Content uploaded by Yundong Gao
Author content
All content in this area was uploaded by Yundong Gao on Jul 28, 2015
Content may be subject to copyright.
A preview of the PDF is not available
Morphological and ecological divergence of Lilium and Nomocharis within the Hengduan Mountains and Qinghai-Tibetan Plateau may result from habitat specialization and hybridization
Abstract and Figures
Several previous studies have shown that some morphologically distinctive, small genera of vascular plants that are endemic to the Qinghai-Tibetan Plateau and adjacent Hengduan Mountains appear to have unexpected and complex phylogenetic relationships with their putative sisters, which are typically more widespread and more species rich. In particular, the endemic genera may form one or more poorly resolved paraphyletic clades within the sister group despite distinctive morphology. Plausible explanations for this evolutionary and biogeographic pattern include extreme habitat specialization and hybridization. One genus consistent with this pattern is Nomocharis Franchet. Nomocharis comprises 7-15 species bearing showy-flowers that are endemic to the H-D Mountains. Nomocharis has long been treated as sister to Lilium L., which is comprised of more than 120 species distributed throughout the temperate Northern Hemisphere. Although Nomocharis appears morphologically distinctive, recent molecular studies have shown that it is nested within Lilium, from which is exhibits very little sequence divergence. In this study, we have used a dated molecular phylogenetic framework to gain insight into the timing of morphological and ecological divergence in Lilium-Nomocharis and to preliminarily explore possible hybridization events. We accomplished our objectives using dated phylogenies reconstructed from nuclear internal transcribed spacers (ITS) and six chloroplast markers.
Our phylogenetic reconstruction revealed several Lilium species nested within a clade of Nomocharis, which evolved ca. 12 million years ago and is itself nested within the rest of Lilium. Flat/open and horizon oriented flowers are ancestral in Nomocharis. Species of Lilium nested within Nomocharis diverged from Nomocharis ca. 6.5 million years ago. These Lilium evolved recurved and campanifolium flowers as well as the nodding habit by at least 3.5 million years ago. Nomocharis and the nested Lilium species had relatively low elevation ancestors (<1000 m) and underwent diversification into new, higher elevational habitats 3.5 and 5.5 million years ago, respectively. Our phylogeny reveals signatures of hybridization including incongruence between the plastid and nuclear gene trees, geographic clustering of the maternal (i.e., plastid) lineages, and divergence ages of the nuclear gene trees consistent with speciation and secondary contact, respectively.
The timing of speciation and ecological and morphological evolutionary events in Nomocharis are temporally consistent with uplift in the Qinghai-Tibetan Plateau and of the Hengduan Mountains 7 and 3-4 million years ago, respectively. Thus, we speculate that the mountain building may have provided new habitats that led to specialization of morphological and ecological features in Nomocharis and the nested Lilium along ecological gradients. Additionally, we suspect that the mountain building may have led to secondary contact events that enabled hybridization in Lilium-Nomocharis. Both the habitat specialization and hybridization have probably played a role in generating the striking morphological differences between Lilium and Nomocharis.
Figures - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
... Sealy, distinguished primarily by the exquisite symmetry of its whorled leaves (Figure 1;Stearn 1956;Liang 1980;Liang and Tamura 2000). The genetic relationship based on internal transcribed spacer (ITS) and chloroplast genome fragments reveals that it belongs to Nomocharis clade, which exhibits the contradiction of high divergence in morphology but with low genetic distancing (Liang and Tamura 2000;Gao et al. 2012Gao et al. , 2013Gao et al. , 2015. More genetic information is needed to resolve their phylogenetic relationships. ...
... Additionally, we included 3 outgroups from closely related genera. The selection of sequences is based on our previous work (Gao et al. 2012(Gao et al. , 2013(Gao et al. , 2015. ...
... To clarify the evolutionary and position of L. paradoxum, we utilized three closely related genus species as outgroups and constructed Bayesian Inference (BI) tree based on 18 representative lilies from different clades in the genus Lilium, and the results (Figure 3) mostly consistent with previous studies (Gao et al. 2012(Gao et al. , 2013(Gao et al. , 2015. The clade Nomocharis and Lophophorum form two well-supported monophyly, and L. paradoxum is in the former, being most closely related to L. gongshanense (Y.D. Gao et X.J. He) Y.D. Gao, L. apertum Franch. ...
Lilium paradoxum, a herb from southeastern Xizang, China, has its first complete chloroplast genome sequenced using next-generation sequencing. The genome is 151,814 bp, consisting of inverted repeats (IRs; 26,323 bp), small single-copy (SSC; 17,524 bp), and large single-copy regions (LSC; 81,644 bp). It encodes 112 unique genes: 78 protein-coding, 30 tRNA, and 4 rRNA genes. Phylogenetic analysis of 22 genomes shows L. paradoxum is closely related to L. gongshanense, L. apertum, and L. souliei. These findings enhance understanding of Lilium's phylogenetic relationships and evolution, particularly the Nomocharis clade in the Hengduan Mountains.
... Nomocharis Franchet (Franchet, 1889) was initially established as an independent genus close to Lilium and Fritillaria in the late 19th century. After much debate about the status of Lilium and Nomocharis (Balfour, 1918;Evans, 1925;Sealy, 1950Sealy, , 1983Liang, 1984), the latter was eventually accommodated and nested in Lilium as a subgenus clade based upon molecular phylogenetics (Nishikawa et al., 1999;Peruzzi et al., 2009;Gao et al., 2013;Du et al., 2014;Gao et al., 2015;Gao and Gao, 2016;Huang et al., 2018;Li et al., 2022a;Zhou et al., 2023). As one of the most complicated and unclear subgroups in Lilium, previous studies have provided us with a superficial understanding of its position within the genus and the possible processes of speciation that were involved (Gao et al., 2012(Gao et al., , 2013(Gao et al., , 2015Zhou et al., 2023), but the detailed phylogenetic framework and evolutionary mechanisms are not clear due to limited sampling. ...
... After much debate about the status of Lilium and Nomocharis (Balfour, 1918;Evans, 1925;Sealy, 1950Sealy, , 1983Liang, 1984), the latter was eventually accommodated and nested in Lilium as a subgenus clade based upon molecular phylogenetics (Nishikawa et al., 1999;Peruzzi et al., 2009;Gao et al., 2013;Du et al., 2014;Gao et al., 2015;Gao and Gao, 2016;Huang et al., 2018;Li et al., 2022a;Zhou et al., 2023). As one of the most complicated and unclear subgroups in Lilium, previous studies have provided us with a superficial understanding of its position within the genus and the possible processes of speciation that were involved (Gao et al., 2012(Gao et al., , 2013(Gao et al., , 2015Zhou et al., 2023), but the detailed phylogenetic framework and evolutionary mechanisms are not clear due to limited sampling. ...
... The group's distribution is narrow and limited to the Hengduan (H-D) Mountains of southwestern China, the eastern Qinghai-Tibetan Plateau (QTP), and adjacent Myanmar and India (Sealy, 1950(Sealy, , 1983Liang, 1984;1995;Liang and Tamura, 2000). These regions were formed relatively recently and underwent quite severe changes in geographic features, and this led previous authors to speculate upon the likelihood of a comparatively short process of speciation and evolution in Nomocharis clade taxa contemporaneously with the uplift of eastern Tibet and the H-D Mountains (Liang, 1984;1995;Gao et al., 2015) and, therefore, that the group had a recent origin. ...
The former genus Nomocharis, which has been merged as a clade within the genus Lilium (Liliaceae), represents one of the most complicated and unclear groups included in the latter. Research on members of the Nomocharis clade has been quite limited due to the sampling difficulties caused by its selective environmental preferences. In this study, we propose a new species within this clade, Lilium liangiae, as a further bridge connecting the former genus Nomocharis with other members of the genus Lilium. We conducted morphological clustering, phylogenetic, and comparative genomics analyses of nuclear internal spacers and the newly generated complete chloroplast genome, in conjunction with previously published sequences, and performed ancestral state reconstruction to clarify the evolutionary pattern of important traits in Lilium. The clustering results of 38 morphological traits indicated that the new species is allied to Nomocharis, further increasing the morphological polymorphism in the latter. The phylogenetic results and morphological clustering both supported L. liangiae belonging to the subclade Ecristata in Nomocharis, its closest affinity being Lilium gongshanense. Inconsistencies in phylogenetic relationships were detected between nuclear and plastid datasets, possibly due to ancient hybridization and ongoing introgression. Comparative genomics revealed the conservation and similarity of their chloroplast genomes, with variations observed in the expansion and contraction of the IR regions. A/T and palindromic repeat sequences were the most abundant. Seven highly variable regions (Pi≥0.015) were identified as potential molecular markers based on the chloroplast genomes of 47 species within Lilium. Both nuclear and plastid genes exhibited very low variability within the Nomocharis clade, contrasting with their highly variable morphological appearance. The ancestral state reconstruction analysis suggests that the campanulate flower form, as in L. liangiae, arose at least three times within the genus Lilium, revealing parallel evolution in the latter. Overall, this study adds important genetic and morphological evidence for understanding the phylogenetic relationships and parallel evolution patterns of species within the genus Lilium.
... Ecological divergence has been found to be an important mechanism of diversity in southwest China (Liu et al., 2014;Gao et al., 2015;Cai et al., 2018;Zhao et al., 2021). Apart from A. jichouense, A. sp. ...
... Cytonuclear discordance is very common in plant groups involved in adaptive radiation, extensive hybridization, and polyploidisation (e.g., Rieseberg and Soltis, 1991;Gao et al., 2015;Ma et al., 2022). In our study, the plastomes of the A. sikkimense complex have their own evolutionary history and failed to resolve the phylogenetic relationships of the different species in the A. sikkimense complex thoroughly. ...
Polyploidization is a process that typically leads to instantaneous reproductive isolation and has, therefore, been considered as one of the major evolutionary forces in the species-rich Hengduan Mountains (HM), yet this topic remains poorly studied in the region. Allium sikkimense and its relatives (about eight species) compose a natural diploid–polyploid complex with the highest diversity in the HM and adjacent areas. A combination of nuclear ribosomal DNA (nrDNA), plastome, transcriptome, and ploidy identification through chromosome counting and flow cytometry is employed to reconstruct the phylogenetic relationships in this complex and to investigate the frequency and the evolutionary significance of polyploidy in the complex. The plastome failed to resolve the phylogenetic relationships of the different species in the A. sikkimense complex, and the phylogenetic tree based on nrDNA also has limited resolution. However, our study reveals a well-resolved phylogenetic framework for species in the A. sikkimense complex using more than 1,000 orthologous genes from the transcriptome data. Previously recognized morphospecies A. sikkimense are non-monophyletic and comprise at least two independently evolved lineages (i.e., cryptic species), each forming a clade with different diploid species in this complex. The embedded pattern of octoploid A. jichouense and tetraploid A. sp. nov. within different polyploid samples of A. sikkimense supports a possible scenario of budding speciation (via niche divergence). Furthermore, our results reveal that co-occurring species in the A. sikkimense complex usually have different ploidy levels, suggesting that polyploidy is an important process for reproductive isolation of sympatric Allium species. Phylogenetic network analyses suggested that the phylogenetic relationships of the A. sikkimense complex, allowing for reticulation events, always fit the dataset better than a simple bifurcating tree. In addition, the included or exserted filaments, which have long been used to delimit species, are highly unreliable taxonomically due to their extensive parallel and convergent evolution.
... At the top, it has numerous whorls of spectacular trumpet-shaped flowers. The colour of the stem can range from green to reddish-purple, depending on genetic differences and environmental conditions (Gao et al., 2015). ...
Natural remedies for treating illnesses were sought after by ancient humans, who employed animals and plants with therapeutic powers out of instinct. Plants were utilized for prevention and therapy up until iatrochemistry was discovered in the 16th century as human understanding increased. People have sought out medicines throughout history to relieve ailments and reduce suffering. Out of eight ashtvarga plants, Lilium polyphyllum is a herbaceous, bulbous herbal medication used in traditional, conventional, and modern medical systems to treat a variety of physiological conditions. It is a significant source of many compound types with a variety of chemical structures and medicinal effects. Present review is compilation of immense medicinal potential of the plant with information on its habit, habitat and conservation status.
... Additionally, chloroplast genomes are high conservation over nuclear and mitochondrial genomes. Therefore, partial chloroplast genome sequences are preferred and suitable to use for phylogenetic studies and species/varieties identification and discrimination with similar morphology characteristics [38][39][40][41][42][43] . DNA barcoding has been deployed in prior studies owing to its effectiveness in plant variety and cultivar identification. ...
More than 200 different cultivars of durian exist worldwide but Durio zibethinus or Musang King (MK) is the most premium and prized durian fruit among the recommended varieties. Early identification of this premium variety is critical to protect from non-authentic MK durian cultivars. However, the MK variety's morphological traits are nearly identical to other varieties. Currently, the identification of durian varieties is mostly performed via evaluation of leaf shape, fruit shape, aroma, taste and seed shape and this requires trained personnel for the morphology observation. To enable the rapid identification of the MK variety, PCR amplification of ten durian varieties using six gene candidates from the chloroplast genome was first performed to obtain DNA probes that were specific to the MK durian variety. PCR amplification of ten durian varieties using primers designed confirmed that the nadhA gene sequence showed an obvious difference in the MK variety from other durian varieties. The unique sequence of MK was used as a DNA probe to develop an electrochemical biosensor for the direct identification of the MK durian variety. The electrochemical biosensor was based on the hybridization response of the immobilized DNA probe with the target DNA from the MK variety and was monitored via differential pulse voltammetry technique. Under optimal conditions, the DNA electrochemical biosensor showed a low detection limit at 10% of MK genomic DNA concentration with a wide linear calibration range of 0.05–1.5 µM (R² = 0.9891) and RSD value of 3.77% (n = 3). The results of the developed DNA biosensor provide high promise for the development of portable sensors employed in the determination of MK variety in the field.
... The exchange of genetic material in plant species is an important driver of plant evolution (Stebbins, 1959;Soltis et al., 2014;Hübner et al., 2019;Steensels et al., 2021), whereas geographic isolation prevents gene exchange among populations (Gao et al., 2015;Di Santo and Hamilton, 2021). Based on the above analysis, we believe that interspecific hybridization caused by outbreeding and the high compatibility of tea-oil Camellia germplasm resources, the obstruction of gene communication caused by geographic distance, and artificial selection are important factors in the evolution of the species in Sect. ...
Tea-oil Camellia is one of the four woody oil crops in the world and has high ecological, economic and medicinal values. However, there are great differences in the classification and merging of tea-oil Camellia Sect. Oleifera species, which brings difficulties to the innovative utilization and production of tea-oil Camellia resources. Here, ISSR, SRAP and chloroplast sequence markers were analyzed in 18 populations of tea-oil Camellia Sect. Oleifera species to explore their phylogenetic relationships and genetic diversity. The results showed that their genetic diversity were low, with mean H and π values of 0.16 and 0.00140, respectively. There was high among-population genetic differentiation, with ISSR and SRAP markers showing an Fst of 0.38 and a high Nm of 1.77 and cpDNA markers showing an Fst of 0.65 and a low Nm of 0.27. The C. gauchowensis, C. vietnamensis and Hainan Island populations formed a single group, showing the closest relationships, and supported being the same species for them with the unifying name C. drupifera and classifying the resources on Hainan Island as C. drupifera. The tea-oil Camellia resources of Hainan Island should be classified as a special ecological type or variety of C. drupifera. However, cpDNA marker-based STRUCTURE analysis showed that the genetic components of the C. osmantha population formed an independent, homozygous cluster; hence, C. osmantha should be a new species in Sect. Oleifera. The C. oleifera var. monosperma and C. oleifera populations clustered into two distinct clades, and the C. oleifera var. monosperma populations formed an independent cluster, accounting for more than 99.00% of its genetic composition; however, the C. oleifera populations contained multiple different cluster components, indicating that C. oleifera var. monosperma significantly differs from C. oleifera and should be considered the independent species C. meiocarpa. Haplotype analysis revealed no rapid expansion in the tested populations, and the haplotypes of C. oleifera, C. meiocarpa and C. osmantha evolved from those of C. drupifera. Our results support the phylogenetic classification of Camellia subgenera, which is highly significant for breeding and production in tea-oil Camellia.
Premise
Species delimitation is an integral part of evolution and ecology and is vital in conservation science. However, in some groups species delimitation is difficult, especially where ancestral relationships inferred from morphological or genetic characters are discordant, possibly due to a complicated demographic history (e.g., recent divergences between lineages). Modern genetic techniques can take account of complex histories to distinguish species at a reasonable cost and are increasingly used in numerous applications. We focus on the scribbly gums, a group of up to five closely related and morphologically similar ‘species’ within the eucalypts.
Methods
Multiple populations of each recognized scribbly gum species were sampled over a wide region across climates and genome‐wide scans used to resolve species boundaries.
Key results
We found that none of the taxa were completely diverged, and there were two genetically distinct entities: the inland distributed Eucalyptus rossii and a coastal conglomerate consisting of four species forming three discernible, but highly admixed groups. Divergence among taxa was likely driven by temporal vicariant processes resulting in partial separation across biogeographic barriers. High interspecific gene flow indicated separated taxa reconnected at different points in time blurring species boundaries.
Conclusions
Our results highlight the need for genetic screening when dealing with closely related taxonomic entities, particularly those with modest morphological differences. We show that the use of high throughput sequencing can be effective at identifying species groupings and processes driving divergence, even in the most taxonomically complex groups, and support it as a standard practice for disentangling species complexes.
This article is protected by copyright. All rights reserved.
Understanding which reproductive barriers contribute to speciation is essential to understanding the diversity of life on earth. Several contemporary examples of strong hybrid seed inviability (HSI) between recently diverged species suggest that HSI may play a fundamental role in plant speciation. Yet, a broader synthesis of HSI is needed to clarify its role in diversification. Here, I review the incidence and evolution of HSI. Hybrid seed inviability is common and evolves rapidly, suggesting that it may play an important role early in speciation. The developmental mechanisms that underlie HSI involve similar developmental trajectories in endosperm, even between evolutionarily deeply diverged incidents of HSI. In hybrid endosperm, HSI is often accompanied by whole-scale gene misexpression, including misexpression of imprinted genes which have a key role in endosperm development. I explore how an evolutionary perspective can clarify the repeated and rapid evolution of HSI. In particular, I evaluate the evidence for conflict between maternal and paternal interests in resource allocation to offspring (i.e., parental conflict). I highlight that parental conflict theory generates explicit predictions regarding the expected hybrid phenotypes and genes responsible for HSI. While much phenotypic evidence supports a role of parental conflict in the evolution of HSI, an understanding of the underlying molecular mechanisms of this barrier is essential to test parental conflict theory. Lastly, I explore what factors may influence the strength of parental conflict in natural plant populations as an explanation for why rates of HSI may differ between plant groups and the consequences of strong HSI in secondary contact.
This objective of this study was to explore the antioxidant, antihyperglycemic, and antiglycation properties of 13 methanolic extracts from 8 different Lilium species by various in vitro assays and their chemometric profiles to provide the evidence-based information for further exploration. Chemometric profiling was performed using the MetaboAnalyst and GNPS: Global Natural Products Social Molecular Networking platforms to explore and identify the metabolites from Lilium metabolomics profiles. In vitro bioactivity studies showed that flower bud extract of L. lancifolium was the most active extract with the strongest radical scavenging activity against DPPH and SO radicals, reducing activity of ferric ions and highest total phenolic contents among tested extracts. Univariate and multivariate statistical analysis revealed that the chemical variation between the sample classes: parts used (bulb vs flower); bioactivity (active vs inactive) and studied species. 13 bioactive metabolites were putatively identified and some of them were confirmed by comparing the MS/MS spectrums with the standards. Results suggest that metabolite profiling along with the bioactivity study could be useful to explore the chemical compositions, functional and developmental potentials of valuable plant species, and lay the foundation for identification, separation and assessment of their potential medicinal applications and species classification.
We sequenced and analyzed the complete chloroplast genomes of Lilium amoenum, Lilium souliei, and Nomocharis forrestii in detail, including the first sequence and structural comparison of Nomocharis forrestii. We found that the lengths and nucleotide composition of the three chloroplast genes showed little variation. The chloroplast genomes of the three Lilium species contain 87 protein coding genes (PCGs), 38 tRNAs, and 8 rRNA genes. The only difference is that Nomocharis forrestii had an additional infA pseudogene. In the sequence analysis of the Lilium chloroplast genomes, 216 SSRs, 143 pairs of long repeats, 571 SNPs, and 202 indels were detected. In addition, we identified seven hypervariable regions that can be used as potential molecular markers and DNA barcodes of Lilium through complete sequence alignment. The phylogenetic tree was constructed from the three chloroplast genome sequences of Lilium obtained here and 40 chloroplast genome sequences from the NCBI database (including 35 Lilium species, 4 Fritillaria species, and one species of Smilax). The analysis showed that the species clustering of the genus Lilium essentially conformed to the classical morphological classification system of Comber, but differences in the classification of individual species remained. In our report, we support the reclassification of Lilium henryi and Lilium rosthorniiy in the genus Lilium. In general, this study not only provides genome data for three Lilium species, but also provides a comparative analysis of the Lilium chloroplast genomes. These advances will help to identify Lilium species, clarify the phylogenetic analysis of the Lilium genus, and help to solve and improve the disputes and deficiencies in the traditional morphological classification.
Androsace L. consists of more than 100 species distributed mainly in northern temperate areas. This genus, typically characterized by having small flowers with a constricted corolla throat, was subdivided into six sections: sect. Samuelia Schlechtd., sect. Mirabiles (Hand. -Mazz.) Yang & Huang, sect. Androsace, sect. Chamaejasme Koch., sect. Aizoidium Hand. -Mazz., and sect. Orthocaulon Hand. -Mazz. In this genus, the species with cushion-like growth belong to sect. Chamaejasme occurring in alpine habitats. There are other three small genera in the tribe Androsaceae. Pomatosace Maxim., with only one species, P. filicula Maxim., endemic to Qinghai-Tibetan Plateau, is traditionally placed in the subtribe Soldanellinae because of its circumscissile capsules, but other morphological characters and the recently reported evidence from cytology indicate that its affinity is closer with Androsace. Douglasia Lindley has eight homogeneous species in Northern American mountains. Vitaliana Sesler, with only one species in the European Alps, is usually considered a congener of Douglasia. These four genera, morphologically similar in having small flowers with a constricted corolla throat and "Androsace pollen", referred here as "Androsace group", am disjunctly distributed from the Qinghai-Tibetan Plateau, to Central- and Northern-Asia, Europe and North America. It is an ideal group to elucidate the origin and dispersal patterns of the Northern Hemisphere flora, which has a distribution center in the eastern Qinghai-Tibetan Plateau and the Hengduan Mountains. In addition, the habits of this group range from annuals to rosette perennials and cushion-like perennials. The cushion-like species are disjunctly distributed in the central Qinghai-Tibetan Plateau and European Alps mountains. Further studies are needed to determine whether they are closely related to each other or it is only the result of convergent evolution under the habitat pressure selection in separate sites. We firstly reported cpDNA trnL-F, nrDNA ITS sequences respectively or both for 29 populations of 14 species in Androsace and Pomatosace mainly from the central Qinghai-Tibetan Plateau. A phylogeny was further constructed for these species, and those distributed in Europe and North America in "Androsace group" and related genera in Primulaceae based on newly reported sequences and those downloaded from GenBank. Phylogenetic inferences indicated that the four genera in "Androsace group" form a well-supported monophyletic clade. Two main clades were discovered in the "Androsace group": a wholly Androsace clade, and the other comprising Pomatosace, Douglasia, Vitaliana and nine Androsace species in all analyses. The grouping and position of three species of sect. Samelia and two species of sect. Androsace varied according to the analyses of the different datasets, trnL-F, ITS or a combination of them. The biogeographical mapping of species distribution revealed that the basal species of each clade sited in southeastern China and the eastern Qinghai-Tibetan Plateau, which obviously supports the origin area hypotheses for "Androsace group" based on morphological studies. From the eastern Qinghai-Tibetan Plateau multiple lineages spread to and colonized Europe in different ages. The crude timing based on ITS sequence differentiation indicated a recent diversification of "Androsace group" within Miocene of the Tertiary. The cushion-like species evolved independently in Asia and Europe. The origin and diversification of cushion-like species in the Qinghai-Tibetan Plateau might be earlier than those in Europe. Their occurrence in the Qinghai-Tibetan Plateau area might be correlated with the large-scale uprising of the plateau and the mountains building since Miocene. But the appearance of the cushion-like species in Europe might result from the fast habitat isolation due to the climate oscillation and the cycles of developing and retreating of the ice sheet in the Northern Hemisphere during the Quaternary. The large-scale colonization of cushion-like Androsace species in the Qinghai-Tibetan Plateau probably occurred in the late Holocene when the global climate re-cooled after the end of glacial cycles. The intraspecific genetic differentiation in some species might partly reflect their retreat and re-colonization in the Qinghai-Tibetan Plateau area during the climate oscillation.
Milula, a monotypic genus endemic to the Qinghai-Tibetan Plateau, was found to be nested deeply within Allium by the molecular phylogeny despite the aberrant morphology. It remains unknown what had contributed to the rapid evolution of morphology and origin of this exceptional species. In contrast to a previous report of its karyotypes with 2n = 16 = 8M+8SM (2SAT), similar to most species of Allium, a rather different karyotype, 2n = 20 = 4M +10SM+6T (2SAT), was found in examined 31 individuals from 6 populations of M. spicata distributed in the central Tibet. Karyotypes of 7 Allium species occurring in the Qinghai-Tibetan Plateau were further reported. The basic number x = 8 was confirmed for all of them and their karyotypes consist mainly of metacentric and submetacentric chromosomes with rare subterminal and terminal chromosomes. The karyotype of M. spicata is distinctly different from that of most Allium species occurring in the plateau through a complete comparison of all available species in this region and adjacent areas. However, the same chromosome number and similar karyotypic structure were found in A. fasciculatum of Sect. Bromatorrhiza, indicating a possible close relationship between them. But this similarity is contradictory to the preliminary molecular phylogenetic analysis that Milula was closely related to A. cyathophorum of Sect. Bromatorrhiza with x=8, but the other species with x=10 and 11 in this section were clearly placed in the other clade. We therefore suggested that the paralleling evolution from x=8 to x=9, 10 and 11 with increasing asymmetry of karyotype possibly due to the chromosomal Robertsonian translocation might occur separately in the two recognized phylogenetic lineages of Allium. In addition to aneuploidy and following change of the chromosomal structures, the habitat isolation due to the recent uplift of the Qinghai-Tibetan Plateau and the Quaternary climatic oscillation, plays a greater role in origin of Milula and other endemic species (genera) with aberrant morphology from their progenitors.