Phylogenomic insights from the complete chloroplast genome of Alnus hirsuta (Betulaceae): Genome structure and lineage relationships within the genus Alnus

Kyu Tae PARK; Jeong-Ki HONG; Yeon-Sik CHOO; KyoungSu CHOI

doi:10.11110/kjpt.2026.56.1.75

Korean J. Pl. Taxon > Volume 56(1); 2026 > Article

PARK, HONG, CHOO, and CHOI: Phylogenomic insights from the complete chloroplast genome of Alnus hirsuta (Betulaceae): Genome structure and lineage relationships within the genus Alnus

Research Article

Korean Journal of Plant Taxonomy 2026; 56(1): 75-84.

Published online: January 31, 2026

DOI: https://doi.org/10.11110/kjpt.2026.56.1.75

Phylogenomic insights from the complete chloroplast genome of Alnus hirsuta (Betulaceae): Genome structure and lineage relationships within the genus Alnus

Kyu Tae PARK^1,²

, Jeong-Ki HONG³

, Yeon-Sik CHOO^1,^*

, KyoungSu CHOI^1,^*

¹Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea

²Bio-resource Research Center, Kyungpook National University, Daegu 41566, Korea

³Diversity Forecast & Evaluation Division, Nakdonggang National Institute of Biological Resources, Sangju 37242, Korea

Corresponding author: Yeon-Sik CHOO, E-mail: yschoo@knu.ac.kr, KyoungSu CHOI, E-mail: choiks010@knu.ac.kr

Editor: Sang-Tae KIM

Received October 21, 2025 Revised December 22, 2025 Accepted January 14, 2026

(open-access):

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The genus Alnus (Betulaceae) comprises approximately 29–35 species that are typically distributed across the temperate Northern Hemisphere, playing key ecological roles through symbiotic nitrogen fixation with Frankia. Although chloroplast genomes have been increasingly utilized to resolve phylogenetic relationships with the genus Alnus, no plastome-level study has focused on A. hirsuta, a widespread Northeast Asian alder of both ecological and economic importance. Here, we aimed to sequence and assemble the complete chloroplast genome of A. hirsuta using Illumina NovaSeq data and conducted comparative plastome analyses using 19 congeners. The plastome of A. hirsuta was 160,879 bp in length and exhibited the typical quadripartite structure, with 112 annotated genes and a GC content of 36.4%. Comparative analyses revealed a highly conserved genomic architecture across the genus Alnus, with only minor lineage-specific variations at the boundary between inverted repeat and small single-copy (SSC) regions. A sliding-window analysis identified divergence hotspots predominantly located in large single-copy/SSC intergenic spacers, particularly cemA–petA, rpl32–trnL, psaC–ndhE, and ycf2, which may serve as informative markers for phylogenetic inference and species delimitation within the genus. Phylogenomic reconstruction based on 76 protein-coding genes strongly supported the monophyly of Alnus, with the subg. Alnobetula forming a distinct lineage. Notably, A. hirsuta was resolved as a sister to A. formosana and closely related to A. fauriei, thereby clarifying previously ambiguous relationships within the subg. Alnus. Our findings provide new genomic resources and phylogenetic insights into the diversification of Alnus, contributing to both evolutionary studies and practical applications such as species identification and conservation.

Keywords: Alnus, Betulaceae, chloroplast genome, cpSSR, divergence hotspot, phylogenetic analysis

INTRODUCTION

The genus Alnus in the family Betulaceae (Fagales) comprises approximately 29–35 species (Gryta et al., 2017). These include tree and shrub species distributed across the northern temperate zone (Benson and Silvester, 1993; Colagar et al., 2016), with the highest species diversity (18–23 species) in Asia (Kennedy et al., 2010; Haq et al., 2021). Because members of the genus Alnus typically occupy soils with limited nitrogen availability, they have evolved a unique ecological strategy to thrive in such environments (Benson and Silvester, 1993). They establish symbiotic associations with the nitrogen-fixing actinomycete Frankia, which induces specialized root nodules that supply nitrogen to the host plant. This symbiotic relationship confers rapid growth and resilience to Alnus species under nutrient-poor conditions, enabling them to colonize and stabilize marginal habitats (Lee and Tsai, 2018; Yuan et al., 2023). Although the genus Alnus is ecologically unified by its Frankia symbiosis, its taxonomic history and phylogenetic relationships remain complex and merit clarification.

The evolutionary relationships within the birch family (Betulaceae) have been clarified through integration of morphological evidence, molecular datasets, and the fossil record (Bousquet et al., 1992; Wang et al., 2013). Natural hybridization with subsequent introgression is common in this family, and polyploidy has been reported in several lineages (Atkinson, 1992). While these processes are important drivers of diversification in the Betuloideae, they also obscure phylogenetic signal and complicate taxonomic circumscription, particularly in the genera Betula and Alnus (Järvinen et al., 2004; Thomson et al., 2015; Gryta et al., 2017). Historically, the genus Alnus was treated as part of Betula but was subsequently recognized as a distinct genus, a separation later reinforced by morphological and molecular studies (Bousquet et al., 1992; Savard et al., 1993). Furlow’s (1979) monograph further stabilized this framework by renaming Alnaster as subg. Alnobetula and Gymnothyrsus as subg. Alnus, and by elevating Clethropsis to subgeneric rank based on a suite of diagnostic traits.

Within the genus Alnus, recent molecular phylogenies consistently support a three-subgenus framework, with a core clade comprising subg. Alnus and subg. Clethropsis sister to subg. Alnobetula (Navarro et al., 2003; Chen and Li, 2004; Ren et al., 2010; Rochet et al., 2011). Subg. Alnus is characterized by stalked shoot buds and flowering from late winter to spring, and it includes widely distributed species such as A. glutinosa. This historical and phylogenetic context explains why species limits and deeper relationships in Alnus remain challenging. Hybridization and polyploidization blur boundaries, while morphological convergence across subgenera can mask evolutionary signal.

Chloroplasts, the organelles responsible for photosynthesis in green plants, possess genomes that are most often uniparentally inherited. In angiosperms, chloroplast genomes generally span ~120–160 kb and display a conserved quadripartite architecture: two inverted repeats (Ira and IRb) flanking the large single-copy (LSC) and small single-copy (SSC) regions (Chumley et al., 2006; Wicke et al., 2011). The advent of next-generation sequencing (NGS) has enabled plastome-scale studies in many lineages, allowing reconstruction of structural variation, well-supported phylogenies, divergence-time estimation, and identification of gene losses (Blazier et al., 2016; Daniell et al., 2016; Mohanta et al., 2020; Yu et al., 2022; Zhang et al., 2022; Liu et al., 2023). Within the genus Alnus, chloroplast (plastome) genome studies have become increasingly common, documenting genome structure and phylogenetic relationships (Lee et al., 2019; Zhang et al., 2021; Yang et al., 2022). However, plastome studies have not been conducted for A. hirsuta.

Alnus hirsuta Turcz. is a Northeast Asian alder distributed across Korea, China, Japan, and Mongolia. It is morphologically distinguished from the closely related A. japonica by its generally broader ovate to suborbicular leaves (Huh and Huh, 1999). Alnus hirsuta has attracted pharmacological interest because diarylheptanoids isolated from this species exhibit antioxidative and anti-inflammatory activities, underscoring its value as a useful plant resource (Hu and Wang, 2011). Owing to its rapid growth, stress tolerance, and soil-stabilizing root system, A. hirsuta is used for roadside shelterbelts and riparian buffers and is also planted as a street tree in many regions. These attributes collectively highlight the species’ ecological and applied significance and justify focused comparative work on its genome and evolution.

In this study, we aimed to assemble and annotate the complete chloroplast genome of A. hirsuta using NGS. We also aimed to comprehensively analyze chloroplast-genome features—including genome size, GC content, gene content, and organization—together with codon usage and repeat sequences, based on the annotated plastome. Furthermore, we aimed to clarify the phylogenetic placement of A. hirsuta and its relationships with congeners by constructing a phylogenetic tree from concatenated protein-coding genes sampled across Alnus and representative Betulaceae. This provides a theoretical basis for understanding Alnus evolution and for species identification.

MATERIALS AND METHODS

Plant sampling, DNA extraction, and sequencing

Fresh leaf tissue of A. hirsuta was collected from Samhyeri, Yean-myeon, Andong-si (36°43’45.3”N, 128°54’10.6”E) and deposited in the herbarium of Kyungpook National University (KNU) (specimen number: KNU_NI_001). The sample was identified by KyoungSu Choi (Department of Biology, Kyungpook National University). Total genomic DNA was extracted from silica-dried leaves using the DNeasy Plant Mini Kits (Qiagen Corporation, Valencia, CA, USA). Sequencing was performed on the Illumina NovaSeq 6000 platform (Illumina, Inc., San Diego, CA, USA) with paired-end 150-bp reads and an average insert size of 500 bp.

Plastome assembly and annotation

The complete chloroplast genome of A. hirsuta was assembled with GetOrganelle v.1.7.7.1 (Jin et al., 2020). Gene annotation was performed with GeSeq, and tRNA loci were identified with tRNAscan-SE v2.0.9 (Chan et al., 2021). The circular genome map was generated with OGDRAW v1.3.1 (Greiner et al., 2019).

Comparative genomics, divergence hotspot, and repeat analysis

We compiled plastomes from 19 Alnus species for comparative analyses of structural variation. IRscope (Amiryousefi et al., 2018) was used to examine inverted-repeat (IR) lengths and junction positions (LSC–IRb, IRb– SSC, SSC–IRa, IRa–LSC) across all plastomes. Whole plastomes were aligned with MAFFT (Katoh, 2002), and nucleotide diversity (Pi) was estimated in DnaSP v6.9 (Rozas et al., 2017) with a 600-bp sliding window and a 200-bp step. Simple sequence repeats (SSRs) were identified with MISA (Beier et al., 2017), using minimum repeat thresholds of 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs, respectively.

Phylogenetic analysis

Phylogenetic inference was based on 76 chloroplast protein-coding genes from the complete chloroplast genomes of 19 Alnus species, with Casuarina glauca as the outgroup. The sequences were aligned with MAFFT v7.4.0.9 (Katoh, 2002) and concatenated into a single data set. A maximum-likelihood (ML) tree was constructed in Geneious Prime (https://www.geneious.com) using RAxML v8.2.11 (Stamatakis, 2014) under the GTRGAMMA model with 1,000 bootstrap replicates.

RESULTS AND DISCUSSION

Plastome feature of A. hirsuta

The nucleotide sequence of A. hirsuta has been deposited in GenBank. The chloroplast genome of A. hirsuta was 160,879 bp in length (Fig. 1) and displayed the typical quadripartite structure, comprising a pair of IRs (26,052 bp), LSC (89,571 bp), and SSC (19,204 bp). Alnus hirsuta exhibited 112 annotated genes, including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. The GC content of the plastome of A. hirsuta was 36.4%.

To provide broader phylogenomic context, we incorporated plastomes from 19 Alnus species from the National Center for Biotechnology Information into our comparative analyses (Table 1). Among these 19 species, plastome lengths ranged from 160,013 bp (A. firma) to 161,117 bp (A. lanata). The plastomes showed no variation in gene number and shared identical GC content. In most angiosperms, chloroplast genomes are typically maternally inherited and undergo limited recombination, which contributes to their highly conserved structures among closely related species (Daniell et al., 2016). Previous studies on Betulaceae (Yang et al., 2022) have demonstrated that chloroplast genomes are highly conserved, and our analyses further confirmed this high level of conservation within the genus Alnus.

IR boundary analysis

Although plastomes remain strongly conserved in overall architecture, shifts at IR boundaries are widely reported. Among closely related species, such boundary movements are typically subtle and involve only the gain or loss of a few genes (Goulding et al., 1996; Yoo et al., 2021; Amenu et al., 2022; Choi et al., 2023; Li et al., 2025; Yan et al., 2025).

We found that the IR boundary analysis of the 19 Alnus plastomes revealed a highly conserved overall structure, with only minor lineage-specific variations (Fig. 2). The LSC/IRb junction was consistently located within the rps19 gene, with approximately 20–37 bp extending into the IRb region. At the IRa/LSC boundary, trnH was adjacent to the junction, whereas rps19 was partially duplicated in IRa.

At the IRb/SSC and SSC/IRa junctions, two distinct boundary configurations were identified, primarily governed by the length and positioning of ndhF and ycf1. In the predominant configuration (Type I), the IRb/SSC boundary lied within ndhF, producing a short ndhF fragment (from single-digit to a few hundred bp) that was duplicated into IRb and overlapped ycf1. The S SC/IRa boundary was typically situated within ycf1, which extended several kilobases into IRa. In the alternative configuration (Type II), the ndhF extension into IRb was markedly reduced or absent, resulting in a slightly different junction architecture. Consistent with this genus-wide pattern, A. hirsuta conformed to Type I—ndhF crossed into IRb and ycf1 extended into IRa—while retaining the conserved LSC/IR boundaries (i.e., rps19 within IRb and trnH adjacent to the IRa/LSC junction). Despite these minor lineage-specific differences, the plastomes of the genus Alnus exhibited an overall stable and conserved IR–SC boundary framework, consistent with previous studies reporting minimal divergence of IR–SC junctions within Betulaceae (Yang et al., 2022).

SSRs analysis

SSRs are tandemly repeated DNA motifs of 1–10 bp per repeat unit, dispersed throughout the genome. Because of their high allelic variability, SSRs are informative molecular markers widely used for genotyping and population-genetic analyses (Fu et al., 2016; Ahmad et al., 2018). We identified 100 SSRs in A. hirsuta, comprising 52 mononucleotides, 21 dinucleotides, 8 trinucleotides, 16 tetranucleotides, 2 pentanucleotides, and 1 hexanucleotide (Fig. 3).

The total number of chloroplast SSRs across the 19 Alnus plastomes ranged from 89 in A. firma to 112 in A. nitida. Mononucleotide repeats were the most abundant, with the highest count in A. ferdinandi-coburgii (58). Dinucleotide repeats reached a maximum of 24 in A. incana. Trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats were comparatively rare, with maximum counts of 12 (A. cremastogyne), 18 (A. alnobetula), 5 (A. nitida, A. rubra, A. ferdinandi-coburgii, A. lanata, and A. cremastogyne), and 3 (A. incana), respectively.

Comparative divergence hotspots

Plastome-wide alignments across the family Betulaceae typically show high similarity, with variability concentrated in the single-copy regions. In the present study, the sliding-window analysis (Fig. 4) using the Alnus dataset also revealed higher nucleotide diversity in the LSC and SSC than in the IRs, and non-coding intervals were more variable than protein-coding regions. Peaks of diversity within the genus Alnus were centered on intergenic spacers adjacent to cemA–petA, ycf2, rpl32–trnL, and psaC–ndhE. These patterns agreed with the broader Betulaceae tendency for divergence hotspots to cluster in non-coding LSC/SSC spacers, though some locus-level differences were evident. Previous family-wide analyses (Yang et al., 2022) highlighted five coding genes with elevated nucleotide polymorphisms (Pi) (psaI, ycf1, rpl22, psaJ, and cemA) and identified rpl22 as a promising barcode owing to its moderate variability and suitable length. They also recovered highly variable non-coding intervals, including trnT_GGT–psbD, trnE_TTC–trnT_GGT, ndhC–trnV_UAC, trnH–psbA, and ycf4–cemA. In contrast, our findings identified cemA–petA and psaC–ndhE as prominent lineage-specific peaks, whereas some previously noted hotspots, including trnT–psbD, were less pronounced. Together, these results indicate that the regional architecture of variability is conserved across Betulaceae, but the exact positions of maxima vary among lineages. Hence, it is important to tailor chloroplast markers to the focal clade. Within the genus Alnus, spacers flanking cemA, psaC/ndhE, and rpl32–trnL, along with ycf2, appear especially informative for phylogenetic inference and species delimitation.

Phylogenetic analysis of Alnus

Previous molecular phylogenetic studies (Navarro et al., 2003; Chen and Li, 2004; Colagar et al., 2016; Gryta et al., 2017) have consistently indicated the monophyly of the genus Alnus, but the taxonomic relationships within subgenus Alnus remained poorly resolved. In nrITS-based phylogenetic analyses (Navarro et al., 2003; Colagar et al., 2016), A. hirsuta exhibited a highly ambiguous taxonomic placement. Although analyses based on the chloroplast genome (Meucci et al., 2021; Zhang et al., 2021; Yang et al., 2022) and nuclear genes have been conducted for the genus Alnus, these analyses and datasets did not include A. hirsuta. The inferred molecular phylogeny shows that all species of the genus form a monophyletic group (Fig. 5). Subgenus Alnobetula (A. firma and A. alnobetula subsp. alnobetula) separates from other Alnus species. However, one species of the subgenus Clethropsis (A. maritima) and two species of subgenus Alnus (A. rubra and A. acuminata) were intermingled (100% bootstrap values). We found that A. hirsuta is closely related to A. fauriei with a 100% bootstrap value and forms a sister group to A. formosana. In conclusion, our findings elucidate the relationships among the close relatives of A. hirsuta, which had previously remained unclear.

NOTES

ACKNOWLEDGMENTS

This research was funded by the Korea Environment Industry & Technology Institute (KEITI) under the Ministry of Environment of the Republic of Korea (Grant number: 2021003420003), which contributes to multi-ministerial national biological research resources. The study was also supported by a grant provided by the Nakdonggang National Institute of Biological Resources (NNIBR), which is funded by the Ministry of Environment (MOE) of the Republic of Korea (NNIBR202001107, NNIBR20252103).

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

Fig. 1

The chloroplast genome of Alnus hirsuta. Genes shown on the inside and outside of the circle are transcribed in clockwise and counterclockwise directions, respectively. The darker gray area in the inner circle represents GC content.

Fig. 2

Comparison of the borders LSC, SSC and IR regions among genus Alnus species. IR, inverted repeats; LSC, large single-copy region; SSC, small single-copy region

Fig. 3

Analysis of repeat sequences in 19 Alnus species.

Fig. 4

Comparison of the nucleotide diversity (Pi) values among 19 Alnus species.

Fig. 5

The maximum likelihood tree constructed using 76 protein-coding genes from 19 Alnus species. The stability of each tree node was tested by bootstrap analysis with 1,000.

Table 1

Comparison of the chloroplast genome features of Alnus species.

Species	Accession no.	Total length (bp)	LSC (bp)	IR (bp)	SSC (bp)	PCGs	tRNA	rRNA	GC contents (%)
Alnus hirsuta	PX512379	160,879	89,571	26,052	19,204	78	30	4	36.4
A. japonica	MF136507	160,788	89,426	26,052	19,258	78	30	4	36.4
A. alnobertula subsp. alnobetula	MF136498	160,473	89,256	26,020	19,177	78	30	4	36.4
A. cordata	MF136500	160,740	89,427	26,031	19,251	78	30	4	36.4
A. nitida	MF136513	160,871	89,530	26,173	18,995	78	30	4	36.4
A. orientalis	MF136514	160,470	89,099	26,092	19,187	78	30	4	36.4
A. rubra	MF136515	160,754	89,517	26,033	19,171	78	30	4	36.4
A. subcordata	MF136516	160,702	89,384	26,054	19,210	78	30	4	36.4
A. incana	MG386364	161,055	89,571	26,108	19,268	78	30	4	36.4
A. nepalensis	MG386365	160,735	89,316	26,189	19,041	78	30	4	36.4
A. cremastogyne	MH628453	160,538	89,034	26,139	19,226	78	30	4	36.4
A. formosana	MW865380	161,029	89,720	26,052	19,205	78	30	4	36.4
A. firma	OL891506	160,013	88,773	26,036	19,168	78	30	4	36.5
A. acuminata	OL891508	160,666	89,408	26,033	19,192	78	30	4	36.4
A. trabeculosa	OL891513	160,744	89,590	25,921	19,312	78	30	4	36.4
A. ferdinandi-coburgii	OL891514	161,108	89,733	26,149	19,077	78	30	4	36.4
A. lanata	OL891517	161,117	89,742	26,149	19,077	78	30	4	36.4
A. fauriei	OL891518	160,934	89,585	26,052	19,245	78	30	4	36.4
A. maritima	OL891520	160,577	89,472	26,032	19,041	78	30	4	36.4

LSC, large single-copy region; IR, inverted repeats; SSC, small single-copy region; PCG, protein-coding genes.

LITERATURE CITED

Ahmad, A., Wang, J.-D. Pan, Y.-B. Sharif, R. and Gao, S.-J. 2018. Development and use of simple sequence repeats (SSRs) markers for sugarcane breeding and genetic studies. Agronomy 8: 260.

Amenu, S. G., Wei, N. Wu, L. Oyebanji, O. Hu, G. Zhou, Y. and Wang, Q. 2022. Phylogenomic and comparative analyses of Coffeeae alliance (Rubiaceae): Deep insights into phylogenetic relationships and plastome evolution. BMC Plant Biology 22: 88.

Amiryousefi, A., Hyvönen, J. and Poczai, P. 2018. IRscope: An online program to visualize the junction sites of chloroplast genomes. Bioinformatics 34: 3030-3031.

Atkinson, M. D. 1992. Betula pendula Roth (B. verrucosa Ehrh.) and B. pubescens Ehrh. Journal of Ecology 80: 837-870.

Beier, S., Thiel, T. Münch, T. Scholz, U. and Mascher, M. 2017. MISA-web: A web server for microsatellite prediction. Bioinformatics 33: 2583-2585.

Benson, D. R. and Silvester, W. B. 1993. Biology of Frankia strains, actinomycete symbionts of actinorhizal plants. Microbiological Reviews 57: 293-319.

Blazier, J. C., Jansen, R. K. Mower, J. P. Govindu, M. Zhang, J. Weng, M.-L. and Ruhlman, T. A. 2016. Variable presence of the inverted repeat and plastome stability in Erodium. Annals of Botany 117: 1209-1220.

Bousquet, J., Strauss, S. H. Doerksen, A. H. and Price, R. A. 1992. Extensive variation in evolutionary rate of rbcL gene sequences among seed plants. Proceedings of the National Academy of Sciences of the United States of America 89: 7844-7848.

Chan, P. P., Lin, B. Y. Mak, A. J. and Lowe, T. M. 2021. tRNA-scan-SE 2: Improved detection and functional classification of transfer RNA genes. Nucleic Acids Research 49: 9077-9096.

Chen, Z. and Li, J. 2004. Phylogenetics and biogeography of Alnus (Betulaceae) inferred from sequences of nuclear ribosomal DNA ITS region. International Journal of Plant Sciences 165: 325-335.

Choi, K., Hwang, Y. Hong, J.-K. and Kang, J.-S. 2023. Comparative plastid genome and phylogenomic analyses of Potamogeton species. Genes 14: 1914.

Chumley, T. W., Palmer, J. D. Mower, J. P. Fourcade, H. M. Calie, P. J. Boore, J. L. and Jansen, R. K. 2006. The complete chloroplast genome sequence of Pelargonium x hortorum: Organization and evolution of the largest and most highly rearranged chloroplast genome of land plants. Molecular Biology and Evolution 23: 2175-2190.

Colagar, A. H., Yousefzadeh, H. Shayanmehr, F. Jalali, S. G. Zare, H. and Tippery, N. P. 2016. Molecular taxonomy of Hyrcanian Alnus using nuclear ribosomal ITS and chloroplast trnHpsbA DNA barcode markers. Systematics and Biodiversity 14: 88-101.

Daniell, H., Lin, C.-S. Yu, M. and Chang, W.-J. 2016. Chloroplast genomes: Diversity, evolution, and applications in genetic engineering. Genome Biology 17: 134.

Fu, P.-C., Zhang, Y.-Z. Ya, H.-Y. and Gao, Q.-B. 2016. Characterization of SSR genomic abundance and identification of SSR markers for population genetics in Chinese jujube (Ziziphus jujuba Mill.). PeerJ 4: e1735.

Furlow, J. J. 1979. The systematics of the American species of Alnus (Betulaceae). Rhodora 81: 1-121.

Goulding, S. E., Olmstead, R. G. Morden, C. W. and Wolfe, K. H. 1996. Ebb and flow of the chloroplast inverted repeat. Molecular and General Genetics 252: 195-206.

Greiner, S., Lehwark, P. and Bock, R. 2019. OrganellarGenome-DRAW (OGDRAW) version 1.1: Expanded toolkit for the graphical visualization of organellar genomes. Nucleic Acids Research 47: W59-W64.

Gryta, H., Van De Paer, C. Manzi, S. Holota, H. Roy, M. and Besnard, G. 2017. Genome skimming and plastid microsatellite profiling of alder trees (Alnus spp., Betulaceae): Phylogenetic and phylogeographical prospects. Tree Genetics and Genomes 13: 118.

Haq, Z., Khan, S. M. Shah, S. A. and Abdullah, 2021. Ecosystem services of Himalayan Alder. Ecological Intensification of Natural Resources for Sustainable Agriculture. Jhariya, M. K., Meena, R. S. and Banerjee, A. (eds.), Springer, Singapore. Pp. 429-459.

Hu, W. and Wang, M.-H. 2011. Antioxidative activity and anti-inflammatory effects of diarylheptanoids isolated from Alnus hirsuta. Journal of Wood Science 57: 323-330.

Huh, M. K. and Huh, H. W. 1999. Genetic diversity and population structure of Alnus hirsuta (Betulaceae) in Korea. Journal of Plant Research 112: 437-442.

Järvinen, P., Palmé, A. Orlando Morales, L. Lännenpää, M. Keinänen, M. Sopanen, T. and Lascoux, M. 2004. Phylogenetic relationships of Betula species (Betulaceae) based on nuclear ADH and chloroplast matK sequences. American Journal of Botany 91: 1834-1845.

Jin, J.-J., Yu, W.-B. Yang, J.-B. Song, Y. dePamphilis, C. W. Yi, T.-S. and Li, D.-Z. 2020. GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biology 21: 241.

Katoh, K., Misawa, K. Kuma, K.-I. and Miyata, T. 2002. MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: 3059-3066.

Kennedy, P. G., Schouboe, J. L. Rogers, R. H. Weber, M. G. and Nadkarni, N. M. 2010. Frankia and Alnus rubra canopy roots: An assessment of genetic diversity, propagule availability, and effects on soil nitrogen. Microbial Ecology 59: 214-220.

Lee, J.-T. and Tsai, S.-M. 2018. The nitrogen-fixing Frankia significantly increases growth, uprooting resistance and root tensile strength of Alnus formosana. African Journal of Biotechnology 17: 213-225.

Lee, S.-I., Nkongolo, K. Park, D. Choi, I.-Y. Choi, A.-Y. and Kim, N.-S. 2019. Characterization of chloroplast genomes of Alnus rubra and Betula cordifolia, and their use in phylogenetic analyses in Betulaceae. Genes and Genomics 41: 305-316.

Li, M., Geng, M. Zhou, Y. Wu, B. Sun, Y. Hu, S. and Li, P. 2025. Integrative chloroplast genomics unravels phylogenetic discordance in Glehnia (Apiaceae). Industrial Crops and Products 236: 121978.

Liu, T.-J., Zhang, S.-Y. Wei, L. Lin, W. Yan, H.-F. Hao, G. and Ge, X.-J. 2023. Plastome evolution and phylogenomic insights into the evolution of Lysimachia (Primulaceae: Myrsinoideae). BMC Plant Biology 23: 359.

Meucci, S., Schulte, L. Zimmermann, H. H. Stoof-Leichsenring, K. R. Epp, L. Bronken Eidesen, P. and Herzschuh, U. 2021. Holocene chloroplast genetic variation of shrubs (Alnus alnobetula, Betula nana, Salix sp.) at the Siberian tundra-taiga eco-tone inferred from modern chloroplast genome assembly and sedimentary ancient DNA analyses. Ecology and Evolution 11: 2173-2193.

Mohanta, T. K., Mishra, A. K. Khan, A. Hashem, A. Abd Allah, E. F. and Al-Harrasi, A. 2020. Gene loss and evolution of the plastome. Genes 11: 1133.

Navarro, E., Bousquet, J. Moiroud, A. Munive, A. Piou, D. and Normand, P. 2003. Molecular phylogeny of Alnus (Betulaceae), inferred from nuclear ribosomal DNA ITS sequences. Plant and Soil 254: 207-217.

Ren, B.-Q., Xiang, X.-G. and Chen, Z.-D. 2010. Species identification of Alnus (Betulaceae) using nrDNA and cpDNA genetic markers. Molecular Ecology Resources 10: 594-605.

Rochet, J., Moreau, P.-A. Manzi, S. and Gardes, M. 2011. Comparative phylogenies and host specialization in the alder ecto-mycorrhizal fungi Alnicola, Alpova and Lactarius (Basidiomycota) in Europe. BMC Evolutionary Biology 11: 40.

Rozas, J., Ferrer-Mata, A. Sánchez-DelBarrio, J. C. Guirao-Rico, S. Librado, P. Ramos-Onsins, S. E. and Sánchez-Gracia, A. 2017. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Molecular Biology and Evolution 34: 3299-3302.

Savard, L., Michaud, M. and Bousquet, J. 1993. Genetic diversity and phylogenetic relationships between birches and alders using ITS, 18S rRNA, and rbcL gene sequences. Molecular Phylogenetics and Evolution 2: 112-118.

Stamatakis, A. 2014. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313.

Thomson, A. M., Dick, C. W. and Dayanandan, S. 2015. A similar phylogeographical structure among sympatric North American birches (Betula) is better explained by introgression than by shared biogeographical history. Journal of Biogeography 42: 339-350.

Wang, N., Thomson, M. Bodles, W. J. A. Crawford, R. M. M. Hunt, H. V. Featherstone, A. W. Pellicer, J. and Buggs, R. J. A. 2013. Genome sequence of dwarf birch (Betula nana) and cross-species RAD markers. Molecular Ecology 22: 3098-3111.

Wicke, S., Schneeweiss, G. M. dePamphilis, C. W. Müller, K. F. and Quandt, D. 2011. The evolution of the plastid chromosome in land plants: Gene content, gene order, gene function. Plant Molecular Biology 76: 273-297.

Yan, K., Li, W. Sun, C. Lu, X. Zhou, X. Wang, Y. and Tian, Y. 2025. Complete chloroplast genome analysis of Casearia kurzii: Gene loss at the IR boundary and monophyletic evolution within Casearia. Plants 14: 1356.

Yang, Z., Ma, W. Yang, X. Wang, L. Zhao, T. Liang, L. Wang, G. and Ma, Q. 2022. Plastome phylogenomics provide new perspective into the phylogeny and evolution of Betulaceae (Fagales). BMC Plant Biology 22: 611.

Yoo, S.-C., Oh, S.-H. and Park, J. 2021. Phylogenetic position of Daphne genkwa (Thymelaeaceae) inferred from complete chloroplast data. Korean Journal of Plant Taxonomy 51: 171-175.

Yu, J., Fu, J. Fang, Y. Xiang, J. and Dong, H. 2022. Complete chloroplast genomes of Rubus species (Rosaceae) and comparative analysis within the genus. BMC Genomics 23: 32.

Yuan, Y., Chen, Z. Huang, X. Wang, F. Guo, H. Huang, Z. and Yang, H. 2023. Comparative analysis of nitrogen content and its influence on actinorhizal nodule and rhizospheric microorganism diversity in three Alnus species. Frontiers in Microbiology 14: 1230170.

Zhang, H., Zhang, X. Sun, Y. Landis, J. B. Li, L. Hu, G. Sun, J. Tiamiyu, B. B. Kuang, T. Deng, T. Sun, H. and Wang, H. 2022. Plastome phylogenomics and biogeography of the subfam Polygonoideae (Polygonaceae). Frontiers in Plant Science 13: 893201.

Zhang, X., Li, M. Wei, Q. Xiao, Y. Qin, Y. Zhong, L. and Qin, Z. 2021. Characterization of the complete chloroplast genome of the Taiwan alder Alnus formosana (Betulaceae) based on next-generation sequencing technology. Mitochondrial DNA Part B Resources 6: 2841-2842.