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.