­­
Figure 6. Maximum likelihood tree of Primula species based on chloroplast genomes. Bootstrap support values over 95% are labeled with asterisks. Outgroups and P. poissonii complex are highlighted with gray and purple shadings, respectively.
4. Discussion
4.1. The general characteristics of Primula chloroplast genomes
As with most angiosperms, the chloroplast genomes were conserved inPrimula species, with similar GC content and typical quadripartite structures, including small and large single copy (SSC and LSC) regions separated by two inverted repeats (IRs) regions [60]. However, gene loss was found here. The infA gene, which encodes translation initiation factor 1 [61], was present in the chloroplast genome of P. poissonii and P. wilsonii , but was not present in the related Primula chloroplast genomes in our study. Additionally, these findings are consistent with the results of somePrimula species and other groups in angiosperm chloroplast genomes in previous studies [62, 63]. Remarkably, the ycf15gene was only missing in the chloroplast genomes of P. wilsonii .ycf15 was located in the IR region and was highly conserved. The absence of ycf15 was also reported in many other plants, such asColchicum genus [64]. However, the function of theycf15 gene remains unclear and needs to be further investigated.
The patterns of gene loss we revealed here could be used for phylogeny reconstruction and species identification. The loss of ycf15 gene in colchicine plants successfully determined the infrageneric relationship in the expanded Colchicum genus [64]. Thus, the non-presence of ycf15 we found here might be a valuable molecular marker to separate P. wilsonii from P. poissonii , which is morphologically similar to P. wilsonii . Both of the two species are perennial herbs with candelabra inflorescence and purple flowers, so some scholars argue P. wilsonii should be merged into P. poissonii or treated as a subspecies of P. poissonii . Here we suggested that the missing ycf15 gene in the P. wilsoniichloroplast genome could be extremely useful for distinguishing the two confusing species at the molecular level.
4.2. The evolution of the chloroplast genomes in Primula
IR regions are highly conserved in most angiosperm chloroplast genomes. However, the contraction and expansion of IR regions are not rare [65]. In this study, gene orders at the boundaries of SC/IR regions were the same among the five chloroplast genomes of Primula . However, the accurate positions of the genes at the SC/IR border were slightly varied, such as the genes rps19 , ndhF ,ycf1 , rpl2 and trnH [63]. In addition, some genes normally located in the SC region, such as ndhF , had moved to IR region due to the expansion of the IR region. It was reported that the chloroplast genomes’ size, the LSC/SSC length, the gene numbers and pseudogene origination could vary among different species due to the expansion and contraction of IR regions [66, 67]. Moreover, the loss of IR regions has been occasionally detected in some taxa [68]. Thiscould be the reason that the chloroplast genome size of P. miyabeana was the largest among the five Primula species with the longest IR region, and the chloroplast genome size of P. secundiflora was the smallest with the shortest IR region. Furthermore, a large number of studies also confirmed that the length of IR region greatly affected the chloroplast genome size [69, 70].
Species of Primula are famous for their ornamental value and heterostyly phenomenon in Southwest China. More genomic resources are needed to deeply investigate the phylogeny, biogeography, genetics and heterostyly evolution of Primula species. In addition, considering that P. wilsonii is a plant species with extremely small populations (PSESP), we need more genetic information for the conservation of germplasm resources. The numbers and distributions of repeat sequences, especially large repeats that are longer than 20 bp and 60 bp, may play important roles in the arrangement and recombination of the plastid genome [71, 72]. A total of 123 repeats were detected in the six Primula chloroplast genomes. All the repeat sequences appeared to be shorter than 60 bp in length. These findings are consistent with the results in other Primula species [63, 73], but not in agreement with the results of some other angiosperm plants, such as herbaceous Alpinia species [74] or woodyAquilaria species [70]. Our study detected very high levels of polymorphism in the large repeat sequences among the sixPrimula species in terms of both the types or numbers. Therefore, these large repeats might be an informative source for developing genetic markers for population genetics and phylogenetic constructions of Primula [75]. SSRs markers are a valuable genetic resource for phylogenetic investigations, population genetics assessment and species discrimination due to their abundant polymorphism and codominant inheritance [70, 76]. The SSRs markers detected here were mostly A/T mono-nucleotide repeats (28/38), similar to the results of otherPrimula species [63] and some other angiosperm species [77, 78]. The vast majority of SSRs loci were in SC regions (78.95% in LSC regions and 15.79% in SSC regions), yet few of them were present in IR regions. Moderate sequence divergence with greater variability in the SC region of Primula chloroplast genomes was displayed, which corresponded with previous studies [79]. Since the hyper-variable regions of the chloroplast genome are useful for phylogenetic construction, population genetics and DNA barcoding, the 17 highly polymorphic loci and the SSRs markers found in our study could serve as potential genetic markers for further phylogenetic and biogeographic analyses, population genetics and conservation analysis ofPrimula species.
4.3. Phylogenetic relationships of Chinese Primula
A total of 60 species representing 20 of 24 sections in ChinesePrimula were sampled in our phylogenetic construction using chloroplast genome sequences based on ML method. Three major clades ofPrimula were detected with high internal support in this study, which was in accordance with previous studies [25, 73, 80]. Several sections did not exhibit monophyletic taxa, such as Sects.Monocarpicae , Crystallophlomis , Obconicolisteri ,Denticulata and Proliferae , which were partly or entirely confirmed by the previous viewpoints [25, 73, 80]. A decision on the monophyly of Sect. Proliferae requires additional consideration. It has been treated as a monophyletic group based on the concatenation of ITS, matK and rbcL sequences [25, 73]. However, the chloroplast transcripts and protein coding sequences from chloroplast genomes analyses strengthen the assumption that Sects.Amethyatina and Petiolares species are nested within Sect.Proliferae [80]. This assumption is additionally supported by the results based on the whole chloroplast genome analysis in our investigation. This is corroborated by morphological traits such as an umbel with multiple flowers, campanulate calyx, and globose capsule. On the one hand, the conflicting phylogenetic diagnoses of nuclear and chloroplast sequences are common in plants [81]. On the other hand, the adaptive radiation caused by heterostyly, polyploidization and natural hybridization, or gene introgression might complicate the phylogenetic relationships under Primula[20-24]. This would explain why quite a few sections inPrimula didn’t belong to monophyletic group according to morphological characters.
P. wilsonii , together with P. poissonii , P. anisodora, and P. miyabeana (endemic to Taiwan) form to P. poissonii complex, which was one of the taxonomically challenging groups in Sect. Proliferae . The close relationship of these species has been revealed in studies, and P. wilsonii was closest to P. miyabeana based on rbcL + matK + ITS sequences, with low support [25]. However, the closest relative species was P. anisodora with very high support based on chloroplast genomic sequences in this study. Therefore, we suggest that the phylogenetic relationships between Primula species need to be further studied based on more genetic information, especially at the genome level, and we may come to the conclusion that chloroplast genomes sequences could provide a valuable resource for phylogenetic constructing of Primula .
5. Conclusions
This study compared the basic characteristics of the chloroplast genomes from several Chinese Primula species. We assessed the variation and IR boundaries evolution among these species. Furthermore, we constructed the phylogenetic relationships of the genus Primulacovering a wide range of samples based on their chloroplast genomic sequences. In addition, we determined the conserved and variable regions in the chloroplast genomes. The large repeat sequences, SSRs loci, and 17 hypervariable regions were detected here, which could be used for population genetics and phylogenetic analysis in the future. Three major clades in Primula were confirmed, yet the sections were not in accordance with morphological traits, reflecting in the non-monophyletic nature of several sections. Therefore, we suggest that chloroplast genomes provide useful genetic and evolutionary information for studies on the phylogeny, population genetics, and conservation ofPrimula species.
Funding: This research was funded by National Natural Science Foundation of China (32100169) and Natural Science Foundation of Anhui Province (2108085QC104), Natural Science Foundation from Educational Commission of Anhui Province (KJ2020B25) to Y.P. Xie, the Henan Province Youth Talent Lift Project of China (212102310242) and the Key Scientific and Technological Project of Henan Province (2021HYTP042) to G.-G. Yang.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: The data presented in the study are depositing in the NCBI repository, and the accession numbers are shown in the article.
Acknowledgments: The authors wish to thank Jian-Li Zhao and Li Li for help in collecting samples and operation of software; Heng-Yi Shao and MPDI for their advices on the language organization and English editing.
Conflicts of Interest: The authors declare no conflict of interest.
References
Barrett, S.C.H.; Shore, J.S. New insights on heterostyly: comparative biology, ecology and genetics. In Self-incompatibility in flowering plants-evolution, diversity and mechanisms ; Franklin-Tong, V.E., Ed.; Springer-Verlag: Berlin, Germany, 2008; pp 3–32.
  1. Chen, M.L.; You, Y.L.; Zhang, X.P. Advances in the research of heterostyly. Acta. Pratacult. Sin. 2010 ,19 , 226–239.
  2. Ganders, F.R. The biology of heterostyly. Nea. Zeal. J. Bot. 1979 , 17 , 607-635.
  3. Watanabe, K.; Yang, T.Y.A.; Nishihara, C.; Huang, T.L.; Nakamura, K.; Peng, C.I.; Sugawara, T. Distyly and floral morphology ofPsychotria cephalophora (Rubiaceae) on the oceanic Lanyu (Orchid) Island, Taiwan. Bot. Stud. 2015 ,56 , 10.
  4. Xu, X.Y., Zhou, L.L., Wang, Z.K., Zhuang, L. Flower distyly and breeding system of Limonium chrysocomum. Bull. Bot. Res. 2015 , 35 , 883-890.
  5. Yang, C.; He, X.; Gou, G. Ophiorrhiza guizhouensis (Rubiaceae), a new species from Guizhou Province, southwestern China.PhytoKeys 2018 , 95 , 121.
  6. Zhang, C.; Wang, L.L.; Duan, Y.W.; Lan, D.; Yang, Y.P. Pollination ecology of Arnebia szechenyi (Boraginaceae), a Chinese endemic perennial characterized by distyly and heteromorphic self-incompatibility. Ann. Bot. Fenn. 2014 ,51 , 297-304.
  7. Webb, C.J.; Lloyd, D.G. The avoidance of interference between the presentation of pollen and stigmas in angiosperms II. Herkogamy. Nea. Zeal. J. Bot. 1986 , 24 , 163-178.
  8. Darwin, C. The different forms of flowers on plants of the same species. John Murray: London, UK, 1877
  9. Barrett, S.C.H.; Cruzan, M.B. Incompatibility in heterostylous plants. In Genetic control of self-incompatibility and reproductive development in flowering plants ; Williams, E.G., Clarke, A.E., Knox, R.B. Eds.; Springer Netherlands: Dordrecht, Holland, 1994; pp 189-219.
  10. Barrett, S.C.H. The evolution of plant sexual diversity.Nat. Rev. Genet. 2002 , 3 , 274-284.
  11. Barrett, S.C.H. Heterostylous genetic polymorphisms: model systems for evolutionary analysis. In Evolution and function of heterostyly ; Barrett, S.C.H., Ed.; Springer-Verlag: Berlin, Germany, 1992; pp 1-29.
  12. Weller, S.G. The different forms of flowers – what have we learned since Darwin? Bot. J. Linn. Soc. 2009 , 160 , 249–261.
  13. Cohen, J.I. A case to which no parallel exists’: the influence of Darwin’s Different Forms of Flowers. Am. J. Bot.2010 , 97 , 701–716.
  14. J. R. Primula (new edition) . Timber Press: Oregon, USA, 2003
  15. Wen, J.; Zhang, J.Q.; Nie, Z.L.; Zhong, Y.; Sun, H. Evolutionary diversifications of plants on the Qinghai-Tibetan Plateau.Front. Genet. 2014 , 5 , 4
  16. Smith, W.W; Fletcher, H.R. XVII. —The genus Primula : SectionsObconica , Sinenses , Reinii , Pinnatae ,Malacoides , Bullatae , Carolinella , Grandis and Denticulata. Trans. R. Soc. Edinb . 1947 , 61, 415–478.
  17. Wendelbo, P. Studies in Primulaceae. II. An account of Primulasubgenus Sphondylia (Syn. Sect. Floribundae ) with a review of the subdivisions of the genus. Matematisk-Naturvitenskapelig Ser. 1961, 11, 1-46.
  18. Hu, C.M., Primulaceae . Science Press: Beijing, China, 1990; Vol. 59, p 2.
  19. De Vos, J.M.; Hughes, C.E.; Schneeweiss, G.M.; Moore, B.R.; Conti, E. Heterostyly accelerates diversification via reduced extinction in primroses. P. Roy. Soc. B-Biol. Sci. 2014 ,281 , 20140075.
  20. Guggisberg, A.; Mansion, G.; Conti, E. Disentangling reticulate evolution in an arctic-alpine polyploid complex. Syst. Biol. 2009 , 58 , 55-73.
  21. Ma, Y.P.; Tian, X.; Zhang, J.; Wu, Z.K.; Sun, W.B. Evidence for natural hybridization between Primula beesiana and P. bulleyana, two heterostylous primroses in NW Yunnan, China. J. Syst. Evol. 2014 , 52 , 500–507.
  22. Xie, Y.P.; Zhu, X.F.; Ma, Y.P.; Zhao, J.L.; Li, L.; Li, Q.J. Natural hybridization and reproductive isolation between two Primulaspecies. J. Integr. Plant Biol. 2017 ,59 , 526-530.
  23. Xie, Y.P.; Zhao, J.L.; Zhu, X.F.; Li, L.; Li, Q.J. Asymmetric hybridization of Primula secundiflora and P. poissoniiin three sympatric populations. Biodiv. Sci.2017 , 25 , 647-653.
  24. Yan, H.F.; Liu, Y.J.; Xie, X.F.; Zhang, C.Y.; Hu, C.M.; Hao, G.; Ge, X.J. DNA barcoding evaluation and its taxonomic implications in the species-rich genus Primula L. in China. PLoS One2015 , 10 , e0122903.
  25. Sun, W.B.; Yang, J.; Dao, Z.L., Study and conservation of plant species with extremely small populations (PSESP) in Yunnan province, China. Science Press: Beijing, China, 2019; p 176
  26. Moore, M.J.; Soltis, P.S.; Bell, C.D.; Burleigh, J.G.; Soltis, D.E. Phylogenetic analysis of 83 plastid genes further resolves the early diversification of eudicots. Proc. Natl. Acad. Sci. U.S.A. 2010 , 107 , 4623– 4628.
  27. Daniell, H.; Lin, C.S.; Yu, M.; Chang, W.J. Chloroplast genomes: diversity, evolution, and applications in genetic engineering.Genome Biol. 2016 , 17 , 1-29.
  28. Daniell, H. Transgene containment by maternal inheritance: Effective or elusive? Proc. Natl. Acad. Sci. U.S.A. 2007 ,104 , 6879–6880.
  29. Wang, L.; Wuyun, T.-N.; Du, H.Y.; Wang, D.P.; Cao, D.M. Complete chloroplast genome sequences of Eucommia ulmoides : genome structure and evolution. Tree Genet. Genomes2016 , 12 , 12.
  30. Palmer, J.D.; Jansen, R.K.; Michaels, H.J.; Chase, M.W.; Manhart, J.R. Chloroplast DNA variation and plant phylogeny. Ann. Mo. Bot. Gard. 1988 , 75 , 1180-1206.
  31. Wambugu, P.W.; Brozynska, M.; Furtado, A.; Waters, D.L.; Henry, R.J. Relationships of wild and domesticated rices (Oryza AA genome species) based upon whole chloroplast genome sequences. Sci. Rep.2015 , 5 , 13957.
  32. Nguyen, V.B.; Park, H.S.; Lee, S.C.; Lee, J.; Park, J.Y.; Yang, T.J. Authentication markers for five major Panax species developed via comparative analysis of complete chloroplast genome sequences. J. Agric. Food Chem. 2017 ,65 , 6298-6306.
  33. Henriquez, C.L.; Arias, T.; Pires, J.C.; Croat, T.B.; Schaal, B.A. Phylogenomics of the plant family Araceae. Mol. Phylogenet. Evol. 2014 , 75 , 91-102.
  34. Zhai, W.; Duan, X.S.; Zhang, R.; Guo, C.C.; Li, L.; Xu, G.X.; Shan, H.Y.; Kong, H.Z.; Ren, Y. Chloroplast genomic data provide new and robust insights into the phylogeny and evolution of the Ranunculaceae. Mol. Phylogenet. Evol. 2019 ,135 , 12-21.
  35. Kirill, A.; Alexander, U.; Maksim, M.; Vladimir, K.; Vera, G. Comparative analysis of chloroplast genomes of seven perennialHelianthus species. Gene 2021 ,774 , 145418.
  36. Shinozaki, K.; Ohme, M.; Tanaka, M.; Wakasugi, T.; Hayashida, N.; Matsubayashi, T.; Zaita, N.; Chunwongse, J.; Obokata, J.; Yamaguchi-Shinozaki, K.; et al. The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J. 1986 , 5 , 2043–2049.
  37. Ohyama, K.; Fukuzawa, H.; Kohchi, T.; Shirai, H.; Sano, T.; Sano, S.; Umesono, K.; Shiki, Y.; Takeuchi, M.; Chang, Z.; et al. Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nat. Rev. Genet.1986 , 322 , 572-574.
  38. Xie, Y.P.; Jiang, X.F.; Yang G.G. The complete plastome ofPrimula wilsonii , a heterostylous ornamental species. MITOCHONDRIAL DNA PART B 2021, 6, 1324-1325.
  39. Doyle, J.J.; Doyle, J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 1987 ,19 , 11-15.
  40. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014 ,30 , 2114–2120.
  41. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol.2012 , 19 , 455-477.
  42. Boetzer, M.; Henkel, C.V.; Jansen, H.J.; Butler, D.; Pirovano, W. Scaffolding preassembled contigs using SSPACE. Bioinformatics2011 , 27 , 578-579.
  43. Boetzer, M.; Pirovano, W. Toward almost closed genomes with GapFiller. Genome Biol. 2012 , 13 , R56
  44. Langmead, B.; Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 2012 , 9 , 357–359.
  45. Hyatt, D.; Chen, G.L.; Locascio, P.F.; Land, M.L.; Larimer, F.W.; Hauser, L.J. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics2010 , 11 , 119.
  46. Wu, S.T.; Zhu, Z.W.; Fu, L.M.; Niu, B.F.; Li, W.Z. WebMGA: a customizable web server for fast metagenomic sequence analysis.BMC Genomics , 2011 , 12 , 444
  47. Laslett, D.; Canback, B. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res.2004 , 32 , 11-16.
  48. Lohse, M.; Drechsel, O.; Kahlau, S.; Bock, R. OrganellarGenomeDRAW - a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets.Nucleic Acids Res. 2013 , 41 , W575–W581.
  49. Kurtz, S.; Choudhuri, J.V.; Ohlebusch, E.; Schleiermacher, C.; Stoye, J.; Giegerich, R. REPuter: The manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res.2001 , 29 , 4633-4642.
  50. Beier, S.; Thiel, T.; Münch, T.; Scholz, U.; Mascher, M. MISA-web: a web server for microsatellite prediction. Bioinformatics2017 , 33 , 2583-2585.
  51. Frazer, K.A.; Pachter, L.; Poliakov, A.; Rubin, E.M.; Dubchak, I. VISTA: Computational tools for comparative genomics. Nucleic Acids Res. 2004 , 32 , 273-279.
  52. Brudno, M.; Do, C.B.; Cooper, G.M.; Kim, M.F.; Davydov, E.; Nisc Comparative Sequencing Program; Green, E.D.; Sidow, A.; Batzoglou, S. LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA. Genome Res. 2003 , 13 , 721-731.
  53. Rozas, J.; Ferrer-Mata, A.; Sánchez-Delbarrio, J.C.; Guirao-Rico, S.; Librado, P.; Ramos-Onsins, S.E.; Sánchez-Gracia, A. DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Mol. Biol. Evol. 2017 , 34 , 3299-3302.
  54. Amiryousefi, A.; Hyvönen, J.; Poczai, P. IRscope: an online program to visualize the junction sites of chloroplast genomes.Bioinformatics 2018 , 34 , 3030-3031.
  55. Chen, Z.D.; Yang, T.; Lin, L.; Lu, L.M.; Li, H.L.; Sun, M.; Liu, B.; Chen, M.; Niu, Y.T.; Ye, J.F.; et al. Tree of life for the genera of Chinese vascular plants. J. Syst. Evol. 2016 , 54, 277-306.
  56. Katoh, K.; Standley, D.M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 2013 , 30 , 772–780.
  57. Darriba, D; Taboada G.L.; Doallo, R.; Posada, D. jModelTest 2: more models, new heuristics and parallel computing. Nat. Methods ,2012 , 9, 772.
  58. Stamatakis, A.; Hoover, P.; Rougemont, J. A rapid bootstrap algorithm for the RAxML web servers. Syst. Biol. 2008 ,57 , 758-771.
  59. Xin, T.Y.; Zhang, Y.; Pu, X.D.; Gao, R.R.; Xu, Z.C.; Song, J.Y. Trends in herbgenomics. Sci. China Life Sci. 2019 ,62 , 288- 308.
  60. Wicke, S.; Schneeweiss, G.M.; Depamphilis, C.W.; Müller, K.F.; Quandt, D. The evolution of the plastid chromosome in land plants: gene content, gene order, gene function. Plant Mol. Biol.2011 , 76 , 273-297.
  61. Gichira, A.W.; Li, Z.Z.; Saina, J.K.; Long, Z.C.; Hu, G.W.; Gituru, R.W.; Wang, Q.F.; Chen, J.M. The complete chloroplast genome sequence of an endemic monotypic genus Hagenia (Rosaceae): Structural comparative analysis, gene content and microsatellite detection. Peer J 2017 , 5 , e2846.
  62. Ren, T.; Yang, Y.C.; Zhou, T.; Liu, Z.L. Comparative plastid genomes of Primula species: sequence divergence and phylogenetic relationships. Int. J. Mol. Sci. 2018 ,19 , 1050.
  63. Nguyen, P.A.T.; Kim, J.S.; Kim, J.H. The complete chloroplast genome of colchicine plants (Colchicum autumnale L. and Gloriosa superba L.) and its application for identifying the genus.Planta 2015 , 242 , 223-237.
  64. Kim, K.J.; Lee, H.L. Complete chloroplast genome sequences from Korean Ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants. DNA Research2004 , 11 , 247-261.
  65. Menezes, A.P.A.; Resende-Moreira, L.C.; Buzatti, R.S.O.; Nazareno, A.G.; Carlsen, M.; Lobo, F.P.; Kalapothakis, E.; Lovato, M.B. Chloroplast genomes of Byrsonima species (Malpighiaceae): comparative analysis and screening of high divergence sequences. Sci. Rep. 2018 , 8 , 1-12.
  66. Saina, J.K.; Li, Z.Z.; Gichira, A.W.; Liao, Y.Y. The complete chloroplast genome sequence of tree of heaven (Ailanthus altissima (mill.)) (sapindales: Simaroubaceae), an important pantropical tree. Int. J. Mol. Sci. 2018 ,19 ,
  67. Yi, X.; Gao, L.; Wang, B.; Su, Y.J.; Wang, T. The complete chloroplast genome sequence of Cephalotaxus oliveri (Cephalotaxaceae): Evolutionary comparison of Cephalotaxus chloroplast DNAs and insights into the loss of inverted repeat copies in Gymnosperms.Genome Biol. Evol. 2013 , 5 , 688–698.
  68. Sun, Y.X.; Moore, M.J.; Lin, N.; Adelalu, K.F.; Meng, A.; Jian, S.G.; Yang, L.S.; Li, J.Q.; Wang, H.C. Complete plastome sequencing of both living species of Circaeasteraceae (Ranunculales) reveals unusual rearrangements and the loss of the ndh gene family. BMC Genomics 2017 , 18 , 592.
  69. Ren, F.M.; Wang, L.Q.; Li, Y.; Zhuo, W.; Xu, Z.C.; Guo, H.J.; Liu, Y.; Gao, R.R.; Song, J.Y. Highly variable chloroplast genome from two endangered Papaveraceae lithophytes Corydalis tomentella andCorydalis saxicola. Ecol. Evol. 2021 ,11 , 4158-4171.
  70. Pombert, J.F.; Lemieux, C.; Turmel, M. The complete chloroplast DNA sequence of the green alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes. BMC Biol. 2006 ,4 , 3.
  71. Guisinger, M.M.; Kuehl, J.V.; Boore, J.L.; Jansen, R.K. Extreme reconfiguration of plastid genomes in the angiosperm family Geraniaceae: rearrangements, repeats, and codon usage.Mol. Biol. Evol. 2011 , 28 , 583–600.
  72. Xu, W.B.; Xia, B.S.; Li, X.W. The complete chloroplast genome sequences of five pinnate-leaved Primula species and phylogenetic analyses. Sci. Rep. 2020 ,10 , 20782.
  73. Li, D.M.; Zhu, G.F.; Xu, Y.C.; Ye, Y.J.; Liu, J.M. Complete chloroplast genomes of three medicinal Alpinia species: genome organization, comparative analyses and phylogenetic relationships in family Zingiberaceae. Plants 2020 , 9 , 286.
  74. Hishamuddin, M.S.; Lee, S.Y.; Ng, W.L.; Ramlee, S.I.; Lamasudin, D.U.; Mohamed, R. Comparison of eight complete chloroplast genomes of the endangered Aquilaria tree species (Thymelaeaceae) and their phylogenetic relationships. Sci. Rep .2020 , 10 , 13034
  75. Gulzar, K.; Zhang, F.Q.; Gao, Q.B.; Fu, P.C.; Zhang, Y.; Chen, S.L. Spiroides shrubs on Qinghai-Tibetan Plateau: multilocus phylogeography and palaeodistributional reconstruction of Spiraea alpina andS. Mongolica (Rosaceae). Mol. Phylogenet. Evol.2018 , 123 , 137-148.
  76. Shen, J.; Zhang, X.; Jacob, B.L.; Zhang, H.J.; Deng, T.; Sun, H.; Wang, H.C. Plastome Evolution in Dolomiaea (Asteraceae, Cardueae) using Phylogenomic and Comparative analyses.Front. Plant Sci. 2020 , 11 , 376.
  77. Wang, L.Y.; Wang, J.; He, C.Y.; Zhang, J.G.; Zeng, Y.F. Characterization and comparison of chloroplast genomes from two sympatric Hippophae species (Elaeagnaceae). J. Forestry Res. 2021 , 32 , 307-318.
  78. Zhu, A.D.; Guo, W.H.; Gupta, S.; Fan, W.S.; Mower, J.P. Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates. New Phytol.2016 , 209 , 1747-1756.
  79. Liu, T.J. A transcriptomic phylogenomic study of Primula L. (Primulaceae). Doctor, South China Agreicultural University, Guangzhou, China, 2018.
  80. Degna, J.H.; Rosenberg, N.A. Gene tree discordance, phylogenetic inference and the multispecies coalescent. Trends Ecol. Evsol. 2009 , 24 , 332-340.