as a feasible cause for the cytonuclear discordance in Lilium phylogeny and the absence of monophyly in these section-level taxonomic units within the genus. Briefly, both LTT and BAMM analyses showed that explosive radiation since the late Miocene ( ca . 9 Ma) may have played an essential role in developing the rich species diversity of Lilium . Such radiative diversification most likely resulted in ancestral allelic polymorphisms being shared between closely related lineages or species [ 44 – 48 ], thereby resulting in phylogenetic incongruence and the non-monophyly of these Lilium sections. This study also provides circumstantial evidence to the speculation that convergent evolution is a non-negligible evolutionary trigger for the blurred taxonomic boundaries of these section-level taxonomic units defined by floral morphologies [ 49 , 50 ]. Pertinently, S-DIVA analysis inferred that nine cross-regional dispersals occurred during the evolutionary process of Lilium , which would result in the gathering of populations of genetically distinct species in new circumstances. Given that the adaptation to new habitat would trigger colonized populations to evolve convergently in morphologically functional traits [ 51 ], the widespread evolutionary convergence would result in distinct Lilium taxa possessing highly similar morphological characteristics [ 49 , 50 ]. Collectively, the lines of evidence suggest that the evolution of Lilium may have experienced frequent hybridization, ILS, and morphologically evolutionary convergence. The co-occurrence of such evolutionarily complicated events imply that accurate subdivision of the genus can be a challenging task. To establish a credible classification system for this genus, taxonomic work based on multidisciplinary evidence is needed. Biogeographic scenario Since the beginning of life, climate changes occurring in various geological eras have profoundly impacted the evolution of organisms [ 52 – 57 ]. Theoretically, large-scale climatic change can lead to geographic shifts in available niches for plants [ 58 – 60 ], thus triggering migration or local extinction, which in turn significantly affects the distribution of plant taxa and communities [ 61 ]. Such biogeographic scenario is well evidenced in the evolution of Lilium , as our data suggest that the Neogene climatic changes played a crucial role in shaping the extant distribution of the genus. Under the context of a robust phylogenetic framework, the historical biogeography of Lilium was explored in this study using molecular dating and S-DIVA analysis. Although previous studies based on nuclear ITS data set proposed the geographic origin of Lilium in Southwest China and the Himalayas [ 15 , 18 ], the S-DIVA analysis showed that the maternal MRCA of Lilium might distribute throughout the Northern Hemisphere, spreading throughout Eurasia and North America. Associated with a dispersal and an extinct event, the crown node of Lilium was dated at 16.82 Ma, coinciding with the MMCO, the warmest interval of the last 23 Ma [ 62 – 64 ]. Within the Northern Hemisphere, the global warming in the MMCO most likely led to the expansion of temperate forest zone toward high latitudes and altitudes, as well as the northward expansion of tropical rainforests [ 65 ]. Given that the winged seeds of Lilium are easily transported by wind over long distances, the substitution of vegetation zones along latitudinal and altitudinal gradients in the MMCO would trigger the northward or upward migration of ancestral populations and local extinction of some ancestral populations encountering physical or ecological barriers, and thus drove the early divergence of Lilium . After MMCO, the global temperatures have gradually decreased since the Middle Miocene Climate Transition (MMCT, 15.97–11.61 Ma) [ 63 , 64 ]. This climatic change resulted in the expansion of temperate biome and aridification in continental interior in the Northern Hemisphere [ 66 – 68 ], which may have triggered in the dispersal, vicariance, and extinction events within these two early diverged Lilium ancestral populations. This scenario can be justified by the results of molecular dating and S-DIVA analysis: The divergence of Clade I that formed the rudiment of the disjunction distribution between North American and Southwest China and the Himalayas, resulting from a combination of dispersal, vicariance, and extinction, occurred at 13.03 Ma; accompanied by a vicariance, the crown age of Clade II was dated at 12.18 Ma. Subsequently, S-DIVA analysis inferred five dispersal events within Clades I. Among them, two intercontinental dispersal events, which were dated to 10.54 Ma and 10.05 Ma, respectively, from Southwest China and the Himalayas to North American were inferred; the remaining three dispersal events, which were dated to 9.8 Ma, 9.17 Ma, and 7.06 Ma, occurred within East Asia. On timescale, all these dispersal events took place in the late Miocene, when the intensification of Asian summer monsoon established a humid climate, and caused a significant expansion of forests in East Asia [ 69 – 74 ]. These climatic and environmental shifts would create favorable habitats that facilitated the eastward and northward spread of Lilium from Southwest China and the Himalayas to East, Central, South China and northern Indochina, as well as to North China and Northeast Asia. Because of the existence of the Bering land bridge (BLB) in the Miocene [ 75 , 76 ], the ancestor of L. philadelphicum could migrate from East Asia to North America via the BLB. Within Clade II, the formation of extent distribution range may also have been driven by the ancient climatic changes describe above. For instance, as the intensification of the Asian monsoon created a connection between forests from low to high latitudes of East Asia around the Oligocene–Miocene transition [ 69 ], the climatic cooling in the late Miocene would drive the southward and westward expansion of the ancestral populations of L. davidii to reach Central and Southwest China. Additionally, along with the expansion of temperate forests toward high latitudes of East Asia in the early Pliocene [ 70 ], the ancestor of L. bulbiferum may have migrated into Europe. New insights into evolutionary diversification of Lilium Posterior to the MMCT, global temperate has been continuously decreased since the late Miocene [ 62 – 64 , 66 , 67 , 77 – 79 ]. This climate change is assumed to have led to the expansion of temperate habitats and subsequently proliferation of temperate biomes, and the broader niches may have triggered rapid species diversification in the temperate regions [ 68 ]. Given the lack of empirical studies, more evidence is needed to confirm whether the global climate cooling since the late Miocene has generally contributed to the diversification of plant taxa adapted to temperate climates in the Northern Hemisphere [ 68 ]. Moreover, the distribution of plant diversity in the Northern Hemisphere is extremely uneven, with much higher diversity in East Asia than in Europe and North America [ 80 – 84 ]. To date, the geological and climatic events triggering this uneven distribution of plant diversity remain poorly elucidated [ 85 ]. Interestingly, extant Lilium species are typically distributed in temperate regions of the Northern Hemisphere, and there are far more Lilium species in East Asia than in Central Asia, Europe, and North America [ 2 ]. Therefore, investigating the evolutionary diversification of the genus may provide insightful evidence for better understanding of the effects of the late Miocene global cooling on species diversification in temperate plant taxa, and to explore the climatic and geological events responsible for the uneven distribution of plant diversity in the Northern Hemisphere. Under time-calibrated phylogenetic framework, both LTT and BAMM analyses identically showed that species diversification rate of Lilium has abruptly increased since 9.0 Ma, around the late Miocene. This shift in species diversification rate appears in parallel with the global cooling posterior to the MMCO [ 62 – 64 , 77 – 79 ], the intensification of monsoonal climate in East Asia [ 69 – 73 , 86 , 87 ], and the further uplift of the QTP [ 88 , 89 ]. This suggests that the acceleration of species diversification rate observed in Lilium may have been jointly triggered by these climatic and geological events. Globally, rich niche and ecological and climatic heterogeneity are the basis for forming species diversity [ 90 – 92 ]. From this perspective, the global cooling since the late Miocene and the resultant expansion of temperate habitats may have provided more niches for species diversification in Lilium . Synchronously, global cooling led to regional aridification of inlands of the Northern Hemisphere, which would fragment habitats of Lilium and promoted vicariance to burst speciation. Regionally, the QTP ulteriorly rose from the late Miocene to the early Pliocene, which further strengthened the monsoonal climate in East Asia [ 88 , 89 , 93 – 95 ]. The uplift of QTP created diverse habitats in East Asia, particularly in Southwest China and the Himalayas [ 96 , 97 ], and the intensification of summer monsoon established favorable humid climate over much of East Asia [ 69 – 73 ]. Such complex geological, ecological, and environmental heterogeneity in East Asia is proposed to have driven rapid diversification of a wide spectrum of plant taxa [ 35 , 69 , 85 , 97 – 100 ], and would facilitate species radiation in Lilium . Oppositely, the uplift of QTP simultaneously led to arid environment in Asia inland [ 69 , 101 – 103 ], and thus would cause local reduction of Lilium species in central Asia. As a result, there are more rich Lilium species diversity in East Asia than in Central Asia, Europe, and North America. As a case study, the inferred historical diversification of Lilium provides new insights into the importance of the uplift of QTP and its induced climatic changes in the formation of uneven distribution of plant diversity in the Northern Hemisphere. Conclusions In this study, robust backbone phylogeny of Lilium was reconstructed based on a large plastome data set, providing a robust phylogenetic framework for exploring classification, historical biogeography, and evolutionary diversification for the genus. The results suggest that the ancestor of Lilium might widely distribute throughout the temperate regions of the Northern Hemisphere, and the early divergence of the genus may have been driven by the global warming in the MMCO. Subsequently, the global cooling since the late Miocene, as well as the uplift of the QTP and the enhancement of monsoonal climate in East Asia from the late Miocene to the Pliocene, jointly shaped the distribution range and driven evolutionary radiation of this genus. Additionally, the inferred evolutionary radiation and distant dispersal events in Lilium imply the evolution of the genus may have experienced ILS and morphological convergence. Together with intersectional hybridization revealed by a previous study [ 17 ], these evolutionarily complicated events may have resulted in the blurred sectional boundaries in this genus. Given that the rapid uplift of QTP and enhancement of monsoonal climate dramatically triggered radiative species divergence, there are higher Lilium species diversity in East Asia than in Central Asia, Europe, and North America. As a case study, our findings suggest that the unique geological and climatic events in the Neogene of East Asia, such as the rapid uplift of QTP and the establishment of monsoonal climate, may have played essential role in formation of uneven distribution of plant diversity in the Northern Hemisphere. Methods Plant samples, DNA extraction and Illumina sequencing In total of 50 currently accepted Lilium species were included in this study. The taxonomic sampling covers 43.48% extant species and the entire distribution range of this genus. Among them, plastomes representing 14 species were newly sequenced in this study ( Additional file 2 : Table S1), the remaining were obtained from publicly available GenBank database (last accessed on Apr. 1, 2022; Additional file 3 : Table S2). The plant materials sampled in this study was identified by Dr. Yunheng Ji. The voucher specimens were deposited at herbarium of Kunming Institute of Botany (Chinese Academy of Sciences), and the original sources of the plant samples and voucher information are presented in Additional file 2 : Table S1. Total genomic DNAs of these newly collected samples were extracted from apporaximately10 mg silica gel dried leaves using the CTAB method [ 104 ]. Paired-end libraries with an average insert size of approximately 400 bp were prepared using a TreSeq DNA Sample Prep Kit (Illumina, Inc., USA) according to the manufacturer’s protocol. Shotgun sequencing was performed on the Illumina Novaseq 6000 platform [ 105 ] to generate approximately four Gb of raw reads for each sample. Plastome assembly and annotation The software Trimmomatic v0.40 [ 106 ] was used to remove adaptors and to filter low-quality Illumina reads with presetting parameters. Based on the clean reads, plastomes were assembled with the pipeline GetOrganelle v1.9.77 [ 107 ], using the complete plastome DNA sequence of L. taliense (GenBank Accession Number: KY009938) as the reference. Assembly graphs were visualized and edited using Bandage v0.8.0 [ 108 ]. Assembled plastomes were annotated with GeSeq [ 109 ]. Positions for start and stop codons and the exon/intron boundaries were checked manually using Geneious v10.2 [ 110 ]. Annotated tRNA genes were further verified with tRNAScan-SE v2.0 [ 111 ]. The multiple genome alignment program Mauve v4.0 [ 112 ] was used to detect structural rearrangements among Lilium plastomes. Phylogenetic analysis Phylogenetic framework of Lilium was reconstructed based on 53 complete plastomes. Inferred from previous phylogenetic studies [ 18 , 20 , 113 ], the complete plastomes of 16 species from Fritillaria , Cardiocrinum , Notholirion and Tulipeae were designed as outgroups. The complete plastome DNA sequences were aligned using the MAFFT program [ 114 ]. Phylogenetic analyses were performed with maximum likelihood (ML) and Bayesian inference (BI) methods. ML phylogeny was reconstructed using IQ-Tree v2.1.3 [ 115 , 116 ] under the TVM + F + R6 model, with 1000 rapid-search replicates to estimate bootstrap (BS) support for each node. BI analysis was performed using MrBayes v3.22 [ 117 ] and the TVM + I + G model recommended by ModelTest v3.7 [ 118 ] with the Akaike information criterion [ 119 ]. The BI analysis run Markov Chain Monte Carlo (MCMC) algorithm of two million generations, and trees were sampled once every 100 generations with the first 25% discarded as burn-in and the effective sample size (ESS) > 200. The posterior probability values (PP) were calculated based on the remaining trees. The resulting ML and BI trees were edited using Figtree v1.4.3 [ 120 ]. Molecular dating and diversification rate estimation Divergence times were estimated using BEAST v1.10.4 [ 121 ]. Given the absence of well-documented fossils in Liliaceae, three calibration points referring to the result of Li et al [ 20 ] were used to calibrate phylogenetic tree as follows: (1) 25.16 Ma for the crown node, (2) 22.89 Ma for the stem age of the Lilium + Fritillaria clade, and (3) 18.6 Ma for the stem age of Lilium . BEAUti v1.10.4 [ 122 ] was used to set criteria for molecular dating. The ML tree was fixed as the topological constraint in the BEAST analysis, using Yule process prior with the uncorrected lognormal relaxed clock model under GTR nucleotide substitution model. The MCMC simulations were run for 400 million generations with sampling every 1,000 generations. The ESS was monitored by Tracer v1.7.1 [ 123 ]. After removing the first 20% as burn-in, independent trees were combined using the TreeAnnotator v1.10.4 [ 124 ]. The rate change of species diversification over time was inferred using the LTT plot method, a visual tool to access patterns of diversity in time scales. The consensus chronogram inferred from the results of molecular dating was computed by APE v5.6-2 package [ 125 ] in an R environment. Additionally, shifts in net diversification rate in Lilium was estimate using the BAMM, an entirely orient method for detecting and quantifying heterogeneity in evolutionary rates [ 126 ]. Based on the time calibrated trees from BEAST, the BAMM analysis was conducted with BAMMtools v2.1.9 [ 126 ]. Ancestral range reconstruction The following five regions were defined for biogeographic analyses based on the distribution of extant Lilium species: (A) Southwest China and Himalayas. (B) East, Central, South China and northern Indochina. (C) North China and Northeast Asia. (D) Central Asia and Europe. (E) North America. The details are provided in Additional file 4 : Table S3. The ancestral range reconstruction was conducted using the S-DIVA method as implemented in the RASP v4 soft [ 127 ]. The tree data from BEAST analysis were used as the input trees, and the maximum number of areas at each node was set to five. To avoid biased inferences caused by uncertainty in the root areas of the outgroups, in advance, we removed outgroups using APE v5.6-2 package [ 125 ]. Abbreviations BAMM: Bayesian Analysis of Macroevolutionary Mixtures; BI: Bayesian Inference; BLB: Bering land bridge; bp: Base pair; BP: Bootstrap percentage; BS: Bootstrap; CTAB: Cetyl trimethylammonium bromide; DNA: Deoxyribonucleic acid; ESS: Effective sample size; Gb: Giga base pairs; HPD: Highest posterior density; ILS: Incomplete lineage sorting; IR: Inverted repeat; ITS: internal transcribed spacer of nuclear ribosomal DNA; QTP: Qinghai-Tibet Plateau; LSC: Large single-copy; LTT: lineage through time; Ma: Mega-annum; MCMC: Markov Chain Monte Carlo; ML: Maximum Likelihood; MMCT: Middle Miocene Climate Transition; MMCO: Middle Miocene Climate Optimum; MRCA: Most recent common ancestor; PP: Posterior probability; rRNA: Ribosomal RNA; S-DIVA: Statistical-Dispersal Vicariance Analysis; SSC: Small single copy; tRNA: Transfer RNA. Declarations Ethics approval and consent to participate Collection of all plant samples in this study completely followed the Regulations on the Protection of Wild Plants of the People's Republic of China, the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. Consent to publication Not applicable. Availability of data and materials The sequences generated in this study are available at GenBank (accession numbers are presented in Additional file 2 : Table S1 and Additional file 3 : Table S2). Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This study was financially supported by Yunnan Revitalization Talent Support Program “Top Team” Project, the National Natural Science Foundation of China (31872673), the NSFC-Joint Foundation of Yunnan Province (U1802287). Authors’ contributions YJ conceived the study; NZ and KM collected and analyzed the data; NZ, KM, and YJ wrote the manuscript; YJ, YH, LJ, JH discussed the results and revised the manuscript. All authors have read and approved the manuscript. Acknowledgements We thank the Germplasm Bank of Wild Species (Kunming Institute of Botany, Chinese Academy of Sciences) and Haicheng An for providing some plant materials in this study; We also grateful to Lifang Yang for her help with data analysis. References Royal Botanic Gardens, Kew. Plants of the World Online. 2022. Available at: https://powo.science.kew.org/. Accessed May 2, 2022. Liang SY, Tamura MN. Lilium L. In: Wu ZY, Raven PH, editors. Flora of China. 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Paradis E, Schliep K. Ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics. 2019;35:526–528. https://doi.org/10.1093/bioinformatics/bty633. Rabosky DL, Grundler M, Anderson C, Title P, Shi JJ, Brown JW, et al. BAMMtools: an R package for the analysis of evolutionary dynamics on phylogenetic trees. Methods Ecol Evol. 2014;5:701–707. https://doi.org/10.1111/2041-210X.12199. Yu Y, Blair C, He X. RASP 4: Ancestral state reconstruction tool for multiple genes and characters. Mol Biol Evol. 2020;37:604–606. https://doi.org/10.1093/molbev/msz257. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1Fig.S1.pdf Additional file 1 (Fig. S1): Mauve alignment of 53 complete plastomes of Lilium , with Lilium amabile as reference. Additionalfile2TableS1.docx Additionalfile3TableS2.docx Additionalfile4TableS3.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2303338","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":157471430,"identity":"7984f689-77c0-47b1-bd3c-312165c9b066","order_by":0,"name":"Nian Zhou","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nian","middleName":"","lastName":"Zhou","suffix":""},{"id":157471431,"identity":"4f99777a-7f3b-4e07-878a-cb4f80198ed4","order_by":1,"name":"Ke Miao","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Miao","suffix":""},{"id":157471432,"identity":"120f35f2-bebc-4359-9188-3c8f4fe23678","order_by":2,"name":"Changkun Liu","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Changkun","middleName":"","lastName":"Liu","suffix":""},{"id":157471433,"identity":"cc6fe46a-2b9d-49ff-95e7-6b1966459365","order_by":3,"name":"Linbo Jia","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Linbo","middleName":"","lastName":"Jia","suffix":""},{"id":157471434,"identity":"72741301-a136-4656-8794-35d015ee0a97","order_by":4,"name":"Jinjin Hu","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinjin","middleName":"","lastName":"Hu","suffix":""},{"id":157471436,"identity":"3b69f878-4744-4cf7-a2f3-33ab81e685e5","order_by":5,"name":"Yongjiang Huang","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yongjiang","middleName":"","lastName":"Huang","suffix":""},{"id":157471438,"identity":"6dcc4f00-9937-4882-807a-d97f0cddb1a2","order_by":6,"name":"Yunheng Ji","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACxmYGNoYPDAw8fED6MNFaGGcAtbARrQUI2Jh5QCQQMhOlnrmdx+yxbY6dDFBL4uECBjt5BvazBwg4jMfcOHdbMshhBw7PYEg2bODJSyCkxUw6dxszUAt7w2EeBuYEBgkeA8JaLLfVw7TUE6mFcdthiMN4GA4To4WtTLJ323EeNma2hMMzDI4btvHk4Ndi2H94m8TPbdX2/Oxtxp8LKqrl+dnPENDSwAFVAI4UA1AEEQDAaHhASM0oGAWjYBSMdAAAJMUydIqtw+UAAAAASUVORK5CYII=","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yunheng","middleName":"","lastName":"Ji","suffix":""}],"badges":[],"createdAt":"2022-11-23 03:29:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2303338/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2303338/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30018509,"identity":"ad7717c6-c177-4ae4-9442-6ba68fe80957","added_by":"auto","created_at":"2022-12-07 16:03:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":211451,"visible":true,"origin":"","legend":"\u003cp\u003ePlastome phylogeny of \u003cem\u003eLilium\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e(a) Maximum likelihood (ML) phylogeny of \u003cem\u003eLilium \u003c/em\u003einferred from complete plastome DNA sequences. (b) Bayesian inference (BI) phylogeny of \u003cem\u003eLilium \u003c/em\u003einferred from complete plastome DNA sequences. Numbers at nodes represent maximum likelihood bootstrap (BS) percentages or Bayesian posterior probabilities (PP), and an asterisk represents the BP or PP is 100/1.00. The sectional treatments of Comber (1949) and the section \u003cem\u003eNomocharis \u003c/em\u003edefined by Gao et al\u003cem\u003e. \u003c/em\u003e(2012) are indicated on the right.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-2303338/v1/4da7adfeb9e51135644cf06d.png"},{"id":30017598,"identity":"4e68ef58-58fa-4d1d-b2eb-c4281447691f","added_by":"auto","created_at":"2022-12-07 15:55:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1248029,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular dating in \u003cem\u003eLilium\u003c/em\u003ebased on complete plastome DNA sequences. Numbers above/under the tree branches represent mean divergent ages and 95% confidence interval of divergence times. Red arrows indicate the calibration points for the molecular dating (see text). Divergence time and the timeline are indicated in mega-annum (Ma).\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-2303338/v1/c4ab40ac4f93d3950c9c9e85.png"},{"id":30017594,"identity":"f2233496-f04f-4223-b000-ed82cf8565d7","added_by":"auto","created_at":"2022-12-07 15:55:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66099,"visible":true,"origin":"","legend":"\u003cp\u003eResult of lineage through time (LTT) plots analysis.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-2303338/v1/f58f8da55dffca7fbf7164da.png"},{"id":30017596,"identity":"bc1ca5d0-5914-4709-aa8c-d3629c7bebcf","added_by":"auto","created_at":"2022-12-07 15:55:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43232,"visible":true,"origin":"","legend":"\u003cp\u003eResult of Bayesian Analysis of Macro-evolutionary Mixtures (BAMM) based on the time-calibrated maximum clade credibility tree from BEAST. Colors on the branch represent the mean of the posterior density of net diversification rate (speciation rate minus extinction rate).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-2303338/v1/c899aba19add91b42e80e16c.png"},{"id":30019605,"identity":"f8431cca-d374-4432-979f-206d9525d1a5","added_by":"auto","created_at":"2022-12-07 16:11:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1847967,"visible":true,"origin":"","legend":"\u003cp\u003eReconstruction of ancestral area of \u003cem\u003eLilium \u003c/em\u003eusing S-DIVA analysis inferred from plastid tree. \u003cem\u003eLilium \u003c/em\u003especies assigned to five areas based on their current distributions: A. Southwest China and Himalayas, B. East, Central, South China and northern Indochina, C. North China and Northeast Asia, D. Central Asia and Europe, E. North America.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-2303338/v1/d6b54b637fa57dc6a57a25f7.png"},{"id":43790606,"identity":"d5d2000f-656a-4224-8445-247faaef5161","added_by":"auto","created_at":"2023-09-27 20:52:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1323614,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2303338/v1/dc77d873-f4a7-4157-9bc3-54a4e0106550.pdf"},{"id":30017602,"identity":"eaa26923-d810-4030-9db6-5712a41b3557","added_by":"auto","created_at":"2022-12-07 15:55:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8512310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1 (Fig. S1):\u003c/strong\u003e Mauve alignment of 53 complete plastomes of \u003cem\u003eLilium\u003c/em\u003e, with \u003cem\u003eLilium amabile\u003c/em\u003e as reference.\u003c/p\u003e","description":"","filename":"Additionalfile1Fig.S1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2303338/v1/26d7d7fe4ba357d0c9b57159.pdf"},{"id":30018510,"identity":"555bb1ca-02a6-4df6-984d-d477514312b7","added_by":"auto","created_at":"2022-12-07 16:03:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13621,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-2303338/v1/8b0217a69bfb1b28761f1a80.docx"},{"id":30018507,"identity":"f90e6041-05cd-412b-9e83-55de918d214b","added_by":"auto","created_at":"2022-12-07 16:03:52","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14815,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-2303338/v1/b3aa559305dbbcaa6a1fec1e.docx"},{"id":30017601,"identity":"7a956c80-3d57-4226-b140-46bc99d7c335","added_by":"auto","created_at":"2022-12-07 15:55:52","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":22853,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile4TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-2303338/v1/f6483d2c9f9a2daba006c2a3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plastome phylogenomics, biogeography, and evolutionary diversification of Lilium (Liliaceae)","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eLilium\u003c/em\u003e L. is a large genus of the monocotyledonous family Liliaceae, which encompasses approximately 115 species of bulbous herbaceous perennials [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] spreading throughout the cold and temperate regions of the Northern Hemisphere [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. East Asia is the species diversity center of the genus [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], as nearly half of currently recognized \u003cem\u003eLilium\u003c/em\u003e species occur in China, chiefly in Southwest China and the Himalayas [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eLilium\u003c/em\u003e species are commercially valued ornamental plants and cultivated widely across the world for the esthetic property of their showy and colorful flowers [\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, the flowers and bulbs of numerous \u003cem\u003eLilium\u003c/em\u003e species are traditionally used as herbal medicines and health food in Eurasia and North America [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]; currently, their great commercial value of \u0026ldquo;medicine food homology\u0026rdquo; is attracting universal attentions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe floral morphologies of \u003cem\u003eLilium\u003c/em\u003e which are used to create subgeneric classification exhibit high levels of interspecific variations, resulting in the constant changes in the taxonomic delimitation of sectional boundaries according to different classification systems [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Among them, the classification proposed by Comber [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], which divided the genus into seven sections (i.e., sect. \u003cem\u003eArchelirion\u003c/em\u003e, sect. \u003cem\u003eDaurolirion\u003c/em\u003e, sect. \u003cem\u003eLeucolirion\u003c/em\u003e, sect. \u003cem\u003eLiriotypus\u003c/em\u003e, sect. \u003cem\u003eMartagon\u003c/em\u003e, sect. \u003cem\u003ePseudolirium\u003c/em\u003e, and sect. \u003cem\u003eSinomartagon\u003c/em\u003e), has been universally adopted. Nevertheless, previous phylogenetic studies identically showed that most of the section-level taxonomic units recognized by Comber [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] are non-monophyletic, and there are still massive controversies over the intrageneric relationships of \u003cem\u003eLilium\u003c/em\u003e [\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, \u003cem\u003eNomocharis\u003c/em\u003e Franch., which was once a segregated genus, was regarded as congeneric with \u003cem\u003eLilium\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and then reduced as a section-level taxonomic unit of \u003cem\u003eLilium\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although the taxonomic proposal that synonymized \u003cem\u003eNomocharis\u003c/em\u003e to \u003cem\u003eLilium\u003c/em\u003e has been well evidenced by molecular phylogeny [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], the monophyletic nature of the sect. \u003cem\u003eNomocharis sensu\u003c/em\u003e Gao and Gao [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] remains uncertain. Such phylogenetic ambiguities impede to better understand the biogeographic scenario and evolutionary diversification of this economically important genus.\u003c/p\u003e \u003cp\u003eTo reconstruct robust phylogenetic relationships, extending taxon sampling and employing alternative data sets with more phylogenetically informative variations are both preferred [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Recently, high-throughput sequencing technologies have been increasingly used to generate genome-scale data for phylogenetic studies. Phylogenetical analysis of sequence data generated by high-throughput sequencing, such as complete plastid genomes (plastome) and genome-wide nuclear sequences, which possess orders of magnitude more sequence variations than single or multiple sequences regions produced by Sanger sequencing, have exhibited greater potential for resolving recalcitrant relationships in a wide spectrum of plant lineages [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31 CR32 CR33 CR34 CR35\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTheoretically, phylogenetic reconstruction based on the uniparentally inherited plastomes recovers only the maternal (or in some cases the paternal) relationships of a certain plant lineage, compared with the relatively integrated evolutionary schemes recovered by analysis of the biparentally inherited nuclear genomes. Even so, with the wide application of plastomes in phylogenetic studies, numerous historically difficult issues in plant phylogenetics have been satisfactorily addressed [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], suggesting that plastomes are as important as nuclear genome data sets and will continue to play an indispensable role in plant phylogenetics.\u003c/p\u003e \u003cp\u003ePreviously, Du et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], Kim et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and Li et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] attempted to apply completed plastome DNA sequences to clarify phylogenetic ambiguities in \u003cem\u003eLilium\u003c/em\u003e. Although analysis of plastome data set largely improve phylogenetic resolution and support, sampling size at the species level of these studies was too low to recover high-resolution and well-supported phylogenetic backbone of the genus. Here, the taxonomic sampling of \u003cem\u003eLilium\u003c/em\u003e is extended to include 50 currently accepted species representing these seven sections recognized by Comber [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] plus the section \u003cem\u003eNomocharis\u003c/em\u003e defined by Gao and Gao [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Based on phylogenomic analyses of a large plastome data set, the primary objectives of the present study are: (1) to recover a robust backbone phylogeny of the economically important plant genus; and (2) to explore biogeographic scenarios and historical diversification for the genus inferred from phylogenetic framework and molecular dating.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic relationships\u003c/h2\u003e \u003cp\u003ePlastomes of \u003cem\u003eLilium\u003c/em\u003e species sampled in this study varied from 151,655 bp (\u003cem\u003eL. bakerianum\u003c/em\u003e) to 153,235 bp (\u003cem\u003eL. fargesii\u003c/em\u003e) in size, which are conserved in gene content and genome rearrangement. These plastomes contained 114 unique genes, including 80 protein-coding genes, 4 rRNA genes, and 30 tRNA genes. As shown by multiple genomes mauve alignment (\u003cb\u003eAdditional file 1\u003c/b\u003e: Fig. S1), \u003cem\u003eLilium\u003c/em\u003e plastomes were consistent in genes order and no structural rearrangement was detected. Based the alignment of complete plastome DNA sequences, the ML and BI analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) generated almost identical tree topologies (the only difference is the placement of \u003cem\u003eL. lancifolium\u003c/em\u003e), with full support for most nodes (BS\u0026thinsp;=\u0026thinsp;100%; PP\u0026thinsp;=\u0026thinsp;1.00). Both ML and BI phylogenies recovered two major clades (BS\u0026thinsp;=\u0026thinsp;100%; PP\u0026thinsp;=\u0026thinsp;1.00), consisting of East Asia\u0026thinsp;+\u0026thinsp;North America species (Clade I) and Eurasia species (Clade II). Additionally, the seven sections recognized by Comber [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and the section \u003cem\u003eNomocharis sensu\u003c/em\u003e Gao and Gao [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] were recovered as monophyletic by neither ML nor BI phylogeny.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dating and historical diversification\u003c/h2\u003e \u003cp\u003eThe BEAST analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) showed that the early divergence of \u003cem\u003eLilium\u003c/em\u003e, corresponding to the splitting of two major clades (Clade I and Clade II), occurred at 16.82 Ma (95% HPD:19.55\u0026ndash;13.60 Ma), around the middle Miocene. The crown ages of Clade I and Clade II were dated at 13.03 Ma (95% HPD: 16.43\u0026ndash;9.51 Ma) and 12.18 Ma (95% HPD:16.26\u0026ndash;7.71 Ma), respectively. Within each clade, the divergence of shallow branches, which resulted in the formation of most extant species, intensively occurred in the late Miocene, Pliocene, and Pleistocene. The lineage through time (LTT) plots analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) proposed that the diversification rate was relatively stable during the early evolution \u003cem\u003eLilium\u003c/em\u003e, but sharply accelerated in the late Miocene (\u003cem\u003eca.\u003c/em\u003e 9 Ma) and kept increasing during the Pliocene and Pleistocene. Consistent with this, the Bayesian Analysis of Macroevolutionary Mixtures (BAMM) identified a sharp shift in net species diversification rate in \u003cem\u003eLilium ca\u003c/em\u003e. 9 Ma, and the trend of continuous growth of net species diversification rate had been maintained in the Pliocene and Pleistocene (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAncestral area reconstruction\u003c/h2\u003e \u003cp\u003eThe Statistical-Dispersal Vicariance Analysis (S-DIVA) proposed that the most recent common ancestor of (MRCA) of \u003cem\u003eLilium\u003c/em\u003e might have widely occurred in Eurasia and North America, and experienced a distant dispersal and an extinction event to evolve the ancestral populations of clade I and clade II (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Although S-DIVA analysis failed to reconstruct the ancestral area of clade I, the preliminary formation of the disjunction distribution between North American and Southwest China and the Himalayas was inferred to be jointly driven by a dispersal, an extinction, and a vicariance event. Next, five dispersal, one extinction, and four vicariance events were inferred as the evolutionary driving force for the formation of the current distribution range of Clade I. Additionally, S-DIVA analysis showed that the ancestral populations of the clade II mostly likely spread throughout Eurasia from northern Indochina to Europe, and three vicariances and two dispersals were inferred during the evolutionary process of this clade.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic inferences\u003c/h2\u003e \u003cp\u003eIn this study, well-supported and highly resolved intrageneric relationship of \u003cem\u003eLilium\u003c/em\u003e was recovered based on a large plastome data set, in which most nodes received full branch support, providing a robust backbone phylogeny for critically exploring the classification of the genus. Similar to previous molecular phylogenetic investigations of \u003cem\u003eLilium\u003c/em\u003e [\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], the plastome-based phylogeny failed to resolve the seven sections recognized by Comber [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and sect. \u003cem\u003eNomocharis\u003c/em\u003e defined by Gao and Gao [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] as monophyletic. Remarkably, as revealed by previous studies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], there were significant conflicts between the nuclear and plastid phylogenies (cytonuclear discordance). As frequent gene flows were observed among \u003cem\u003eLilium\u003c/em\u003e sections [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], the cytonuclear discordance in \u003cem\u003eLilium\u003c/em\u003e phylogeny, as well as the non-monophyly of these section-level taxonomic units within the genus, were proposed to have been caused by inter-sectional hybridization rather than by ILS [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNevertheless, our data imply that the ILS caused by evolutionary radiation cannot be ruled out as a feasible cause for the cytonuclear discordance in \u003cem\u003eLilium\u003c/em\u003e phylogeny and the absence of monophyly in these section-level taxonomic units within the genus. Briefly, both LTT and BAMM analyses showed that explosive radiation since the late Miocene (\u003cem\u003eca\u003c/em\u003e. 9 Ma) may have played an essential role in developing the rich species diversity of \u003cem\u003eLilium\u003c/em\u003e. Such radiative diversification most likely resulted in ancestral allelic polymorphisms being shared between closely related lineages or species [\u003cspan additionalcitationids=\"CR45 CR46 CR47\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], thereby resulting in phylogenetic incongruence and the non-monophyly of these \u003cem\u003eLilium\u003c/em\u003e sections.\u003c/p\u003e \u003cp\u003eThis study also provides circumstantial evidence to the speculation that convergent evolution is a non-negligible evolutionary trigger for the blurred taxonomic boundaries of these section-level taxonomic units defined by floral morphologies [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Pertinently, S-DIVA analysis inferred that nine cross-regional dispersals occurred during the evolutionary process of \u003cem\u003eLilium\u003c/em\u003e, which would result in the gathering of populations of genetically distinct species in new circumstances. Given that the adaptation to new habitat would trigger colonized populations to evolve convergently in morphologically functional traits [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], the widespread evolutionary convergence would result in distinct \u003cem\u003eLilium\u003c/em\u003e taxa possessing highly similar morphological characteristics [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCollectively, the lines of evidence suggest that the evolution of \u003cem\u003eLilium\u003c/em\u003e may have experienced frequent hybridization, ILS, and morphologically evolutionary convergence. The co-occurrence of such evolutionarily complicated events imply that accurate subdivision of the genus can be a challenging task. To establish a credible classification system for this genus, taxonomic work based on multidisciplinary evidence is needed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiogeographic scenario\u003c/h2\u003e \u003cp\u003eSince the beginning of life, climate changes occurring in various geological eras have profoundly impacted the evolution of organisms [\u003cspan additionalcitationids=\"CR53 CR54 CR55 CR56\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Theoretically, large-scale climatic change can lead to geographic shifts in available niches for plants [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], thus triggering migration or local extinction, which in turn significantly affects the distribution of plant taxa and communities [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Such biogeographic scenario is well evidenced in the evolution of \u003cem\u003eLilium\u003c/em\u003e, as our data suggest that the Neogene climatic changes played a crucial role in shaping the extant distribution of the genus.\u003c/p\u003e \u003cp\u003eUnder the context of a robust phylogenetic framework, the historical biogeography of \u003cem\u003eLilium\u003c/em\u003e was explored in this study using molecular dating and S-DIVA analysis. Although previous studies based on nuclear ITS data set proposed the geographic origin of \u003cem\u003eLilium\u003c/em\u003e in Southwest China and the Himalayas [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], the S-DIVA analysis showed that the maternal MRCA of \u003cem\u003eLilium\u003c/em\u003e might distribute throughout the Northern Hemisphere, spreading throughout Eurasia and North America. Associated with a dispersal and an extinct event, the crown node of \u003cem\u003eLilium\u003c/em\u003e was dated at 16.82 Ma, coinciding with the MMCO, the warmest interval of the last 23 Ma [\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Within the Northern Hemisphere, the global warming in the MMCO most likely led to the expansion of temperate forest zone toward high latitudes and altitudes, as well as the northward expansion of tropical rainforests [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Given that the winged seeds of \u003cem\u003eLilium\u003c/em\u003e are easily transported by wind over long distances, the substitution of vegetation zones along latitudinal and altitudinal gradients in the MMCO would trigger the northward or upward migration of ancestral populations and local extinction of some ancestral populations encountering physical or ecological barriers, and thus drove the early divergence of \u003cem\u003eLilium\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAfter MMCO, the global temperatures have gradually decreased since the Middle Miocene Climate Transition (MMCT, 15.97\u0026ndash;11.61 Ma) [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. This climatic change resulted in the expansion of temperate biome and aridification in continental interior in the Northern Hemisphere [\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], which may have triggered in the dispersal, vicariance, and extinction events within these two early diverged \u003cem\u003eLilium\u003c/em\u003e ancestral populations. This scenario can be justified by the results of molecular dating and S-DIVA analysis: The divergence of Clade I that formed the rudiment of the disjunction distribution between North American and Southwest China and the Himalayas, resulting from a combination of dispersal, vicariance, and extinction, occurred at 13.03 Ma; accompanied by a vicariance, the crown age of Clade II was dated at 12.18 Ma.\u003c/p\u003e \u003cp\u003eSubsequently, S-DIVA analysis inferred five dispersal events within Clades I. Among them, two intercontinental dispersal events, which were dated to 10.54 Ma and 10.05 Ma, respectively, from Southwest China and the Himalayas to North American were inferred; the remaining three dispersal events, which were dated to 9.8 Ma, 9.17 Ma, and 7.06 Ma, occurred within East Asia. On timescale, all these dispersal events took place in the late Miocene, when the intensification of Asian summer monsoon established a humid climate, and caused a significant expansion of forests in East Asia [\u003cspan additionalcitationids=\"CR70 CR71 CR72 CR73\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. These climatic and environmental shifts would create favorable habitats that facilitated the eastward and northward spread of \u003cem\u003eLilium\u003c/em\u003e from Southwest China and the Himalayas to East, Central, South China and northern Indochina, as well as to North China and Northeast Asia. Because of the existence of the Bering land bridge (BLB) in the Miocene [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e], the ancestor of \u003cem\u003eL. philadelphicum\u003c/em\u003e could migrate from East Asia to North America via the BLB.\u003c/p\u003e \u003cp\u003eWithin Clade II, the formation of extent distribution range may also have been driven by the ancient climatic changes describe above. For instance, as the intensification of the Asian monsoon created a connection between forests from low to high latitudes of East Asia around the Oligocene\u0026ndash;Miocene transition [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], the climatic cooling in the late Miocene would drive the southward and westward expansion of the ancestral populations of \u003cem\u003eL. davidii\u003c/em\u003e to reach Central and Southwest China. Additionally, along with the expansion of temperate forests toward high latitudes of East Asia in the early Pliocene [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], the ancestor of \u003cem\u003eL. bulbiferum\u003c/em\u003e may have migrated into Europe.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNew insights into evolutionary diversification of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eLilium\u003c/span\u003e\u003c/p\u003e \u003cp\u003ePosterior to the MMCT, global temperate has been continuously decreased since the late Miocene [\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan additionalcitationids=\"CR78\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. This climate change is assumed to have led to the expansion of temperate habitats and subsequently proliferation of temperate biomes, and the broader niches may have triggered rapid species diversification in the temperate regions [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Given the lack of empirical studies, more evidence is needed to confirm whether the global climate cooling since the late Miocene has generally contributed to the diversification of plant taxa adapted to temperate climates in the Northern Hemisphere [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Moreover, the distribution of plant diversity in the Northern Hemisphere is extremely uneven, with much higher diversity in East Asia than in Europe and North America [\u003cspan additionalcitationids=\"CR81 CR82 CR83\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. To date, the geological and climatic events triggering this uneven distribution of plant diversity remain poorly elucidated [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Interestingly, extant \u003cem\u003eLilium\u003c/em\u003e species are typically distributed in temperate regions of the Northern Hemisphere, and there are far more \u003cem\u003eLilium\u003c/em\u003e species in East Asia than in Central Asia, Europe, and North America [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, investigating the evolutionary diversification of the genus may provide insightful evidence for better understanding of the effects of the late Miocene global cooling on species diversification in temperate plant taxa, and to explore the climatic and geological events responsible for the uneven distribution of plant diversity in the Northern Hemisphere.\u003c/p\u003e \u003cp\u003eUnder time-calibrated phylogenetic framework, both LTT and BAMM analyses identically showed that species diversification rate of \u003cem\u003eLilium\u003c/em\u003e has abruptly increased since 9.0 Ma, around the late Miocene. This shift in species diversification rate appears in parallel with the global cooling posterior to the MMCO [\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan additionalcitationids=\"CR78\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e], the intensification of monsoonal climate in East Asia [\u003cspan additionalcitationids=\"CR70 CR71 CR72\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e], and the further uplift of the QTP [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. This suggests that the acceleration of species diversification rate observed in \u003cem\u003eLilium\u003c/em\u003e may have been jointly triggered by these climatic and geological events.\u003c/p\u003e \u003cp\u003eGlobally, rich niche and ecological and climatic heterogeneity are the basis for forming species diversity [\u003cspan additionalcitationids=\"CR91\" citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. From this perspective, the global cooling since the late Miocene and the resultant expansion of temperate habitats may have provided more niches for species diversification in \u003cem\u003eLilium\u003c/em\u003e. Synchronously, global cooling led to regional aridification of inlands of the Northern Hemisphere, which would fragment habitats of \u003cem\u003eLilium\u003c/em\u003e and promoted vicariance to burst speciation.\u003c/p\u003e \u003cp\u003eRegionally, the QTP ulteriorly rose from the late Miocene to the early Pliocene, which further strengthened the monsoonal climate in East Asia [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e, \u003cspan additionalcitationids=\"CR94\" citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. The uplift of QTP created diverse habitats in East Asia, particularly in Southwest China and the Himalayas [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e], and the intensification of summer monsoon established favorable humid climate over much of East Asia [\u003cspan additionalcitationids=\"CR70 CR71 CR72\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Such complex geological, ecological, and environmental heterogeneity in East Asia is proposed to have driven rapid diversification of a wide spectrum of plant taxa [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e, \u003cspan additionalcitationids=\"CR98 CR99\" citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e], and would facilitate species radiation in \u003cem\u003eLilium\u003c/em\u003e. Oppositely, the uplift of QTP simultaneously led to arid environment in Asia inland [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan additionalcitationids=\"CR102\" citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e], and thus would cause local reduction of \u003cem\u003eLilium\u003c/em\u003e species in central Asia. As a result, there are more rich \u003cem\u003eLilium\u003c/em\u003e species diversity in East Asia than in Central Asia, Europe, and North America. As a case study, the inferred historical diversification of \u003cem\u003eLilium\u003c/em\u003e provides new insights into the importance of the uplift of QTP and its induced climatic changes in the formation of uneven distribution of plant diversity in the Northern Hemisphere.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, robust backbone phylogeny of \u003cem\u003eLilium\u003c/em\u003e was reconstructed based on a large plastome data set, providing a robust phylogenetic framework for exploring classification, historical biogeography, and evolutionary diversification for the genus. The results suggest that the ancestor of \u003cem\u003eLilium\u003c/em\u003e might widely distribute throughout the temperate regions of the Northern Hemisphere, and the early divergence of the genus may have been driven by the global warming in the MMCO. Subsequently, the global cooling since the late Miocene, as well as the uplift of the QTP and the enhancement of monsoonal climate in East Asia from the late Miocene to the Pliocene, jointly shaped the distribution range and driven evolutionary radiation of this genus. Additionally, the inferred evolutionary radiation and distant dispersal events in \u003cem\u003eLilium\u003c/em\u003e imply the evolution of the genus may have experienced ILS and morphological convergence. Together with intersectional hybridization revealed by a previous study [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], these evolutionarily complicated events may have resulted in the blurred sectional boundaries in this genus. Given that the rapid uplift of QTP and enhancement of monsoonal climate dramatically triggered radiative species divergence, there are higher \u003cem\u003eLilium\u003c/em\u003e species diversity in East Asia than in Central Asia, Europe, and North America. As a case study, our findings suggest that the unique geological and climatic events in the Neogene of East Asia, such as the rapid uplift of QTP and the establishment of monsoonal climate, may have played essential role in formation of uneven distribution of plant diversity in the Northern Hemisphere.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePlant samples, DNA extraction and Illumina sequencing\u003c/h2\u003e \u003cp\u003eIn total of 50 currently accepted \u003cem\u003eLilium\u003c/em\u003e species were included in this study. The taxonomic sampling covers 43.48% extant species and the entire distribution range of this genus. Among them, plastomes representing 14 species were newly sequenced in this study (\u003cb\u003eAdditional file 2\u003c/b\u003e: Table S1), the remaining were obtained from publicly available GenBank database (last accessed on Apr. 1, 2022; \u003cb\u003eAdditional file 3\u003c/b\u003e: Table S2). The plant materials sampled in this study was identified by Dr. Yunheng Ji. The voucher specimens were deposited at herbarium of Kunming Institute of Botany (Chinese Academy of Sciences), and the original sources of the plant samples and voucher information are presented in \u003cb\u003eAdditional file 2\u003c/b\u003e: Table S1.\u003c/p\u003e \u003cp\u003eTotal genomic DNAs of these newly collected samples were extracted from apporaximately10 mg silica gel dried leaves using the CTAB method [\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e]. Paired-end libraries with an average insert size of approximately 400 bp were prepared using a TreSeq DNA Sample Prep Kit (Illumina, Inc., USA) according to the manufacturer\u0026rsquo;s protocol. Shotgun sequencing was performed on the Illumina Novaseq 6000 platform [\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e] to generate approximately four Gb of raw reads for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePlastome assembly and annotation\u003c/h2\u003e \u003cp\u003eThe software Trimmomatic v0.40 [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e] was used to remove adaptors and to filter low-quality Illumina reads with presetting parameters. Based on the clean reads, plastomes were assembled with the pipeline GetOrganelle v1.9.77 [\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e], using the complete plastome DNA sequence of \u003cem\u003eL. taliense\u003c/em\u003e (GenBank Accession Number: KY009938) as the reference. Assembly graphs were visualized and edited using Bandage v0.8.0 [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e]. Assembled plastomes were annotated with GeSeq [\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e]. Positions for start and stop codons and the exon/intron boundaries were checked manually using Geneious v10.2 [\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e]. Annotated tRNA genes were further verified with tRNAScan-SE v2.0 [\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e]. The multiple genome alignment program Mauve v4.0 [\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e] was used to detect structural rearrangements among \u003cem\u003eLilium\u003c/em\u003e plastomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003ePhylogenetic framework of \u003cem\u003eLilium\u003c/em\u003e was reconstructed based on 53 complete plastomes. Inferred from previous phylogenetic studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e], the complete plastomes of 16 species from \u003cem\u003eFritillaria\u003c/em\u003e, \u003cem\u003eCardiocrinum\u003c/em\u003e, \u003cem\u003eNotholirion\u003c/em\u003e and \u003cem\u003eTulipeae\u003c/em\u003e were designed as outgroups. The complete plastome DNA sequences were aligned using the MAFFT program [\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e]. Phylogenetic analyses were performed with maximum likelihood (ML) and Bayesian inference (BI) methods. ML phylogeny was reconstructed using IQ-Tree v2.1.3 [\u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e, \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e] under the TVM\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;R6 model, with 1000 rapid-search replicates to estimate bootstrap (BS) support for each node. BI analysis was performed using MrBayes v3.22 [\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e] and the TVM\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G model recommended by ModelTest v3.7 [\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e] with the Akaike information criterion [\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e]. The BI analysis run Markov Chain Monte Carlo (MCMC) algorithm of two million generations, and trees were sampled once every 100 generations with the first 25% discarded as burn-in and the effective sample size (ESS)\u0026thinsp;\u0026gt;\u0026thinsp;200. The posterior probability values (PP) were calculated based on the remaining trees. The resulting ML and BI trees were edited using Figtree v1.4.3 [\u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dating and diversification rate estimation\u003c/h2\u003e \u003cp\u003eDivergence times were estimated using BEAST v1.10.4 [\u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e121\u003c/span\u003e]. Given the absence of well-documented fossils in Liliaceae, three calibration points referring to the result of Li et al [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] were used to calibrate phylogenetic tree as follows: (1) 25.16 Ma for the crown node, (2) 22.89 Ma for the stem age of the \u003cem\u003eLilium\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eFritillaria\u003c/em\u003e clade, and (3) 18.6 Ma for the stem age of \u003cem\u003eLilium\u003c/em\u003e. BEAUti v1.10.4 [\u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e122\u003c/span\u003e] was used to set criteria for molecular dating. The ML tree was fixed as the topological constraint in the BEAST analysis, using Yule process prior with the uncorrected lognormal relaxed clock model under GTR nucleotide substitution model. The MCMC simulations were run for 400\u0026nbsp;million generations with sampling every 1,000 generations. The ESS was monitored by Tracer v1.7.1 [\u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e123\u003c/span\u003e]. After removing the first 20% as burn-in, independent trees were combined using the TreeAnnotator v1.10.4 [\u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e124\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe rate change of species diversification over time was inferred using the LTT plot method, a visual tool to access patterns of diversity in time scales. The consensus chronogram inferred from the results of molecular dating was computed by APE v5.6-2 package [\u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e] in an R environment. Additionally, shifts in net diversification rate in \u003cem\u003eLilium\u003c/em\u003e was estimate using the BAMM, an entirely orient method for detecting and quantifying heterogeneity in evolutionary rates [\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e]. Based on the time calibrated trees from BEAST, the BAMM analysis was conducted with BAMMtools v2.1.9 [\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAncestral range reconstruction\u003c/h2\u003e \u003cp\u003eThe following five regions were defined for biogeographic analyses based on the distribution of extant \u003cem\u003eLilium\u003c/em\u003e species: (A) Southwest China and Himalayas. (B) East, Central, South China and northern Indochina. (C) North China and Northeast Asia. (D) Central Asia and Europe. (E) North America. The details are provided in \u003cb\u003eAdditional file 4\u003c/b\u003e: Table S3. The ancestral range reconstruction was conducted using the S-DIVA method as implemented in the RASP v4 soft [\u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e127\u003c/span\u003e]. The tree data from BEAST analysis were used as the input trees, and the maximum number of areas at each node was set to five. To avoid biased inferences caused by uncertainty in the root areas of the outgroups, in advance, we removed outgroups using APE v5.6-2 package [\u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBAMM: Bayesian Analysis of Macroevolutionary Mixtures; BI: Bayesian Inference; BLB: Bering land bridge; bp: Base pair; BP: Bootstrap percentage; BS: Bootstrap; CTAB: Cetyl trimethylammonium bromide; DNA: Deoxyribonucleic acid; ESS: Effective sample size; Gb: Giga base pairs; HPD: Highest posterior density; ILS: Incomplete lineage sorting; IR: Inverted repeat; ITS: internal transcribed spacer of nuclear ribosomal DNA; QTP: Qinghai-Tibet Plateau; LSC: Large single-copy; LTT: lineage through time; Ma: Mega-annum; MCMC: Markov Chain Monte Carlo; ML: Maximum Likelihood; MMCT: Middle Miocene Climate Transition; MMCO: Middle Miocene Climate Optimum; MRCA: Most recent common ancestor; PP: Posterior probability; rRNA: Ribosomal RNA; S-DIVA: Statistical-Dispersal Vicariance Analysis; SSC: Small single copy; tRNA: Transfer RNA.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollection of all plant samples in this study completely followed the\u0026nbsp;Regulations on the Protection of Wild Plants of the People\u0026apos;s Republic of China, the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequences generated in this study\u0026nbsp;are available at GenBank (accession numbers are presented in \u003cstrong\u003eAdditional file 2\u003c/strong\u003e: Table S1 and \u003cstrong\u003eAdditional file 3\u003c/strong\u003e: Table S2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by\u0026nbsp;Yunnan Revitalization Talent Support Program \u0026ldquo;Top Team\u0026rdquo; Project, the National Natural Science Foundation of China (31872673), the NSFC-Joint Foundation of Yunnan Province (U1802287).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYJ conceived the study; NZ and KM collected and analyzed the data; NZ, KM, and YJ wrote the manuscript; YJ, YH, LJ, JH discussed the results and revised the manuscript.\u0026nbsp;All authors have read and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Germplasm Bank of Wild Species (Kunming Institute of Botany, Chinese Academy of Sciences) and Haicheng An for providing some plant materials in this study; We also grateful to Lifang Yang for her help with data analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRoyal Botanic Gardens, Kew. 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Mol Biol Evol. 2020;37:604\u0026ndash;606. https://doi.org/10.1093/molbev/msz257.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Asian monsoon, climatic changes, distribution range, evolutionary convergence, radiative diversification, species diversity, Qinghai-Tibetan Plateau (QTP)","lastPublishedDoi":"10.21203/rs.3.rs-2303338/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2303338/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLilium\u003c/em\u003e (Liliaceae) is an economically important genus with great medicinal, ornamental, and edible values, however evolutionary history of the genus remains poorly understood due to the lack of robust phylogeny. Based on a large plastome data set, this study aims to recover a robust backbone phylogeny of the genus to infer its historical biogeography and evolutionary diversification.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eComplete plastomes representing 50 currently accepted species in the genus \u003cem\u003eLilium\u003c/em\u003e were sampled for phylogenetic analysis, among which, plastomes representing 14 species were newly sequenced in this study. Under time-calibrated phylogenetic framework, biogeographic scenarios and evolutionary diversification of \u003cem\u003eLilium\u003c/em\u003e were explored. Phylogenetic analysis recovered a backbone phylogeny of \u003cem\u003eLilium\u003c/em\u003e, in which most nodes were fully supported; however, failed to resolve all intrageneric sections as monophyletic. Ancestral area reconstruction proposed that the ancestor of \u003cem\u003eLilium\u003c/em\u003e might widely distribute throughout the temperate regions of the Northern Hemisphere, and has experienced multiple dispersal, extinction, and vicariance events during the evolutionary course. The rate of species diversification has sharply accelerated since the late Miocene (\u003cem\u003eca.\u003c/em\u003e 9 Ma) and kept increasing in the Pliocene and Pleistocene.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe results suggest that ancient climatic changes and geological tectonic activities, such as the Middle Miocene Climate Optimum (MMCO), the late Miocene global cooling, as well as the successive uplift of the Qinghai-Tibetan Plateau (QTP) and the enhancement of monsoonal climate in East Asia during the late Miocene and the Pliocene, jointly shaped the distribution range and drove evolutionary radiation of \u003cem\u003eLilium\u003c/em\u003e. Resulted from radiative divergence and distant dispersal events, the genus may have experienced incomplete lineage sorting (ILS) and morphological convergence. The uplift of QTP and enhancement of monsoonal climate dramatically triggered radiative divergence of species, accounting for the higher \u003cem\u003eLilium\u003c/em\u003e species diversity in East Asia than in Central Asia, Europe, and North America. The findings shed light on the crucial role of the unique Neogene geological and climatic events in East Asia, such as the uplift of QTP and the establishment of monsoonal climate, in shaping the uneven distribution of plant diversity in the Northern Hemisphere.\u003c/p\u003e","manuscriptTitle":"Plastome phylogenomics, biogeography, and evolutionary diversification of Lilium (Liliaceae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-07 15:55:47","doi":"10.21203/rs.3.rs-2303338/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7a0e3fbe-04c4-4a8c-a929-cb64e706c516","owner":[],"postedDate":"December 7th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-09-27T20:44:13+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-07 15:55:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2303338","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2303338","identity":"rs-2303338","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}