Evaluating the utility of deep genome skimming for phylogenomic analyses: A case study in the species-rich genus Rhododendron

doi:10.1016/j.pld.2025.04.006

Plant Diversity ›› 2025, Vol. 47 ›› Issue (04): 593-603.DOI: 10.1016/j.pld.2025.04.006

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Evaluating the utility of deep genome skimming for phylogenomic analyses: A case study in the species-rich genus Rhododendron

Zhi-Qiong Mo (莫智琼)^a,b,c, Chao-Nan Fu (付超男)^b,d, Alex D. Twyford^e,f, Pete M. Hollingsworth^f, Ting Zhang (张挺)^b, Jun-Bo Yang (杨俊波)^b, De-Zhu Li (李德铢)^b,d,g, Lian-Ming Gao (高连明)^a,d,g

a. State Key Laboratory of Plant Diversity and Specialty Crops, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China;
b. Germplasm Bank of Wild Species & Yunnan Key Laboratory of Crop Wild Relatives Omits, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China;
c. University of Chinese Academy of Sciences, Beijing 100049, China;
d. Center for Interdisciplinary Biodiversity Research & College of Forestry, Shandong Agricultural University, Tai'an 271018, Shandong, China;
e. Institute of Ecology and Evolution, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FL, Scotland, UK;
f. Royal Botanic Garden Edinburgh, Edinburgh EH3 5LR, Scotland, UK;
g. Lijiang Forest Biodiversity National Observation and Research Station, Kunming Institute of Botany, Chinese Academy of Sciences, Lijiang 674100, Yunnan, China

Received:2025-02-25 Revised:2025-04-17 Online:2025-08-13 Published:2025-08-13
Contact: Lian-Ming Gao (高连明),E-mail:gaolm@mail.kib.ac.cn
Supported by:
This study was supported by the National Key Research and Development Program of China (2023YFA0915800), the Science and Technology Basic Resources Investigation Program (2021FY100200), the Key Basic Research program of Yunnan Province, China (202101BC070003), the Large-scale Scientific Facilities of the Chinese Academy of Sciences (2017-LSFGBOWS-02), and the Chinese Academy of Sciences President's International Fellowship Initiative (2025PD0021). We are grateful to curator of KUN to access the herbarium specimens, and Jiayun Zou, Zhirong Zhang, Jing Yang, Hongtao Li and Chunxia Zeng, for their help with sample collection, laboratory work and data analysis. Molecular experiments and data analysis were performed at the Laboratory of Molecular Biology and iFlora High-Performance Computing Center of Germplasm Bank of Wild Species, Kunming Institute of Botany, CAS.

Evaluating the utility of deep genome skimming for phylogenomic analyses: A case study in the species-rich genus Rhododendron

a. State Key Laboratory of Plant Diversity and Specialty Crops, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China;
b. Germplasm Bank of Wild Species & Yunnan Key Laboratory of Crop Wild Relatives Omits, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China;
c. University of Chinese Academy of Sciences, Beijing 100049, China;
d. Center for Interdisciplinary Biodiversity Research & College of Forestry, Shandong Agricultural University, Tai'an 271018, Shandong, China;
e. Institute of Ecology and Evolution, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FL, Scotland, UK;
f. Royal Botanic Garden Edinburgh, Edinburgh EH3 5LR, Scotland, UK;
g. Lijiang Forest Biodiversity National Observation and Research Station, Kunming Institute of Botany, Chinese Academy of Sciences, Lijiang 674100, Yunnan, China

通讯作者: Lian-Ming Gao (高连明),E-mail:gaolm@mail.kib.ac.cn
基金资助:
This study was supported by the National Key Research and Development Program of China (2023YFA0915800), the Science and Technology Basic Resources Investigation Program (2021FY100200), the Key Basic Research program of Yunnan Province, China (202101BC070003), the Large-scale Scientific Facilities of the Chinese Academy of Sciences (2017-LSFGBOWS-02), and the Chinese Academy of Sciences President's International Fellowship Initiative (2025PD0021). We are grateful to curator of KUN to access the herbarium specimens, and Jiayun Zou, Zhirong Zhang, Jing Yang, Hongtao Li and Chunxia Zeng, for their help with sample collection, laboratory work and data analysis. Molecular experiments and data analysis were performed at the Laboratory of Molecular Biology and iFlora High-Performance Computing Center of Germplasm Bank of Wild Species, Kunming Institute of Botany, CAS.

Abstract

Abstract: Deep genome skimming (DGS) has emerged as a promising approach to recover orthologous nuclear genes for large-scale phylogenomic analyses. However, its reliability with low DNA quality and quantity typical of archival specimens, such as herbarium material, remains largely unexplored. We used Rhododendron as a case study to evaluate best practices for DGS in phylogenetic analyses at both deep and shallow scales. We first investigated locus recovery variation with sequencing depth, before evaluating the phylogenetic utility of different sets of loci, including Angiosperms353, target nuclear exons, and extended exon-flanking regions. We found DGS effectively recovered nuclear genes from herbarium specimens, with ～15×coverage performing similarly to deeper sequencing. The recovery of target exon and flanking regions was improved by using supercontigs as a reference, offering a potential solution to limited sequencing depth. The high-integrity nuclear sequences recovered robust phylogenetic relationships within Rhododendron. Notably, exon-flanking regions showed significant potential for resolving relationships at shallow scales. Genes recovered with taxon-specific references had less missing data than those recovered by Angiosperms353 and generated higher-resolution phylogenetic trees. This study demonstrates the utility of DGS data for obtaining numerous nuclear genes from herbarium specimens for phylogenetic studies, and makes recommendations for best practices regarding sequencing coverage, locus selection, and bioinformatic approaches.

Key words: Herbarium specimens, Degraded DNA, Deep genome skimming, Target enrichment, Non-targeted exon-flanking regions, Phylogenomics

摘要： Deep genome skimming (DGS) has emerged as a promising approach to recover orthologous nuclear genes for large-scale phylogenomic analyses. However, its reliability with low DNA quality and quantity typical of archival specimens, such as herbarium material, remains largely unexplored. We used Rhododendron as a case study to evaluate best practices for DGS in phylogenetic analyses at both deep and shallow scales. We first investigated locus recovery variation with sequencing depth, before evaluating the phylogenetic utility of different sets of loci, including Angiosperms353, target nuclear exons, and extended exon-flanking regions. We found DGS effectively recovered nuclear genes from herbarium specimens, with ～15×coverage performing similarly to deeper sequencing. The recovery of target exon and flanking regions was improved by using supercontigs as a reference, offering a potential solution to limited sequencing depth. The high-integrity nuclear sequences recovered robust phylogenetic relationships within Rhododendron. Notably, exon-flanking regions showed significant potential for resolving relationships at shallow scales. Genes recovered with taxon-specific references had less missing data than those recovered by Angiosperms353 and generated higher-resolution phylogenetic trees. This study demonstrates the utility of DGS data for obtaining numerous nuclear genes from herbarium specimens for phylogenetic studies, and makes recommendations for best practices regarding sequencing coverage, locus selection, and bioinformatic approaches.

关键词: Herbarium specimens, Degraded DNA, Deep genome skimming, Target enrichment, Non-targeted exon-flanking regions, Phylogenomics

Zhi-Qiong Mo (莫智琼), Chao-Nan Fu (付超男), Alex D. Twyford, Pete M. Hollingsworth, Ting Zhang (张挺), Jun-Bo Yang (杨俊波), De-Zhu Li (李德铢), Lian-Ming Gao (高连明). Evaluating the utility of deep genome skimming for phylogenomic analyses: A case study in the species-rich genus Rhododendron[J]. Plant Diversity, 2025, 47(04): 593-603.

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Evaluating the utility of deep genome skimming for phylogenomic analyses: A case study in the species-rich genus Rhododendron

Evaluating the utility of deep genome skimming for phylogenomic analyses: A case study in the species-rich genus Rhododendron

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