Backbone phylogeny of Salix based on genome skimming data

doi:10.1016/j.pld.2024.09.004

Abstract

Abstract: The genus Salix is a common component of the Northern Hemisphere dendroflora with important ecological and economic value. However, taxonomy and systematics of Salix is extremely difficult and relationships between main lineages, especially deep phylogenies, remain largely unresolved. In this study, we used genome-skimming, plastome assembly, and single-copy orthologs (SCOs) from 66 Salix accessions, along with publicly available plastome and sequence read archive (SRA) datasets to obtain a robust backbone phylogeny of Salix, clarify relationships between its main lineages, and gain a more precise understanding of the origin and diversification of this species-rich genus. The plastome and SCO datasets resolved Salix into two robust clades, with plastome-based phylogenies lacking inner resolution and SCO offering fully resolved phylogenies. Our results support the classification of Salix into five subgenera: Salix, Urbaniana, Triandrae, Longifoliae and Vetrix. We observed a significant acceleration in the diversification rate within the Chamaetia-Vetrix clade, while Salix exhibited increased rates of diversification spanning from the early Oligocene to the late Miocene. These changes coincided with contemporaneous tectonic and climate change events. Our results provide a foundation for future systematic and evolutionary studies of Salix. Additionally, we showed that genome skimming data is an efficient, rapid, and reliable approach for obtaining extensive genomic data for phylogenomic studies, enabling the comprehensive elucidation of Salix relationships.

Key words: Salix, Genome skimming, Phylogenomics, Orthologous coding sequencing, Subgeneric classification

Kai-Yun Chen, Jin-Dan Wang, Rui-Qi Xiang, Xue-Dan Yang, Quan-Zheng Yun, Yuan Huang, Hang Sun, Jia-Hui Chen. Backbone phylogeny of Salix based on genome skimming data[J]. Plant Diversity, 2025, 47(02): 178-188.

References

Abdollahzadeh, A., Osaloo, S.K., Maassoumi, A., 2011. Molecular phylogeny of the genus Salix (Salicaceae) with an emphasize to its species in Iran. Iran. J. Bot., Le 17, 244-253.
Argus, G.W., Eckenwalder, J.E., Kiger, R.W., 2010. Salicaceae.
Azuma, T., Kajita, T., Yokoyama, J., et al., 2000. Phylogenetic relationships of Salix (Salicaceae) based on rbcL sequence data. Am. J. Bot. 87, 67-75.
Bankevich, A., Nurk, S., Antipov, D., et al., 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455-477.
Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120.
Boucher, L.D., Manchester, S.R., Judd, W.S., 2003. An extinct genus of Salicaceae based on twigs with attached flowers fruits, and foliage from the Eocene Green River Formation of Utah and Colorado, USA. Am. J. Bot. 90,1389-1399.
Buchler, W., 1996. Phyllotaxis and morphology of proximal leaves on vegetative axillary shoots of Salix and their systematic implications. Bot. Helv. 106, 31-44.
Capella-Gutierrez, S., Silla-Martinez, J.M., Gabaldon, T., 2009. TrimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972-1973.
Chen, J.H., Huang, Y., Brachi, B., et al., 2019. Genome-wide analysis of Cushion willow provides insights into alpine plant divergence in a biodiversity hotspot. Nat. Commun. 10, 5230.
Chen, J.H., Sun, H., Wen, J., et al., 2010. Molecular phylogeny of Salix L. (Salicaceae) inferred from three chloroplast datasets and its systematic implications. Taxon 59, 29-37.
Chernomor, O., von Haeseler, A., Minh, B.Q., 2016. Terrace aware data structure for phylogenomic inference from supermatrices. Syst. Biol. 65, 997-1008.
Collinson, M.E., 1992. The early fossil history of Salicaceae - a brief review. Proc. Royal Soc. B- Biol. Sci. 98, 155-167.
Dai, X.G., Hu, Q.J., Cai, Q.L., et al., 2014. The willow genome and divergent evolution from poplar after the common genome duplication. Cell Res. 24, 1274-1277.
Dierckxsens, N., Mardulyn, P., Smits, G., 2017. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 45, e18.
Dorn, R.D., 1976. A synopsis of American Salix. Can. J. Bot. 54, 2769-2789.
Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19, 11-15.
Dylus, D., Altenhoff, A., Majidian, S., et al., 2024. Inference of phylogenetic trees directly from raw sequencing reads using Read2Tree. Nat. Biotechnol. 42, 139-147.
Emms, D.M., Kelly, S., 2019. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 20, 238.
Fang, Z., Zhao, S., Skvortsov, A., 1999. Salicaceae. In: Wu, Z, et al. (eds), Flora of China. Science Press, Beijing, pp. 139-274.
Favre, A., Packert, M., Pauls, S.U., et al., 2015. The role of the uplift of the Qinghai-Tibetan Plateau for the evolution of Tibetan biotas. Biol. Rev. 90, 236-253.
Feliner, G.N., Rossello, J.A., 2007. Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants. Mol. Phylogenet. Evol. 44, 911-919.
Guindon, S., Dufayard, J.F., Lefort, V., et al., 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307-321.
Gulyaev, S., Cai, X.-J., Guo, F.-Y., et al., 2022. The phylogeny of Salix revealed by whole genome re-sequencing suggests different sex-determination systems in major groups of the genus. Ann. Bot. 129, 485-498.
Hardig, T., Anttila, C., Brunsfeld, S., 2010. A phylogenetic analysis of Salix (Salicaceae) based on matK and ribosomal DNA sequence data. J. Bot., Le 2010, 197696.
He, L., Wagner, N.D., Horandl, E., 2021. Restriction-site associated DNA sequencing data reveal a radiation of willow species (Salix L., Salicaceae) in the Hengduan Mountains and adjacent areas. J. Systemat. Evol. 59, 44-57.
Hoang, D.T., Chernomor, O., von Haeseler, A., et al., 2018. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518-522.
Huang, Y., Wang, J., Yang, Y.P., et al., 2017. Phylogenomic analysis and dynamic evolution of chloroplast genomes in Salicaceae. Front. Plant Sci. 8, 1050.
Hughes, C.E., Atchison, G.W., 2015. The ubiquity of alpine plant radiations: from the Andes to the Hengduan Mountains. New Phytol. 207, 275-282.
Johnson, M.G., Gardner, E.M., Liu, Y., et al., 2016. HybPiper: extracting coding sequence and introns for phylogenetics from high-throughput sequencing reads using target enrichment. Appl. Plant Sci. 4, 1600016.
Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., et al., 2017. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587-589.
Kapli, P., Yang, Z., Telford, M.J., 2020. Phylogenetic tree building in the genomic age. Nat. Rev. Genet. 21, 428-444.
Karp, A., Shield, I., 2008. Bioenergy from plants and the sustainable yield challenge. New Phytol. 179, 15-32.
Katoh, K., Standley, D.M., 2016. A simple method to control over-alignment in the MAFFT multiple sequence alignment program. Bioinformatics 32, 1933-1942.
Kimura, A., 1988. De salicis subgenere Pleuradenia commentatio. Sci Rep Tohoku Univ (4 Ser Biol) 39, 143-147.
Kozlov, A., Stamatakis, A., 2020. Using RAxML-NG in Practice. In: Phylogenetics in the Genomic Era. In: Scornavacca, C. (Ed.). No commercial publisher | Authors open access book, p. 1.3:1-1.3:25.
Lauron-Moreau, A., Pitre, F.E., Argus, G.W., et al., 2015. Phylogenetic relationships of American willows (Salix L., Salicaceae). PLoS One 10, e0121965.
Leache, A.D., Banbury, B.L., Felsenstein, J., et al., 2015. Short tree, long tree, right tree, wrong Tree: new acquisition bias corrections for inferring SNP phylogenies. Syst. Biol. 64, 1032-1047.
Leache, A.D., Oaks, J.R., 2017. The utility of single nucleotide polymorphism (SNP) data in phylogenetics. Annu. Rev. Ecol. Evol. Syst. 48, 69-84.
Lemoine, F., Domelevo Entfellner, J.-B., Wilkinson, E., et al., 2018. Renewing Felsenstein's phylogenetic bootstrap in the era of big data. Nature 556, 452-456.
Leskinen, E., Alstrom-Rapaport, C., 1999. Molecular phylogeny of Salicaceae and closely related Flacourtiaceae: evidence from 5.8 S, ITS1 and ITS2 of the rDNA. Plant Systemat. Evol. 215, 209-227.
Li, H., Durbin, R., 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754-1760.
Li, W.Q., Shi, M.M., Huang, Y., et al., 2019. Climatic change can influence species diversity patterns and potential habitats of Salicaceae plants in China. Forests 10, 220.
Liu, B.-B., Ma, Z.-Y., Ren, C., et al., 2021. Capturing single-copy nuclear genes, organellar genomes, and nuclear ribosomal DNA from deep genome skimming data for plant phylogenetics: a case study in Vitaceae. J. Systemat. Evol. 59, 1124-1138.
Manchester, S.R., Judd, W.S., Handley, B., 2006. Foliage and fruits of early poplars (Salicaceae: Populus) from the Eocene of Utah, Colorado, and Wyoming. Int. J. Plant Sci. 167, 897-908.
Marincek, P., Leveille-Bourret, E., Heiduk, F., et al., 2024. Challenge accepted: evolutionary lineages versus taxonomic classification of North American shrub willows (Salix). Am. J. Bot. 111, e16361.
Minh, B.Q., Schmidt, H.A., Chernomor, O., et al., 2020. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530-1534.
Nakamura, T., Yamada, K.D., Tomii, K., et al., 2018. Parallelization of MAFFT for large-scale multiple sequence alignments. Bioinformatics 34, 2490-2492.
Nevill, P.G., Zhong, X., Tonti-Filippini, J., et al., 2020. Large scale genome skimming from herbarium material for accurate plant identification and phylogenomics. Plant Methods 16, 1-8.
Nieto Feliner, G., Gutierrez Larena, B., Fuertes Aguilar, J., 2004. Fine-scale Geographical Structure, intra-individual polymorphism and recombination in nuclear ribosomal internal transcribed spacers in Armeria (Plumbaginaceae). Ann. Bot. 93, 189-200.
Ohashi, H., 2000. A systematic enumeration of Japanese Salix (Salicaceae). J. Jpn. Bot. 75, 1-41.
Ohashi, H., 2001. Salicaceae of Japan. Sci. Rep. Tohoku Univ. (4 Ser Biol) 40, 269-396.
Pei, M., Hunter, T., Royle, D., 1999. Host-pathogen relationship between Salix and Melampsora sheds light on the parentage of some biomass willows. New Phytol. 141, 155-160.
Percy, D.M., Argus, G.W., Cronk, Q.C., et al., 2014. Understanding the spectacular failure of DNA barcoding in willows (Salix): Does this result from a trans-specific selective sweep? Mol. Ecol. 23, 4737-4756.
Pineiro, C., Abuin, J.M., Pichel, J.C., 2020. Very Fast Tree: speeding up the estimation of phylogenies for large alignments through parallelization and vectorization strategies. Bioinformatics 36, 4658-4659.
Pittermann, J., Stuart, S.A., Dawson, T.E., et al., 2012. Cenozoic climate change shaped the evolutionary ecophysiology of the Cupressaceae conifers. Proc. Natl. Acad. Sci. U.S.A. 109, 9647-9652.
Rabosky, D.L., 2014. Automatic detection of key innovations, rate shifts, and diversity-dependence on phylogenetic trees. PLoS One 9, e89543.
Rechinger, K.H., 1992. Salix taxonomy in Europe - problems, interpretations, observations. Proc. Royal Soc. B-Biol. Sci. 98, 1-12.
Samuels, J.X., Hopkins, S.S.B., 2017. The impacts of Cenozoic climate and habitat changes on small mammal diversity of North America. Global Planet. Change 149, 36-52.
Sanderson, B.J., Gambhir, D., Feng, G., et al., 2023. Phylogenomics reveals patterns of ancient hybridization and differential diversification that contribute to phylogenetic conflict in willows, poplars, and close relatives. Syst. Biol. 72, 1220-1232.
Sayyari, E., Mirarab, S., 2016. Fast coalescent-based computation of local branch support from quartet frequencies. Mol. Biol. Evol. 33, 1654-1668.
Skvortsov, A.K., 1999. Willows of Russia and Adjacent Countries. University of Joensuu, Joensuu, Finland.
Slater, G.S.C., Birney, E., 2005. Automated generation of heuristics for biological sequence comparison. BMC Bioinf. 6, 31.
Sun, H., 2002. Evolution of arctic-Tertiary flora in Himalayan-Henduan mountains. Acta Bot. Yunnanica 24, 671-688.
Thibault, J., 1998. Nuclear DNA amount in pure species and hybrid willows (Salix): a flow cytometric investigation. Can. J. Bot. 76, 157-165.
Tillich, M., Lehwark, P., Pellizzer, T., et al., 2017. GeSeq - versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 45, W6-W11.
Wagner, N.D., Gramlich, S., Horandl, E., 2018. RAD sequencing resolved phylogenetic relationships in European shrub willows (Salix L. subg. Chamaetia and subg. Vetrix) and revealed multiple evolution of dwarf shrubs. Ecol. Evol. 8, 8243-8255.
Wagner, N.D., He, L., Horandl, E., 2020. Phylogenomic relationships and evolution of polyploid Salix species revealed by RAD sequencing data. Front. Plant Sci. 11, 1077.
Wagner, N.D., Volf, M., Horandl, E., 2021. Highly diverse shrub willows (Salix L.) share highly similar plastomes. Front. Plant Sci. 12, 662715.
Wang, J., Yu, Z., Yao, X., et al., 2022. The complete chloroplast genome sequence of Salix kochiana Trautv. and its phylogenetic analysis. Mitochondrial DNA B Resour. 7, 1123-1125.
Wu, J., Nyman, T., Wang, D.-C., et al., 2015. Phylogeny of Salix subgenus Salix s.l. (Salicaceae): delimitation, biogeography, and reticulate evolution. BMC Evol. Biol. 15, 31.
Wu, S., Wang, Y., Wang, Z., et al., 2022. Species divergence with gene flow and hybrid speciation on the Qinghai-Tibet Plateau. New Phytol. 234, 392-404.
Yang, Z.H., 2007. Paml 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, 1586-1591.
Zachos J.C., Dickens G.R., Zeebe R.E., 2008. An early Cenozoic perspective on greenhouse warming and carbon-cycle dynamics. Nature, 451, 279-283.
Zhang, C., Rabiee, M., Sayyari, E., et al., 2018a. ASTRAL-III: polynomial time species tree reconstruction from partially resolved gene trees. BMC Bioinf. 19, 153.
Zhang, L., Xi, Z.X., Wang, M.C., et al., 2018b. Plastome phylogeny and lineage diversification of Salicaceae with focus on poplars and willows. Ecol. Evol. 8, 7817-7823.
Zhang, Z., Xie, P., Guo, Y., et al., 2022. Easy353: a tool to Get Angiosperms353 genes for phylogenomic research. Mol. Biol. Evol. 39, msac261.

[1]	Tian-Rui Wang, Xin Ning, Si-Si Zheng, Yu Li, Zi-Jia Lu, Hong-Hu Meng, Bin-Jie Ge, Gregor Kozlowski, Meng-Xiao Yan, Yi-Gang Song. Genomic insights into ecological adaptation of oaks revealed by phylogenomic analysis of multiple species [J]. Plant Diversity, 2025, 47(01): 53-67.
[2]	Zheng-Yu Zuo, Germinal Rouhan, Shi-Yong Dong, Hong-Mei Liu, Xin-Yu Du, Li-Bing Zhang, Jin-Mei Lu. A revised classification of Dryopteridaceae based on plastome phylogenomics and morphological evidence, with the description of a new genus, Pseudarachniodes [J]. Plant Diversity, 2025, 47(01): 34-52.
[3]	Liansheng Xu, Zhuqiu Song, Tian Li, Zichao Jin, Buyun Zhang, Siyi Du, Shuyuan Liao, Xingjie Zhong, Yousheng Chen. New insights into the phylogeny and infrageneric taxonomy of Saussurea based on hybrid capture phylogenomics (Hyb-Seq) [J]. Plant Diversity, 2025, 47(01): 21-33.
[4]	Yongli Wang, Yan-Da Li, Shuo Wang, Erik Tihelka, Michael S. Engel, Chenyang Cai. Modeling compositional heterogeneity resolves deep phylogeny of flowering plants [J]. Plant Diversity, 2025, 47(01): 13-20.
[5]	Han-Ning Duan, Yin-Zi Jiang, Jun-Bo Yang, Jie Cai, Jian-Li Zhao, Lu Li, Xiang-Qin Yu. Skmer approach improves species discrimination in taxonomically problematic genus Schima (Theaceae) [J]. Plant Diversity, 2024, 46(06): 713-722.
[6]	Xiang-Zhou Hu, Cen Guo, Sheng-Yuan Qin, De-Zhu Li, Zhen-Hua Guo. Deep genome skimming reveals the hybrid origin of Pseudosasa gracilis (Poaceae: Bambusoideae) [J]. Plant Diversity, 2024, 46(03): 344-352.
[7]	Rivontsoa A. Rakotonasolo, Soejatmi Dransfield, Thomas Haevermans, Helene Ralimanana, Maria S. Vorontsova, Meng-Yuan Zhou, De-Zhu Li. New insights into intergeneric relationships of Hickeliinae (Poaceae: Bambusoideae) revealed by complete plastid genomes [J]. Plant Diversity, 2023, 45(02): 125-132.
[8]	Juan Chen, Sijin Zeng, Linya Zeng, Khang Sinh Nguyen, Jiawei Yan, Hua Liu, Nianhe Xia. Parahellenia, a new genus segregated from Hellenia (Costaceae) based on phylogenetic and morphological evidence [J]. Plant Diversity, 2022, 44(04): 389-405.
[9]	Yong Yang, David Kay Ferguson, Bing Liu, Kang-Shan Mao, Lian-Ming Gao, Shou-Zhou Zhang, Tao Wan, Keith Rushforth, Zhi-Xiang Zhang. Recent advances on phylogenomics of gymnosperms and a new classification [J]. Plant Diversity, 2022, 44(04): 340-350.
[10]	Zeng-Qiang Xia, Zuo-Ying Wei, Hui Shen, Jiang-Ping Shu, Ting Wang, Yu-Feng Gu, Amit Jaisi, Yue-Hong Yan. Lycophyte transcriptomes reveal two whole-genome duplications in Lycopodiaceae: Insights into the polyploidization of Phlegmariurus [J]. Plant Diversity, 2022, 44(03): 262-270.
[11]	Gang Yao, Bine Xue, Kun Liu, Yuling Li, Jiuxiang Huang, Junwen Zhai. Phylogenetic estimation and morphological evolution of Alsineae (Caryophyllaceae) shed new insight into the taxonomic status of the genus Pseudocerastium [J]. Plant Diversity, 2021, 43(04): 299-307.
[12]	Yu-Long Yu, Hui-Chun Wang, Zhi-Xiang Yu, Johann Schinnerl, Rong Tang, Yu-Peng Geng, Gao Chen. Genetic diversity and structure of the endemic and endangered species Aristolochia delavayi growing along the Jinsha River [J]. Plant Diversity, 2021, 43(03): 225-233.
[13]	Feng-Wei Lei, Ling Tong, Yi-Xuan Zhu, Xian-Yun Mu, Tie-Yao Tu, Jun Wen. Plastid phylogenomics and biogeography of the medicinal plant lineage Hyoscyameae (Solanaceae) [J]. Plant Diversity, 2021, 43(03): 192-197.
[14]	Luxian Liu, Yonghua Zhang, Pan Li. Development of genomic resources for the genus Celtis (Cannabaceae) based on genome skimming data [J]. Plant Diversity, 2021, 43(01): 43-53.
[15]	Xin-Yu Du, Jin-Mei Lu, De-Zhu Li. Extreme plastid RNA editing may confound phylogenetic reconstruction: A case study of Selaginella (lycophytes) [J]. Plant Diversity, 2020, 42(05): 356-361.