https://orcid.org/0000-0001-8251-6331
Correction
9 May 2024: The PLOS ONE Staff (2024) Correction: Complete chloroplast genomes of Asparagus aethiopicus L., A. densiflorus (Kunth) Jessop ‘Myers’, and A. cochinchinensis (Lour.) Merr.: Comparative and phylogenetic analysis with congenerics. PLOS ONE 19(5): e0303739. https://doi.org/10.1371/journal.pone.0303739 View correction
Figures
Abstract
Asparagus species are widely used for medicinal, horticultural, and culinary purposes. Complete chloroplast DNA (cpDNA) genomes of three Asparagus specimens collected in Hong Kong—A. aethiopicus, A. densiflorus ‘Myers’, and A. cochinchinensis—were de novo assembled using Illumina sequencing. Their sizes ranged from 157,069 to 157,319 bp, with a total guanine–cytosine content of 37.5%. Structurally, a large single copy (84,598–85,350 bp) and a small single copy (18,677–18,685 bp) were separated by a pair of inverted repeats (26,518–26,573 bp). In total, 136 genes were annotated for A. aethiopicus and A. densiflorus ‘Myers’; these included 90 mRNA, 38 tRNA, and 8 rRNA genes. Further, 132 genes, including 87 mRNA, 37 tRNA, and 8 rRNA genes, were annotated for A. cochinchinensis. For comparative and phylogenetic analysis, we included NCBI data for four congenerics, A. setaceus, A. racemosus, A. schoberioides, and A. officinalis. The gene content, order, and genome structure were relatively conserved among the genomes studied. There were similarities in simple sequence repeats in terms of repeat type, sequence complementarity, and cpDNA partition distribution. A. densiflorus ‘Myers’ had distinctive long sequence repeats in terms of their quantity, type, and length-interval frequency. Divergence hotspots, with nucleotide diversity (Pi) ≥ 0.015, were identified in five genomic regions: accD-psaI, ccsA, trnS-trnG, ycf1, and ndhC-trnV. Here, we summarise the historical changes in the generic subdivision of Asparagus. Our phylogenetic analysis, which also elucidates the nomenclatural complexity of A. aethiopicus and A. densiflorus ‘Myers’, further supports their close phylogenetic relationship. The findings are consistent with prior generic subdivisions, except for the placement of A. racemosus, which requires further study. These de novo assembled cpDNA genomes contribute valuable genomic resources and help to elucidate Asparagus taxonomy.
Citation: Wong K-H, Kong BL-H, Siu T-Y, Wu H-Y, But GW-C, Shaw P, et al. (2022) Complete chloroplast genomes of Asparagus aethiopicus L., A. densiflorus (Kunth) Jessop ‘Myers’, and A. cochinchinensis (Lour.) Merr.: Comparative and phylogenetic analysis with congenerics. PLoS ONE 17(4): e0266376. https://doi.org/10.1371/journal.pone.0266376
Editor: Branislav T. Šiler, Institute for Biological Research, University of Belgrade, SERBIA
Received: October 22, 2021; Accepted: March 19, 2022; Published: April 25, 2022
Copyright: © 2022 Wong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The data that support the findings of this study are openly available in GenBank (https://www.ncbi.nlm.nih.gov) with the accession number MZ337394, MZ337395 & MZ424304.
Funding: The research work was supported by a donation fund from Wu Jieh Yee Charitable Foundation Limited. The fund has no formal grant number. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Asparagus, a genus with ca. 300 species [1–6], originated in southern Africa, particularly in the Cape of Good Hope. Some members are now distributed throughout tropical Africa, Eurasia, and Australia [4–11], mostly in arid and sub-arid regions [4–6]. Asparagus species have evolved their characteristic morphology as an adaptation to drought and arid environments [2, 4]. Their “true leaves” have been reduced to scales or spines, with the stem-derived organs (“cladodes”) performing photosynthesis [2, 7, 8, 12, 13]. Cladode shape is variable, ranging from acicular, filiform, linear to cordate [2–4, 6, 11, 14–17]. Most species store nutrients and water in rhizomes or root tubers [2, 15–17].
Asparagus species are commercially important worldwide [2, 7, 9, 10, 15, 18, 19], and many are widely used, particularly in medicinal, culinary, and horticultural applications. Here, we first summarise the anthropocentric uses and environmental impacts of some Asparagus species and then elucidate the complexity on generic subdivisions and nomenclature of the studied Asparagus species.
Medicinal application
Many Asparagus species have medicinal value [19–29]. The root tubers of A. cochinchinensis (Lour.) Merr., ‘Tiandong’ in Traditional Chinese Medicine, are renowned for their therapeutical functions in nourishing yin, moistening dryness, clearing the heat and engendering fluid [30, 31]. A. officinalis L. [20–22, 24–27], A. setaceus (Kunth) Jessop [20], A. filicinus Buch.-Ham. ex D. Don [24, 28], A. racemosus Willd. [19, 21, 25, 27, 29], and A. schoberioides Kunth [29] have been used as herbal drugs in different regions for various functions. Root tubers of A. filicinus are used as adulterants of Stemonae Radix to cure tracheitis, pneumonia, coughing, and whooping cough [32–36]. In South African, several Asparagus species have been used to treat pulmonary tuberculosis, gonorrhoea, and infertility, while some Asparagus species have been used as charm to increase fertility, ensure victory, or fight against witchcraft [21].
Culinary application
Asparagus species are an important culinary resource. Although young shoots of A. officinalis L., garden asparagus, are widely sold as a vegetable [1–4, 10, 27, 37], its gene pool is relatively limited [38–40]. It is susceptible to multiple biotic and abiotic stresses, including Fusarium rot [41, 42], Puccinia asparagi rust [43, 44], purple spot caused by Stemphylium [45–47], and stem blight caused by Phomopsis asparagi [48], negatively affecting its production and economic value. Attempts to cross A. officinalis with its wild relatives, to enhance tolerance to drought, disease, salinity, and acidity [49], have revealed that dioecious, but not monoecious, species could hybridize with it [44, 46, 50–54].
Young shoots of A. acutifolius L., A. aphyllus L., and A. albus L. are also eaten as vegetables [55]. The fruits of A. racemosus are edible [56].
Horticultural application
Owing to their distinct morphology, Asparagus species, including A. setaceus, A. aethiopicus L., and A. densiflorus (Kunth) Jessop ‘Myers’, have been widely used as ornamental plants [1, 3, 57]. The European Garden Flora [57], published in 1986, mentions 24 Asparagus species, including A. setaceus, A. aethiopicus, A. officinalis, A. densiflorus, A. filicinus, A. asparagoides (L.) Druce, A. falcatus L., and A. racemosus. The New Royal Horticultural Society Dictionary of Gardening [3], published in 1992, reports the same number of species.
The xeromorphic adaptations of Asparagus species are beneficial to the establishment of “Xeroscaping” [58–60], a kind of landscaping that minimises the need for irrigation. The Pictorial Guide to Plant Resources for Skyrise Greenery in Hong Kong (Developmental Bureau of the Hong Kong Special Administrative Region Government) [61–63] recommends three Asparagus species—A. cochinchinensis, A. aethiopicus (recorded as A. densiflorus ‘Sprengeri’), and A. densiflorus ‘Myers’—as skyrise greenery.
Environmental impacts
Global cultivation of Asparagus species has promoted the invasiveness of the species, particularly of the horticultural species. The berries of Asparagus species are a food source for birds, further promoting their seed dispersal [64]. The invasiveness of Asparagus species has been widely recorded in, for instance, Australia [10, 65–67] and the USA [60, 64].
Genus-level taxonomical complexity
Linnaeus first described the genus Asparagus in 1753 [68]. Since the publication of the genus Mysiphyllum by Willdenow in 1808 [14], generic circumscription of the genus Asparagus have been disputed [67, 69, 70]. Based on morphological characters, taxonomists have divided the genus Asparagus sensu lato into three genera: genus Protasparagus [16, 17, 72] (also known as Asparagopsis, an illegitimate homonym [71, 73]); genus Asparagus sensu stricto [16, 17, 71–73]; and genus Myrsiphyllum [16, 17, 71–73]. The genus Asparagus sensu lato has also been divided into three subgenera (subgenus Asparagopsis, Euasparagus, and Myrsiphyllum) [7], or even multiple sections or races [7–9, 15] (S1 Fig). The key morphological characteristics for generic subdivision include the sexual strategy (monoecy or dioecy), perianth segments (free or connate), filaments (free or connate into column), number of ovules per locule (2 or more), cladode shape and arrangement, and presence or absence of spines.
Later evidences and analysis revealed that these subdivisions were not clear-cut. While Malcomber and Demissew [69] advocated to combine these subdivisions into two subgenera under the genus Asparagus (subgenus Asparagus and subgenus Myrsiphyllum), Fellingham and Meyer [70] suggested eliminating the generic subdivisions. It has been stated that “until the phylogenetic relationships within Asparagus are investigated in more details, the recognition of any infrageneric groups is problematic” [4].
Norup et al. [6] utilised chloroplast and nuclear genome barcode regions (trnH-psbA, trnD-trnT, 3′ ndhF, and PHYC) in their classification: using 211 accessions representing 119 species, they divided the genus Asparagus into six major clades and multiple subclades (S1 Fig).
Species and infraspecific taxonomical complexity
Only one Asparagus species, A. cochinchinensis, has been recorded as native to Hong Kong. Exotic species that are common in Hong Kong include Sprenger’s asparagus (A. aethiopicus), foxtail asparagus (A. densiflorus ‘Myers’), lace fern (A. setaceus), and garden asparagus (A. officinalis). The nomenclature of Sprenger’s asparagus and foxtail asparagus is controversial.
Sprenger’s asparagus.
The nomenclature of this species is unclear. In 1890, Regel published the name Asparagus sprengeri based on cultivated plants growing in Natal, Africa [74, 75]. The epithet sprengeri is after Mr. Sprenger, the co-owner of Dammann & Co., which produced this cultivated plant. The name A. sprengeri Regel was adopted by Baker (1875) [7] and Geiner (1919) [9]. In 1966, Jessop [15] synonymised A. sprengeri Regel under the new combination A. densiflorus (Kunth) Jessop, based on morphology and geographical distribution. Since then, it has been commonly recorded as A. densiflorus, based on Jessop [1, 3, 5, 10, 11, 57, 64, 70]. It has even been considered a cultivar (‘Sprengeri’) [1, 57, 64] or a group (the “Sprengeri group”) [3] of A. densiflorus.
The name A. aethiopicus dates from 1767 (S1 Table), when Linnaeus published it in Species Plantarum [68]. Eighty-three years later, Kunth [71] transferred the species to the genus Asparagopsis. It was later subdivided under the genus Asparagus by Baker (in 1875 and 1896) [7, 8] and Jessop (in 1966) [15]. In 1983, Obermeyer [16] transferred it to a new genus Protasparagus, because Asparagopsis is an illegitimate homonym. Malcomber and Demissew [69] combined the genera Protasparagus and Asparagus into genus Asparagus subgenus Asparagus in 1992. Fellingham and Meyer [70], however, cancelled all generic subdivisions three years later, moving it back to the genus Asparagus.
Aspararagopsis aethiopica (and later Asparagus aethiopicus) and Asparagopsis densiflora were adopted in parallel for 116 years, from 1850 to 1965. In 1996, Jessop [15] classified both species in the genus Asparagus (S1 Table). However, these species are considered highly variable [4, 15]. According to Green (1989) [76], Jessop (1966) [15], Judd (2001) [4], and Straley and Utech (2004) [77], the growth habit of A. aethiopicus is more variable, ranging from arching herbs of ca. 1 m in length to scrambling climbers of ca. 7 m in length. In 1986, Green [76] disagreed with Jessop’s treatment [15] of A. sprengeri as A. densiflorus, which is a small-sized species. Green ascribed Jessop’s treatment to the omission of A. densiflorus from Regel’s protologue in Gartenflora [75] and to misidentification of cultivated materials, which rarely reach their full potential size as potted plants. Following Judd in 2001 [4], Straley and Utech, in Flora of North America North of Mexico (2004) [77], also adopted A. aethiopicus for Sprenger’s asparagus, stating “Asparagus densiflorus (Kunth) Jessop has been misapplied to this species”. They considered Sprenger’s asparagus to be a cultivar, suggesting the combination as A. aethiopicus ‘Sprengeri’. On the contrary, Conran, in Horticultural Flora of South-eastern Australia [78], treated it as “Sprengeri Group” of A. aethiopicus.
The voucher specimens of our research materials were authenticated based on the latest Asparagus monograph, The Genus Asparagus in South Africa [15], and the Flora of Hong Kong [79]. The voucher specimen of Sprenger’s asparagus (K. H. Wong 109), collected in Hong Kong, fit the circumscription of A. aethiopicus L. in the monograph, based on their habitats, growth habit, and reproductive characteristics. Therefore, we have adopted A. aethiopicus L. for Sprenger’s asparagus in this study.
Foxtail asparagus.
This cultivated plant was named for its foxtail-like branches, which are in narrow cones, assembled by orderly branchlets, densely surrounding the main stem, and gradually elongating from the stem apex [1, 3, 57, 64, 80]. Because of its popularity as an ornamental plant of good performance, the cultivar was named A. densiflorus ‘Myersii’ in the Royal Horticultural Society’s Award of Garden Merit list [81].
The first binomial name of foxtail asparagus, Asparagus myersii, was raised anonymously at an unknown time, while Asparagopsis densiflora was validly published in 1850 by Kunth (S1 Table) [71]. The species epithet was named after Mr. Meyers, a nurseryman from East London, for the introduction of this plant [82]. In 1966, Jessop [15] mentioned that Asparagus myersii Hort. “had never been validly published”, treating it as nomen nudum. At that time, he combined Kunth’s Asparagopsis into Asparagus L., deeming this cultivated plant to be a form of A. densiflorus. In 1976, this plant was recorded as A. densiflorus ‘Myers’ by L. H. Bailey Hortorium in Hortus III, [1], treating it as a cultivar of A. densiflorus. Since then, this taxonomic treatment has been widely accepted by many taxonomists, horticulturalists, and scientists [3–5, 11, 27, 57, 64, 83].
The spelling of this cultivar epithet occurs in several forms, including the Latin form ‘Myersii’ [57, 80, 81] derived from the species epithet of its nomen nudum, the non-Latin form ‘Myers’ [1, 4, 5, 11, 51, 64, 76, 83] and ‘Meyers’ [82, 84, 85]. According to Article 21.6 of the International Code of Nomenclature of Cultivated Plants (ICNCP), “the epithet of any name in Latin form published before 1 January 1959, even if it is not validly published under the International Code of Nomenclature for Algae, Fungi and Plants (ICN), that meets the requirements for establishment as a cultivar name under this Code (Art. 27.1), may be used as the cultivar epithet, if the plants to which it was applied are now considered to represent a cultivar” [86]. Because these spellings exhibited no ambiguous indication to the same Asparagus cultivar as foxtail asparagus, we follow the treatment of some taxonomists and scientists [1, 4, 5, 11, 51, 64, 76, 83], adopting A. densiflorus (Kunth) Jessop ‘Myers’ for foxtail asparagus throughout this study.
Provocative molecular evidence: The complete chloroplast genome
Past technical limitations restricted the molecular evidence for classification to short genomic fragments. Technological advancements have made the acquisition of complete genomes, and especially chloroplast genomes, more practicable, affordable, and popular. The chloroplast genome, described as a super-barcode [87–89], is important in studying phylogeny and resolving taxonomical problems [89–92].
Prior to the availability of complete chloroplast DNA (cpDNA) genomes, construction of physical maps of Asparagus cpDNA was attempted via Southern hybridisation of total DNA [93, 94]. Lee et al. [93] estimated the length of the A. officinalis ‘Mary Washington 500W’ cpDNA genome at ca. 155 kb, with two inverted repeats (IRs) of 23 kb each, separated by a 90 kb large single copy (LSC) and a 19 kb small single copy (SSC). The same group constructed the physical maps of cpDNA for another seven Asparagus species, A. schoberioides, A. cochinchinensis, A. plumosus, A. falcatus, A. aethiopicus (recorded as A sprengeri), A. virgatus, and A. asparagoides [94]. Their results suggest close relationships between these eight species. Despite the high similarity among these species, the cpDNA of A. falcatus, A. sprengeri, and A. asparagoides showed gain of the HindIII restriction site and loss of the XhoI restriction sites. Nucleotide deletion in rbcL was detected in A. cochinchinensis cpDNA [94].
The first Apsaragus cpDNA genome (NC_034777.1 = KY364194.1) was reported by Sheng et al. in 2017 [95], who assembled and annotated the cpDNA genome of A. officinalis ‘Atlas’ (length 156,699 bp); this revealed a quadripartite structure, including a pair of IRs (26,531 bp each), separated by an 84,999 bp LSC and 18,638 bp SSC, very similar to those reported by Lee et al. [93]. In 2019, Li et al. [96] reported the cpDNA genome of A. setaceus (NC_047458.1 = MK950153.1) of 156,978 bp, also quadripartite, and with a pair of IRs (26,513 bp each) separated by 85,311 bp LSC and 18,641 bp SSC. The cpDNA genome of A. setaceus is similar to that of A. officinalis ‘Atlas’ in terms of structure, gene order, and GC content.
GenBank (National Center for Biotechnology Information; NCBI) currently contains the cpDNA genomes of eight Asparagus species: A. officinalis (NC_034777.1 = KY364194.1, MT712156.1, LN896355.1, LN896356.1, MT712153.1, MT712155.1, and MT712154.1), A. setaceus (NC_047458.1 = MK950153.1 and MT712152.1), A. cochinchinensis (MW970105.1 and MW447164.1), A. densiflorus (MT740250.1), A. dauricus (MT712151.1), A. schoberioides (NC_035969.1 = KX790361.1), A. racemosus (NC_047472.1 = MN736960.1), and A. filicinus (NC_046783.1 = MK920078.1). This constitutes a small fraction of the genus, leaving a large knowledge gap in the molecular study of Asparagus.
We therefore aimed to revisit the phylogenetic relationships between two nomenclaturally confusing species A. aethiopicus and A. densiflorus ‘Myers’, using complete cpDNA genomes. This information will be useful in crossbreeding programmes, environmental remediation, and authentication of medicinal materials. Using Illumina sequencing, we de novo-assembled the complete chloroplast genomes of A. aethiopicus, A. densiflorus ‘Myers’, and A. cochinchinensis. We performed comparative and phylogenetic analysis, including congenerics, using four cpDNA genomes from GenBank: A. officinalis (NC_034777), A. racemosus (NC_047472), A. schoberioides (NC_035969), and A. setaceus (NC_047458). The intra-generic relationships among these seven species were examined and compared to previous generic subdivision. Our analysis helps to elucidate and resolve the taxonomic positions and nomenclature of A. aethiopicus, A. densiflorus ‘Myers’, and other congenerics.
Materials and methods
Ethics statement
This study was conducted in accordance with Hong Kong Special Administrative Region legislation. Sample collection did not negatively affect the environment in any way.
Plant material and DNA extraction
Individuals of the studied species were collected from the Chinese University of Hong Kong (Table 1 and Fig 1). Fresh and healthy cladodes were stored at −80°C in a freezer immediately after collection. Voucher specimens were deposited at the Shiu-Ying Hu Herbarium (herbarium code: CUHK).
A,B: A. aethiopicus. A. Plant climbing under Ficus microcarpa L. f. and twining with Passiflora suberosa L. B. Flowers and cladodes. C,D: A. cochinchinensis. C. Plant straggling on ground. D. Cladodes. E,F,G: A. densiflorus ‘Myers’. E. Plant growing in a concrete pot. F. Flowers and cladodes. G. Fruits and branch apices.
Total genomic DNA was extracted from 0.2 g of frozen cladode using the DNeasy Plant Pro Kit (Qiagen Co., Hilden, Germany) according to the manufacturer’s instructions. Prior to the sequencing conducted by Novogene Bioinformatic Technology Co. Ltd. (http://en.novogene.com/, Beijing, China), DNA quantity and quality were assessed using a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, MA, USA) and 1% agarose gel electrophoresis, respectively.
cpDNA genome sequencing, assembly, and annotation
A paired-end library with an insert-size of 150 bp was constructed and sequenced on a NovaSeq 6000 platform (Illumina Inc. San Diego, CA, USA). Raw reads were quality-trimmed using CLC Assembly Cell 5.1.1 (CLC Inc., Denmark), with Phred < 33. The trimmed reads were assembled into contigs using the CLC de novo assembler. Gaps were filled using Gapcloser in SOAPdenovo 3.23 to form contigs, then retrieved and ordered using NUCmer 3.0 [97]. The ordered contigs were aligned against reference chloroplast genomes. Based on phylogenetic proximity, A. setaceus (NC_047458) was selected as the reference genome for A. aethiopicus and A. densiflorus ‘Myers’, whereas A. schoberioides (NC_035969) was used for A. cochinchinensis. The aligned contigs were assembled into a complete cpDNA genome for each species.
Gene annotation of cpDNA was performed on the GeSeq platform (https://chlorobox.mpimp-golm.mpg.de/geseq.html) [98] based on the GenBank chloroplast genomes. A. aethiopicus and A. densiflorus ‘Myers’ were annotated in reference to A. setaceus (NC_047458) and A. racemosus (NC_047472), while A. cochinchinensis was annotated in reference to A. schoberioides Kunth (NC_035969) and A. officinalis L. (NC_034777). Manual adjustments, including editing the start and stop positions of genes and introns, were made where necessary. The circular genomic map was visualised by OrganellarGenomeDRAW (OGDRAW, https://chlorobox.mpimp-golm.mpg.de/OGDraw.html) [99]. The assembled and annotated chloroplast genomes of A. aethiopicus, A. densiflorus ‘Myers’, and A. cochinchinensis were submitted to GenBank (accession numbers MZ337394, MZ337395, and MZ424304, respectively).
Repeat-sequence analysis
To compare the three newly assembled cpDNA genomes with chloroplast genomes of other Asparagus species, four cpDNA genomes (NC_034777, NC_047472, NC_035969, and NC_047458) were downloaded from GenBank. Repeat motifs, including simple sequence repeats (SSRs) and long sequence repeats (LSRs), were sequentially identified using the MIcroSAtellite identification tool (MISA, https://webblast.ipk-gatersleben.de/misa/index.php?action=1) [100] and REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer) [101]. We screened for SSRs with at least 10, 5, 4, 3, 3, and 3 repeats, respectively, for mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides. LSRs, including forward, reverse, complement, and palindromic sequences, were detected with a maximum computed repeat size of 50 bp and minimal repeat size of 30 bp.
Comparative genome analysis
For structural comparison of the seven cpDNA genomes, we used mVISTA software (https://genome.lbl.gov/vista/mvista/submit.shtml) [102] to visualise the full alignment with annotation, using the A. aethiopicus cpDNA genome as the reference. The shuffle-LAGAN alignment programme [103] was used.
To compare the size and type of IR border genes, IRScope (https://irscope.shinyapps.io/irapp/) [104] was used to visualise the junction sites of the seven cpDNA genomes. Junction gene positions and sizes were verified, and the diagram was redrawn manually.
To investigate divergence hotspots, the seven studied cpDNA genomes were first aligned using MAFFT 7 (https://mafft.cbrc.jp/alignment/server/) [105]. Sliding window analysis was conducted using DNA Sequence Polymorphism (DnaSP) 6.12.03 [106], which calculates the nucleotide diversity value (Pi) of the aligned cpDNA. The window length and step size were set to 600 and 200 bp, respectively.
Phylogenetic analysis
The complete cpDNA genomes of the seven Asparagus species, with one outgroup species, Hyacinthoides non-scripta (L.) Chouard ex Rothm. (NC_046498), were used to construct maximum likelihood (ML) phylogenetic trees using the MEGA-X software [107], with 1000 bootstrap replicates for each tree. The best-fit model of nucleotide substitution, with the lowest Bayesian Information Criterion (BIC) scores, was calculated via ML model selection in MEGA-X. Respective trees were constructed from the aligned sequences of (i) complete cpDNA genome, (ii) protein coding (CDS) regions (excluding introns), (iii) LSC, (iv) SSC, and (v) IRs.
Results
Asparagus cpDNA genomes features
Illumina NovaSeq 6000 sequencing generated 3.2 Gb, 3.1 Gb, and 2.8 Gb raw data for A. aethiopicus, A. densiflorus ‘Myers’, and A. cochinchinensis, respectively. The cpDNA genomes were assembled with a coverage of 173x for A. aethiopicus, 164x for A. densiflorus ‘Myers’, and 381x for A. cochinchinensis.
The three newly assembled cpDNA genomes were relatively conserved in terms of length, gene order, gene content, and structure. The cpDNA genome of A. densiflorus ‘Myers’ was the largest (157,139 bp), followed by A. aethiopicus (157,069 bp), and A. cochinchinensis (156,319 bp; Table 2 and Fig 2). The cpDNA genomes exhibited the quadripartite structure typical of angiosperms. Their LSCs ranged from 84,598 to 85,350 bp in length and their IRs from 26,518 to 26,573 bp. The SSC was 18,677 bp for both A. aethiopicus and A. densiflorus ‘Myers’, and 18,685 bp for A. cochinchinensis.
Genes are colour-coded based on their functions shown in the key. Genes located outside of the outer circle are transcribed anticlockwise, while those inside are transcribed clockwise. In the inner circle, the gradient in dark grey represents GC content, whereas light grey represents AT content.
Identical numbers and types of genes were annotated in A. aethiopicus and A. densiflorus ‘Myers’. One hundred and thirty-six genes were successfully annotated, including 90 protein-coding (mRNA) genes, 38 transcription- and translation-related RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. For A. cochinchinensis, 132 genes were annotated, including 87 mRNA genes, 37 tRNA genes, and 8 rRNA genes. The genes were classified into three categories and 18 functions (Table 3).
The pseudogene ycf1 occurred in A. aethiopicus and A. densiflorus ‘Myers’ but was not detected in A. cochinchinensis. A. densiflorus ‘Myers’ and A. cochinchinensis had 21 intron-containing genes, whereas A. aethiopicus had 20. All three cpDNA genomes had two genes comprising two introns (Table 4). For A. aethiopicus and A. densiflorus ‘Myers’, 20 genes were duplicated in IRs. In contrast, only 19 genes were duplicated in the IRs for A. cochinchinensis, because ycf68 was absent from this genome.
The cpDNA genomes of the three species were comparable in terms of GC content (Table 2). In total, 37.5% of the GC bases were detected in all three cpDNA genomes; 35.4–35.5%, 31.3–31.4%, and 42.9% of the GC content was detected in LSCs, SSCs, and IRs, respectively. Among the three cpDNA genomes, A. cochinchinensis had the highest GC content (37.54%), with 35.54% in LSCs and 31.38% in SSCs, whereas A. aethiopicus had the highest IR GC content (42.94%).
Simple sequence repeat analysis
The SSR number, type, content, and distribution were similar in the seven cpDNA genomes. The number of SSRs ranged from 80 (A. schoberioides) to 88 (A. aethiopicus and A. officinalis) (Fig 3).
Each cpDNA sample contained mono-, di-, tri-, or tetra-nucleotides. Three of the seven cpDNA genomes contained pentanucleotides, whereas the other four contained hexanucleotides. The most common class of SSRs was mononucleotides, ranging from 47 in A. densiflorus ‘Myers’ to 57 in A. officinalis. Dinucleotides were the second most common, ranging from 12 in A. racemosus to 15 in A. aethiopicus and A. densiflorus ‘Myers’. Tetranucleotides were the third most common, ranging from 10 in A. schoberioides to 13 in A. aethiopicus and A. densiflorus ‘Myers’. Trinucleotides repeats were the least common, with five each in A. cochinchinensis, A. officinalis, A. racemosus, and A. schoberioides, and seven each in the other species. One or two pentanucleotide or hexanucleotide repeats were found in each of the seven genomes.
Considering sequence complementarity, most of the SSRs were A/T (adenosine/thymine) repeats. ranging from 46 in A. densiflorus ‘Myers’ to 55 in A. officinalis (Fig 4). AT/AT repeats were the second most common, from 9 in A. racemosus to 12 in A. aethiopicus and A. densiflorus ‘Myers’. AAAT/ATTT repeats were the third most common, at 4 in A. officinalis, 6 in A. schoberioides, and 7 in the other cpDNA genomes.
For the seven genomes, 87.59% of the SSRs comprised entirely adenosine and thymine, with at most 2 bp of guanine and cytosine in the GC-containing SSRs. The dominance of A/T base pairs and low frequency of G/C base pairs in SSRs are consistent with the observations made by Sheng et al. [95].
The cpDNA genomes demonstrated similar proportional distributions of SSRs within the quadripartite structure (Fig 5), with most (ca. two-thirds) found in LSC regions and one-fifth and one-tenth, respectively, found in SSC and IR regions.
The percentages for each region are shown in the middle of each bar. The numbers in brackets are the actual numbers of SSRs distributed in the indicated cpDNA regions.
Long sequence repeat analysis
The species differed significantly in the LSR analysis, particularly for A. densiflorus ‘Myers’ (Figs 6 and 7): for the other six genomes, there were 2 LSRs (A. officinalis and A. schoberioides) to 5 LSRs (A. cochinchinensis), whereas A. densiflorus ‘Myers’ had 34 LSRs, almost 10-fold the average in the others.
All four types of LSRs (forward, reverse, palindromic, and complement repeat) were detected. Notably, the genomes contained from 1 (A. officinalis) to 3 (A. densiflorus ‘Myers’) types of LSRs. Palindromic repeats were the most common LSR type: of the 29 palindromic repeats, A. densiflorus ‘Myers’ had 17. Forward repeats were second, occurring in five of the species, excluding A. officinalis and A. racemosus. Of the 22 forward repeats, A. densiflorus ‘Myers’ had 16. A. densiflorus ‘Myers’ and A. racemosus had 1 reverse repeat and 1 complement repeat, respectively.
The minimum repeat size was set to 30 bp. The longest LSR detected by REPuter was 56 bp. LSRs were detected at lengths of 30, 31, 32, 33, 34, 35, 36, 38, 39, 46, 47, 49, 52, 54, and 56 bp. Fig 7 represents their frequencies in three intervals: (i) 30–39 bp, (ii) 40–49 bp, and (iii) 50–56 bp. LSRs of 30–39 bp and 50–56 bp occurred in all three species, whereas only A. densiflorus ‘Myers’ has LSRs of 40–49 bp (six, in total). LSRs of 30–39 bp were the most common, with 39 detected. A. densiflorus ‘Myers’ had the most in this class, at 26. Each of the three species had at least one 50–56 bp LSR, while A. densiflorus ‘Myers’ had two.
Comparative genome analysis
The IR boundaries of the seven genomes were relatively conserved, with some minor variations (contractions and deletions) (Fig 8).
Numbers in bold indicate the size of the gene (or gene section) within the specified regions. The numbers next to the dashed arrows indicate distances from the specified junctions. Numbers within the coloured bands indicate the lengths of the respective regions. The direction of gene transcription is presented by the obtuse angles of the pentagons. Ψ, pseudogene. Not to scale.
In the LSC/IRB border, rpl22 extended into the LSC by 2–5 bp from the junction, for all species except A. cochinchinensis, in which it extended it by 24 bp. For A. officinalis, rpl22 was 360 bp long, 3 bp shorter than in the others. rps19 in the IRB also exhibited variation, with lengths of 210 bp for A. aethiopicus, A. densiflorus ‘Myers’, and A. racemosus, and 279 bp for the other four species; it extended by 263–332 bp from the LSC/IRB junction into the IRB.
The ycf1 pseudogenes was retained in the border IRB/SSC for all species, except A. cochinchinensis and A. officinalis; its length was 912 bp for all species except A. schoberioides, in which a 110 bp fragment of the SSC was deleted. ndhF in the SSC was 2229 bp long for A. aethiopicus and A. densiflorus ‘Myers’, and 2223 bp long for the other species; it extended from IRB/SSC junction by 3 bp for A. aethiopicus and A. densiflorus ‘Myers’, 7 bp for A. cochinchinensis, and 9 bp for the others.
Functional ycf1 genes (5624–5460 bp long) were located at the SSC/IRA border for all species except A. officinalis, in which an IRA portion was lost to the SSC, leaving a contracted pseudogene of 3824 bp in length. Further, in A. setaceus, the functional ycf1 extended into the SSC by 307 bp from the SSC/IRA junction, unlike in the other species.
At the IRA/LSC border, rps19 (137–279 bp long) in IRA extended by 32–196 bp from the junction, with A. offcinalis having the shortest extension as a contracted pseudogene.
In the sliding-window analysis, five regions—trnS-trnG, ndhC-trnV, accD-psaI, ccsA, and ycf1—were identified as divergence hotspots with Pi ≥ 0.015 (Fig 9). accD-psaI was the most variable (Pi = 0.023), followed by ccsA (Pi = 0.020), and trnS-trnG (Pi = 0.17). These regions represent potential molecular markers for the phylogenetic and population genetics studies of Asparagus species. The sequence identity plot, using A. aethiopicus as a reference (S2 Fig), revealed different identity level (of <50%) among these five regions between the seven species, with “cracks” among the bars.
X-axis: window midpoint; Y-axis: nucleotide diversity value (Pi) for each window. Divergence hotspots (Pi > 0.015) are labelled in red above the corresponding position.
Gene order and gene content were highly conserved among the seven species. The sequence identity plot (S2 Fig) revealed highly similar exon (purple) and intron (blue) regions. UTRs (red) in the non-coding regions clearly illustrate the diversity. The average Pi of 0.004 indicates that the sequence diversity of these species is relatively low.
No structural rearrangement was observed. IRs were more conserved than LSCs or SSCs, as illustrated by the high IR similarity in the sequence identity plot and supported by the sliding window analysis. The LSC and SSC regions contained most of the Pi peaks. In contrast, IRs had low nucleotide diversity (Pi < 0.01), except for the ycf1 divergence hotspot at the SSC/IR border. The other four divergence hotspots were within LSCs (trnS-trnG, ndhC-trnV, and accD-psaI) and SSC (ccsA).
Phylogenetic analysis
Congeneric relationships in the genus Asparagus were examined using three newly assembled cpDNA genomes and four cpDNA genomes from GenBank. ML trees derived from the complete cpDNA genomes, LSC, SSC, and CDS sequences shared the same topology (Fig 10) but different node bootstrap values. A. setaceus was sister to the other six Asparagus species. The branch containing A. aethiopicus and A. densiflorus ‘Myers’ had the highest bootstrap value (100) in all four ML trees, supporting the close relationship between these two species. A. cochinchinensis and A. racemosus formed a sister clade to A. officinalis and A. schoberioides (bootstrap values of 100 for complete cpDNA genomes, LSC, and SSC, and 84 for CDS). The close relationship between A. cochinchinensis and A. racemosus was well supported (bootstrap values of 100 for complete cpDNA genomes and LSC, and 99 for SSC and CDS). This new grouping differs from both traditional taxonomical classifications and molecular phylogenies [5, 6, 11]. We expected A. racemosus, a monoecious species, to group with the three other monoecious species from South Africa. Instead, it was nested within the group of dioecious and Eurasian species in the ML trees, with high bootstrap values.
Numbers next to the nodes: bootstrap values based on complete cpDNA genomes/LSC/SSC/CDS sequences. The topologies are identical. Bold taxa: the three newly assembled cpDNA genomes.
The ML tree based on IR sequences also exhibited unexpected grouping (Fig 11): A. racemosus was still nested with the dioecious species, which were sister to A. officinalis and A. schoberioides, with moderate support (bootstrap value = 71).
Numbers next to the nodes: bootstrap values on IRA and IRB. Bold taxa: the three newly assembled cpDNA genomes.
The close relationship between A. aethiopicus and A. densiflorus ‘Myers’ was supported by the ML trees based on complete cpDNA genomes, LSC, SSC, and CDS sequences (Fig 10) and was further validated by the IR-based tree, with bootstrap values of 100.
Discussion
Molecular insights for nomenclatural confusion
A. aethiopicus and A. densiflorus ‘Myers’ are nomenclaturally controversial. Batchelor and Scott (2006) [67] questioned the taxonomic identity of the cultivar ‘Myers’ (foxtail asparagus), which is often recorded as a cultivar of A. densiflorus [1, 3, 5, 11, 15, 51, 57, 64, 67, 80, 83]. In contrast, some have suggested placing the Asparagus cultivars ‘Sprengeri’ and ‘Myers’ under A. aethiopicus [4, 67, 76, 77]. The Royal Botanic Gardens Victoria [78, 108] has adopted the name A. aethiopicus ‘Myersii’ for foxtail asparagus.
A. densiflorus and A. aethiopicus differ primarily in their growth habit, with the former not a climber and rarely over 1 m tall and the latter an erect herb of 1 m or more and climbing up to 7 m [4, 76]. From our observations, the Asparagus cultivar ‘Myers’ never climbs, even when it is not pot-bound. This growth habit does not correspond with the circumscription of A. aethiopicus emphasised by Green (1986) [76] and Judd (2001) [4]. We agree with Batchelor and Scott (2006) that foxtail asparagus should be considered a cultivar of A. densiflorus [67], and hence the legitimate name should be Asparagus densiflorus (Kunth) Jessop ‘Myers’.
Our findings show that A. aethiopicus and A. densiflorus ‘Myers’ are phylogenetically close, despite their morphological and growth habit differences, with bootstrap values of up to 100 for ML trees based on complete cpDNA genomes, LSC, SSC, IR, or CDS (Figs 10 and 11). Their gene numbers, GC content (Table 2), genome structure (Fig 2), and IR border (Fig 8) are similar. This supports the traditional classifications, which consistently place them under the same generic circumscription: genus Asparagopsis [71], genus Asparagus section Falcati [8], genus Asparagus section Racemosi [15], or genus Protasparagus [16] (S1 Fig and S1 Table). Using short-length DNA regions, Norup et al. [6] suggested placing the two species in an Asparagus–Racemose clade–Racemose 1 clade. Our phylogenetic results, which group the cpDNA genomes of A. aethiopicus and A. densiflorus ‘Myers’, are consistent with this.
The two species showed minor differences. In terms of LSR number and type, A. densiflorus ‘Myers’ differed significantly from A. aethiopicus and the other species. The cpDNA genome of A. densiflorus ‘Myers’ had the most LSRs, and this was the only species with reverse repeats and 40–49 bp LSRs (Figs 6 and 7). SSRs have been used to identify cultivars of potatoes [109, 110], apples [111], and sunflowers [112]. However, these Asparagus species did not differ significantly in SSRs. Nonetheless, the distinctive LSR patterns of A. densiflorus ‘Myers’ could provide a molecular authentication marker.
Our phylogenetic analysis revealed the close relationship between A. aethiopicus and A. densiflorus ‘Myers’ but did not elucidate the species origin of the cultivar. According to Article 21.1 of ICNCP, “The name of a cultivar is a combination of the correct name of the genus or lower taxon to which it is assigned under the ICN, or its unambiguous common name, with a cultivar epithet” [86]. We suggest two treatments to clarify A. densiflorus ‘Myers’ nomenclature: first, to combine only the genus name with the cultivar epithet, as Asparagus ‘Myers’, since this cultivar epithet has not been used for other cultivars being assigned to other Asparagus species; second, to combine the common name and the cultivar epithet, as asparagus ‘Myers’, since the common name of the genus Asparagus is unambiguous and is identical to the genus name.
Unexpected placement of A. racemosus
Taxonomists have attempted to divide the genus Asparagus into three major groups. The first, characterised by flattened and leaf-like cladodes, basally connate perianth segments, and filaments connated into tubes, was classified as the genus Myrsiphyllum by Willdenow (1808) [14], Kunth (1850) [71], and Obermeyer (1984) [17], and as the genus Asparagus subgenus Myrsiphyllum by Baker (1875) [7]. The second and third groups comprise the species with filiform to linear cladodes: the second, comprising monoecious and African species with free perianth segments and filaments, was classified as the genus Asparagopsis by Kunth (1850) [71], the genus Asparagus subgenus Asparagopsis by Baker (1875) [7], and the genus Protasparagus by Obermeyer (1983) [17]; the third, comprising dioecious and Eurasian species with basally connate perianth segments, was classified as the genus Asparagus by Kunth (1850) [71] and the genus Asparagus subgenus Euasparagus by Baker (1875) [7].
A. racemosus, a monoecious species widespread throughout Africa, Asia, and Australia [10, 113], has traditionally been classified into the second group. Fukuda et al. [5] and Kubota et al. [11] placed A. racemosus in genus Asparagus subgenus Protasparagus, whereas Norup et al. [6] placed it in the Asparagus–Racemose clade–Racemose 2 clade.
We expected A. racemosus to cluster with its relatives in the same group, i.e. A. aethiopicus, A. densiflorus ‘Myers’, and A. setaceus. However, one A. racemosus specimens (NC_047472) unexpectedly clustered with the dioecious species A. cochinchinensis, A. officinalis, and A. schoberioides in the ML trees (Figs 9 and 10), using both complete cpDNA genomes and sequence portions. This is contrary to Lee et al. (1997) [114] who, using restriction fragment length polymorphism cpDNA analysis, showed that no monoecious species were clustered within the monophyletic group of dioecious species (A. officinalis, A. schoberiodes, or A. cochinchinensis) [114].
Short cpDNA regions of A. racemosus (ca. 300–1000 bp) were reported by Fukuda et al. (petB intron and petD-rpoA) [5], Kubota et al. (rpl32-trnL, trnQ-5′rps16, ndhF-rpl32, psbD-trnT, 3′rps16-5′trnK) [11], and Norup et al. (3′ ndhF, psbA-trnH, trnD-trnT) [6]. We attempted to determine the start and stop positions of these regions in NC_047472. Ten extracted sequences of the corresponding length (S2 Table) were screened using the NCBI Basic Local Alignment Search Tool, and only trnQ-rps16 (sequence identity 98.70%), psbA-trnH (97.11% and 96.84%), rpl32-trnL (96.49%), petD-rpoA (96.86%), and trnD-trnT (97.71%) matched the respective regions of A. racemosus. GenBank did not contain any voucher information for NC_047472. Because of this lack of voucher information, we are unable to further verify this unanticipated and unlikely grouping. Our intra-generic analyses were constrained by the limited sample size. Further studies on A. racemosus phylogeny are recommended.
Conclusion
Complete cpDNA genomes of three Asparagus specimens collected in Hong Kong were de novo assembled, annotated, and compared with those of congenerics. The seven genomes were relatively conserved in terms of gene content, gene order, and genome structure. A. densiflorus ‘Myers’ differed significantly from the others in LSR number and type. Five divergence hotspots were identified in the sliding-window analysis (Pi ≥ 0.015). Our phylogenetic analysis elucidates the generic subdivision and the nomenclatural complexity of A. aethiopicus and A. densiflorus ‘Myers’. The novel placement of A. racemosus, contrary to previous morphological and molecular classifications, requires further verification. We suggest two ICNCP-compliant names for A. densiflorus ‘Myers’, namely Asparagus ‘Myers’ and asparagus ‘Myers’. These de novo assembled cpDNA genomes provide potential genomic resources, elucidating Asparagus taxonomy, application, and conservation.
Supporting information
S1 Fig. The historical changes on the generic subdivision of the genus Asparagus.
https://doi.org/10.1371/journal.pone.0266376.s001
(PDF)
S2 Fig. Visualisation of the alignments of 7 Asparagus chloroplast genomes using A. aethiopicus as a reference.
https://doi.org/10.1371/journal.pone.0266376.s002
(PDF)
S3 Fig. Specimen photos of voucher K. H. Wong 092, 107, and 109.
https://doi.org/10.1371/journal.pone.0266376.s003
(PDF)
S1 Table. The historical changes on taxonomical status of the 7 studied Asparagus species.
https://doi.org/10.1371/journal.pone.0266376.s004
(XLSX)
S2 Table. Extracted sequences from cpDNA of Asparagus racemosus (NC_047472.1).
https://doi.org/10.1371/journal.pone.0266376.s005
(XLSX)
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