Complete chloroplast genomes of Asparagus aethiopicus L., A. densiflorus (Kunth) Jessop 'Myers', and A. cochinchinensis (Lour.) Merr.: Comparative and phylogenetic analysis with congenerics.

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.

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).

Table 1

Information about the Asparagus specimens deposited at the Shiu-Ying Hu Herbarium.

Species	Collector no.	Inventory no.	Sheet no.	GPS location
Asparagus aethiopicus L.	K. H. Wong 109	CUSLSH2801	CUHK05891	22.420786, 114.208312
Asparagus densiflorus (Kunth) Jessop ‘Myers’	K. H. Wong 092	CUSLSH2773	CUHK05890	22.419994, 114.207354
Asparagus cochinchinensis (Lour.) Merr.	K. H. Wong 107	CUSLSH2799	CUHK05892	22.421524, 114.207135

Fig 1

Photos of three Asparagus plants collected at the Chinese University of Hong Kong.

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.

Table 2

Summary on the cpDNA genome structure of the seven Asparagus species.

	A. aethiopicus	A. densiflorus ‘Myers’	A. cochinchinensis	A. officinalis	A. racemosus	A. schoberioides	A. setaceus
Accession no.	MZ337394	MZ337395	MZ424304	NC_034777	NC_047472	NC_035969	NC_047458
Total length (bp)	157,069	157,139	156,319	156,699	156,742	156,875	156,978
LSC (bp)	85,246	85,350	84,598	84,999	84,989	84,928	85,311
SSC (bp)	18,677	18,677	18,685	18,638	18,619	18,685	18,641
IR (bp)	26,573	26,556	26,518	26,531	26,567	26,631	26,513
Total number of genes	136	136	132	133	130	132	135
mRNA	90	90	87	88	86	88	90
tRNA	38	38	37	37	36	36	37
rRNA	8	8	8	8	8	8	8
Pseudogene (Ψ)	1a	1a	0	7b	1a	1a	1a
1-intron gene	20	21	21	21	21	20	19
2-introns gene	2	2	2	2	2	2	2
Total GC content (%)	37.49	37.49	37.54	37.59	37.55	37.57	37.48
GC content in LSC (%)	35.44	35.43	35.54	35.60	35.53	35.55	35.46
GC content in SSC (%)	31.30	31.31	31.38	31.50	31.43	31.51	31.45
GC content in IR (%)	42.94	42.93	42.90	42.92	42.92	42.93	42.85

a ycf1

b ycf1, ycf15 (x2), ycf68 (x2), infA, rps19.

Fig 2

Chloroplast genome map of A. aethiopicus L., A. densiflorus (Kunth) Jessop ‘Myers’, and A. cochinchinensis (Lour.) Merr.

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).

Table 3

Genes annotated in the complete cpDNA genomes of A. aethiopicus L., A. densiflorus (Kunth) Jessop ‘Myers’, and A. cochinchinensis (Lour.) Merr.

Gene category	Gene functions	Gene names
Photosynthesis-related genes	Rubisco	rbcL
	Photosystem I	psaA, psaB, psaC, psaI, psaJ
	Assembly/ stability of photosystem I	pafI, pafII, pbf1
	Photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbT, psbZ
	ATP synthase	atpA, atpB, atpE, atpF, atpH, atpI
	Cytochrome b/f complex	petA, petB, petD, petG, petL, petN
	Cytochrome c synthesis	ccsA
	NADPH dehydrogenase	ndhA, ndhB%, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Transcription- and translation-related genes	Transcription	rpoA, rpoB, rpoC1, rpoC2
	Ribosomal protein	rpl2%, rpl14, rpl16, rpl20, rpl22, rpl23%, rpl32, rpl33, rpl36, rps2, rps3, rps4, rps7%, rps8, rps11, rps12%, rps14, rps15, rps16, rps18, rps19%
	Translation initiation factor	infA
RNA genes	Ribosomal RNA	rrn16%, rrn23%, rrn4.5%, rrn5%
	Transfer RNA	trnA-UGC%, trnC-GCA, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC*, trnH-GUG%, trnI-CAU%, trnI-GAU%, trnK-UUU, trnL-CAA%, trnL-UAA, trnL-UAG, trnM-CAU$, trnN-GUU%, trnN-GUC, trnP-UGG, trnQ-UUG, trnR-ACG%, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC%, trnV-UAC, trnW-CCA, trnY-GUA
Miscellaneous group	Maturase	matK
	Inner membrane protein	cemA
	ATP-dependent protease	clpP1
	Acetyl-CoA carboxylase	accD
	Unknown functions	ycf1@, ycf2%, ycf68#

% Duplicated in inverted repeat regions

* Duplicated in large single copies of A. densiflorus ‘Myers’ and A. cochinchinensis; appeared once in A. aethiopicus

$ Duplicated in large single copies of A. aethiopicus and A. densiflorus ‘Myers’; appeared once in A. cochinchinensis

@ ycf1 was functional in all three species, but the ycf1 pseudogene was absent from A. cochinchinensis

# Duplicated in inverted repeat regions of A. aethiopicus and A. densiflorus ‘Myers’; absent from A. cochinchinensis.

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.

Table 4

Intron-containing genes in the chloroplast genomes of seven Asparagus species.

	A. aethiopicus	A. densiflorus ‘Myers’	A. cochinchinensis	A. officinalis	A. racemosus	A. schoberioides	A. setaceus	Location
Accession no.	MZ337394	MZ337395	MZ424304	NC_034777	NC_047472	NC_035969	NC_047458	/
trnK-UUU	0	1	1	1	1	1	0	LSC
rps16	1	1	1	1	1	1	1	LSC
trnG-UCC B	1	1	1	1	1	1	ABS	LSC
atpF	1	1	1	1	1	1	1	LSC
rpoC1	1	1	1	1	1	1	1	LSC
ycf3/ pafI C	2	2	2	2	2	2	2	LSC
trnL-UAA	1	1	1	1	1	ABS	1	LSC
trnV-UAC	1	1	1	1	1	1	1	LSC
clpP	2A	2A	2A	2	2	2	2	LSC
petB	1	1	1	1	1	1	1	LSC
petD	1	1	1	1	1	1	1	LSC
rpl16	1	1	1	1	1	1	1	LSC
rpl2 *2	1	1	1	1	1	1	1	IRA + IRB
ndhB *2	1	1	1	1	1	1	1	IRA + IRB
rps12 *2	1	1	1	1	1	1	1	IRA + IRB + LSC
trnI-GAU *2	1	1	1	1	1	1	1	IRA + IRB
trnA-UGC *2	1	1	1	1	1	1	1	IRA + IRB
ndhA	1	1	1	1	1	1	1	SSC

0—No intron; 1–1 intron; 2–2 introns; ABS—Gene absent.

A Annotated as clpP1.

B Located in the region 9167–9994 bp; for NC 047458, trnG-UCC, at 36924–36994 bp, had no intron.

C pafI was annotated in A. aethiopicus, A. densiflorus ‘Myers’, and A. cochinchinensis.

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).

Fig 3

Simple sequence repeat class distribution.

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.

Fig 4

Simple sequence repeat frequency related to sequence complementarity.

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.

Fig 5

Simple sequence repeat distribution in the quadripartite cpDNA structure.

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.

Fig 6

Types of long sequence repeats.

Fig 7

Frequency of long sequence repeats in specified length intervals.

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.

The IR boundaries of the seven genomes were relatively conserved, with some minor variations (contractions and deletions) (Fig 8).

Fig 8

Large single copy (LSC), small single copy (SSC), and inverted repeat (IR) boundary comparison for the seven Asparagus cpDNA genomes.

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.

Fig 9

Complete cpDNA genome nucleotide diversity for the seven Asparagus species.

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).

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.

Fig 10

Maximum likelihood (ML) trees based on Asparagus cpDNA genomes.

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).

Fig 11

Maximum likelihood (ML) trees based on inverted repeats (IRs) for Asparagus.

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.

The historical changes on the generic subdivision of the genus Asparagus.

(PDF)

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Visualisation of the alignments of 7 Asparagus chloroplast genomes using A. aethiopicus as a reference.

(PDF)

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Specimen photos of voucher K. H. Wong 092, 107, and 109.

(PDF)

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The historical changes on taxonomical status of the 7 studied Asparagus species.

(XLSX)

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Extracted sequences from cpDNA of Asparagus racemosus (NC_047472.1).

(XLSX)

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17 Nov 2021

28 Jan 2022

Dear Dr. Šiler, Reviewer 1 and Dr. Sheng,

Rebuttal Letter

I am sincerely writing to you to response all your comments in the Decision Letter in 18th November 2021. Our replies are listed as below.

Comment 1. As suggested by Reviewer #1, literature update is highly recommended, as the authors did not present the latest findings related to the scientific matter. Moreover, an in depth authors' elaboration of the present text regarding the literature update is expected.

Reply: Agreed. We have added the relevant articles in the reference list and elaborated their findings in the sections of introduction, result and discussion.

Comment 2. The whole text would hugely benefit if proofread by a native English speaker or a professional editing agency…

Reply: Agreed. We will employ a professional editing agency to proofread the article after all parties agreed with the content.

Comment 3. L36: "species" should not stand italicized.

Reply: Agreed and revised.

Comment 4. L39: "Asparagus have been widely applied..." - a colloquial expression. Suggestion: "Many species belonging to the Asparagus genus have been widely applied..."

Reply: Agreed. Amendment was made as follows:

“Many species belonging to the genus Asparagus have been widely applied in different aspects of human society”.

The genus name was italicized and put after the word “genus” according to the rule of ICN.

Comment 5. L41, L58 and elsewhere in the text: "anthropocentric usage of Asparagus", "while some Asparagus were used"... - the same comment as the previous.

Reply: Agreed and amended as follows:

L60-61: Adapting to drought conditions, Asparagus species have evolved characteristic morphology.

L71-72: Below we briefly discussed the anthropocentric usage of some Asparagus species…

L89: …while some Asparagus species were used as charm to increase fertility…

Comment 6. L47 and further in the text: Please use "A." to abbreviate the genus name when introducing a species name.

Reply: We agreed with the guidelines of PLOS One. We had adopted abbreviated genus name (A.) for most of the binominal names in the article. However, we would like to keep the presentation of full scientific name for the sentences below, in order to (i) obey the rules of ICN/ICNCP, (ii) accurately present the nomenclatural treatment, and (iii) present the original record of literatures.

L165-167: In 1890, Regel published the name Asparagus sprengeri based on the cultivated plants growing in Natal

L168-169: The name Asparagus sprengeri Regel hence is the scientific name of sprengeri asparagus. [Remarks: the species epithets “sprengeri” is amended in lower case instead of “Sprengeri” in the first submission]

L171-172: Jessop synonymized A. sprengeri Regel under the new combination Asparagus densiflorus (Kunth) Jessop based on morphology and geographical distribution

L178-179: In 1767, Linnaeus published the name Asparagus aethiopicus L. in the Species Plantarum

L187-188: Both names Aspararagopsis aethiopica (and later, Asparagus aethiopicus) and Asparagopsis densiflora were adopted in parallel for 116 years, from 1850 to 1965.

L197-200: Straley and Utech also adopted the scientific name A. aethiopicus for sprengeri asparagus, and stated “Asparagus densiflorus (Kunth) Jessop has been misapplied to this species” in Flora of North America North of Mexico (2004)

L207-210: …in terms of its habitats, growing habits and reproductive characters, fits the circumscription of Asparagus aethiopicus L. in the monograph. Therefore, we adopt the scientific name Asparagus aethiopicus L. for sprengeri asparagus throughout this study.

L218-220: The first binomial name of foxtail asparagus, Asparagus myersii, was raised anonymously in an unknown time, while the name Asparagopsis densiflora was validly published in 1850 by Kunth (S2 Table)

L222-223: In 1966, Jessop mentioned the name Asparagus myersii Hort. “had never been validly published”

L239-241: …, we follow the treatment of some taxonomists and scientists [1,4-5,11,51,64,76,83], adopting the scientific name Asparagus densiflorus Jessop (Kunth) ‘Myers’ for foxtail asparagus throughout this study.

L306: Remains the full name of Asparagus aethiopicus L., Asparagus densiflorus (Kunth) Jessop 'Myers' and Asparagus cochinchinensis (Lour.) Merr. were used in Table 1 which indicates the authentication of specimens.

L614-616: We agreed with Batchelor & Scott (2006) that foxtail asparagus should be a cultivar of A. densiflorus [67], and hence the legitimate name should be Asparagus densiflorus (Kunth) Jessop ‘Myers’.

L647-648: One is to combine only the genus name with the cultivar epithet, as Asparagus ‘Myers’…

L649-651: Another treatment is the combination of the common name and the cultivar epithet, as Asparagus ‘Myers’… [In this case “Asparagus” is not italic and not a genus name]

Comment 7. L77 and elsewhere in the text: Do not capitalize common plant names such as "Garden Asparagus". Write "garden asparagus".

Reply: Agreed. All are amended.

Comments from Reviewer 1:

The Authors have carried out a study based on the sequencing of the Complete Chloroplast Genome of three species (A. aethiopicus, A. densiflorus ‘Myers’ and A. cochinchinensis) belonging to the Asparagus genus. The results obtained were assembled with chloroplast genomes of other four Asparagus species (A. setaceus, A. racemosus, A. schoberioides Asparagus officinalis L.) on NCBI. The manuscript could bring a significative impact for further studies aimed to widen the knowledge of the Asparagus genus and the phylogenetic relationships among Asparagus spp. In my opinion, the paper could be published after a few modifications that might improve this manuscript.

The main criticism to this paper is that I have lacked the mention of some studies employing cpDNA that have been previously developed in the Asparagus genus ( Lee et al 1996; Lee et al 1997; Kanno et al 1997; Seng et al 2017 and Li et al 2019). I was impressed about the comprehensive and detailed revision on the taxonomic classifications in the asparagus genus that have been published since the eighteen century to date. However, previous studies employing cpDNA of different Asparagus spp. are not mentioned in the manuscript. In my opinion these studies should be mentioned in the Introduction section of the manuscript. I think that at least the studies developed by Lee et al. (1997) and Seng et al. (2017) are recommended to be also used in the discussion of the manuscript because the findings of the present study can be compared to a certain extent with the obtained by these two studies.

Reply: Thank you so much for your appreciation! We agreed with your suggestions and advices. The suggested literatures were already included in the sections of introduction, result and discussion with appropriate elaboration.

Other comments and suggestions for the Authors:

> Abstract: The authors wrote: “Conducting comparative and phylogenetic analysis with congeneric species, four cpDNA on NCBI were included in this study”. The scientific name of these four species (A. setaceus, A. racemosus, A. schoberioides Asparagus officinalis L.) they should be included in this section of the manuscript.

Reply: Agreed. The scientific names of the four species were included accordingly.

> lines 35-37: In these lines is written the following sentence: “These aforementioned characteristics were evolved by Asparagus species to adapt to arid environment”. I think that this sentence must be support by a cite/s

Reply: Agreed. The reference source (Dahlgren et al., 1985 and Judd, 2001) were cited accordingly.

> lines 43-44: It is written: “of the two studied Asparagus species were discussed in detail” Is it correct? Two (A. aethiopicus, A. densiflorus ‘Myers’) or three species (A. aethiopicus, A. densiflorus ‘Myers’, A. cochinchinensis)?

Reply: Agreed. The word “two” was deleted from this sentence.

> Lines 78- 79. The authors wrote: “However, the gene pool of A. officinalis is relatively limited” In my opinion there are other studies such as Geoffriau et al 1992, Moreno et al 2006 or Mercati et al 2015 that must be cited instead of the study carried out by Stajner et al 2002

Reply: Agreed. We cited your suggested source of literature instead of the one of Stajner et al., 2002.

> lines 79-80: I suggest modifying the following sentence: “The species is susceptible to multiple diseases” by the species is susceptible to multiple biotic and abiotic stresses…

Reply: Agreed. Amendment was done.

Comments from Reviewer 2:

The introduction part is chaotic in this manuscript, which shoule be rewritten；Please pay attention to some mistakes in your grammar and spelling, and I have labelled these shortcomings in this paper; the content is complete, and the result is clear; and I suggest this paper should be accepted after minor revison.

Reply: Thank you for your comments. We have rewritten the introduction. For the grammar and spelling, we have revised it accordingly, and will sent the manuscript to a professional agency for further proofread after all parties agreed with the content.

Other comments and suggestions for the Authors:

Line 72. Hong Kongwhere

Reply: Agreed and amended. Space between “Kong” and “where” was added.

Line 233. S2 Table

Reply: The sentence in Line 233 had been deleted in the latest version. The usage of S2 Table in other parts of the manuscript were put in brackets.

Line 354. The pseudogene ycf1...

Reply: Agreed. The gene name (ycf1) was italicized.

Line 471. ranging from 5624 to 5460 bp ...

Reply: Agreed. The unit was given for each number as “5624 bp to 5460 bp”.

Line 481. trnS-trnG (Pi = 0.17)

Reply: Agreed. The sentence was revised as “Among these hotspots, accD-psaI was the most variable region (Pi=0.023), followed by ccsA (Pi=0.020) and then trnS-trnG (Pi = 0.17).”

Line 549. [4,Error! Reference source not found.,65,74].

Reply: Agreed. The reference source (Straley & Utech, 2003) was updated.

Line 604. The second group consisting…

Reply: Agreed. The sentence was revised as "The second group consisting of".

Line 628. S4 Table

Reply: Sorry. We have no idea of this amendment. Please further advice.

Comments from the Academic Editor:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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Reply: Agreed. We have updated our manuscript according to the style requirements.

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Reply: The research work was supported by a donation fund from Wu Jieh Yee Charitable Foundation Limited. The fund has no formal grant number.

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Reply: Agreed. This phrase was removed as the data are not a core part of the research.

4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Reply:

The following references were newly added along with the amended manuscript:

38. Geoffriau E, Denoue D, Rameau C. Assessment of genetic variation among asparagus (Asparagus officinalis L.) populations and cultivars: agromorphological and isozymic data. Euphytica. 1992; 61:169-179. doi:10.1007/BF00039655

39. Mercati F, Riccardi P, Harkess A. Single nucleotide polymorphism-based parentage analysis and population structure in garden asparagus, a worldwide genetic stock classification. Mol Breeding. 2015. 35(59):1-12. doi:10.1007/s11032-015-0217-5

40. Moreno R, Espejo JA, Cabrera A, Millán T, Gil J. Ploidic and Molecular Analysis of ‘Morado de Huetor’ asparagus (Asparagus officinalis L.) population; a Spanish tetraploid landrace. 2006; 53:729–736. Genet Resour Crop Evol. doi:10.1007/s10722-004-4717-0

82. Obermeyer AA, Immelamn KL, Bos JJ. (1992). Asparagaceae. In: Leistner OA, du Plessis E, editors. Flora of Southern Africa. Volumn 5, Part 3. Dracaenaceae, Asparagaceae, Luzuriagaceae and Smilacaceae. Pretoria: National Botanical Institute; 1992. pp. 11-82.

93. Lee YO, Kanno A, Kameya T. The physical map of the chloroplast DNA from Asparagus officinalis L. Theor Appl Genet. 1996;92:10-14.

94. Kanno A, Lee YO, Kameya T. The structure of the chloroplast genome in members of the genus Asparagus. Theor Appl Genet. 1997;95:1196-1202.

95. Sheng W, Chai X, Rao Y, Tu X & Du S. Complete chloroplast genome sequence of Asparagus (Asparagus officinalis L.) and its phylogenetic position within Asparagales. J Plant Breed Genet. 2017; 5(3):121-128.

96. Li JR, Li SF, Wang J, Dong R, Zhu HW, Li N, et al. Characterization of the complete chloroplast genome of Asparagus setaceus. Mitochondrial DNA B Resour, 2019; 4(2):2639-2640. doi: 10.1080/23802359.2019.1643798

114. Lee YO, Kanno A, Kameya T. Phylogenetic relationships in the genus Asparagus based on the restriction enzyme analysis of the chloroplast DNA. Japanese Journal of Breeding. 1997; 47:375-378.

The following reference was deleted according to the comment from Reviewer 1:

45. Stajner N, Bohanec B, Javornik B. Genetic variability of economically important Asparagus species as revealed by genome size analysis and rDNA ITS polymorphisms. Plant Sci. 2002; 162(6):931-937. doi: 10.1016/S0168-9452(02)00039-0

Thank you very much for your kindly reviews and consideration.

Yours sincerely,

Dr. David TW LAU

Curator of the Shiu-ying Hu Herbarium

School of Life Sciences

The Chinese University of Hong Kong

Submitted filename: Rebuttal Letter.docx

Click here for additional data file.

31 Jan 2022

16 Mar 2022

Dear Dr. Šiler,

Rebuttal Letter

I am sincerely writing to you to response all your comments in the Decision Letter in 31st January 2022. Our replies are listed as below.

Original comments from Dr. Šiler:

Following the rules of ICN (not ICNCP, since the studied taxa are not cultivated) is absolutely necessary in scientific literature and I fully support it. However, please bear in mind that abbreviating genus name does not oppose the ICN rules (the current version is Shenzhen Code, published by IAPT - https://www.iapt-taxon.org/nomen/main.php; for genera, please see https://www.iapt-taxon.org/nomen/pages/main/art_20.html). It is a common scientific practice to write a scientific name in full when it is first used or when several species from the same genus are being listed or discussed in the same paper or report. For subsequent uses, the genus can be abbreviated to its first letter followed by a period. You can abbreviate the genus name after its first use even when describing a different species within that genus, as long as there is no risk of confusing it for another genus or genera (which is not the case here, since only the genus Asparagus circulates throughout the manuscript). Therefore, by abbreviating the genus name (i) obeying the rules of ICN is not compromised, ii) nomenclatural treatment is presented accurately (no confusion to other genera starting with "A."), and iii) if a species is described in a source article as e.g., "Asparagus aethiopicus", I do not see the reason why in consequent articles dealing with the same species it wouldn't be abbreviated as "A. aethiopicus" if the genus name was already mentioned earlier in the text. The same applies to the main title: reprising the genus name three times is space-consuming and quite exhausting for reading. I suggest abbreviating here the genus name for the second and the third species as well (also, replace "&" with "and" here and elsewhere in the text). The exceptions might be L178-179 (cites the original text), L197-200 (cites the original text), L218-220 (only for mentioning Asparagus myersii, since a binomial name has been announced), L222-223 (cites the original text), Table 1, L614-616 (a full name has been announced), L647-648 (as it complies with the ICN rules), L649-651 (but being the common name, "asparagus” should stand in lowercase).

Moreover, vernacular expressions such as L101 (circumscription of Asparagus), L117 (relationships within Asparagus), L125 (only one native Asparagus), L126 (Other common exotic Asparagus), L232 (7 Asparagus were examined) (and in many other places) were not clarified as required in the previous review round.

102-107: The terms "genus" and "subgenus" should stand in lowercase.

L127-129 and elsewhere in the text: I must repeat: common names should be written in lowercase, i.e., "sprengeri asparagus" (however, I'm not aware of this Latinized common name, but "Sprenger's asparagus" might be acceptable, after Carl Ludwig Sprenger), "lace fern", etc.

The authors are urged to meticulously check the text once again for proper English usage while following the comments stated above. I still strongly encourage the authors to have the MS proofread by a native English speaker or professional editing agency.

Reply: Thank you so much for your detailed clarification. Please see the reply of the subtracted comments from your comments:

Comment 1: Therefore, by abbreviating the genus name (i) obeying the rules of ICN is not compromised, ii) nomenclatural treatment is presented accurately (no confusion to other genera starting with "A."), and iii) if a species is described in a source article as e.g., "Asparagus aethiopicus", I do not see the reason why in consequent articles dealing with the same species it wouldn't be abbreviated as "A. aethiopicus" if the genus name was already mentioned earlier in the text.

Reply: Agreed. Please see the amendment in the manuscript.

Comment 2: The same applies to the main title: reprising the genus name three times is space-consuming and quite exhausting for reading. I suggest abbreviating here the genus name for the second and the third species as well …

Reply: Agreed. Please see the amendment in the manuscript.

Comment 3: (also, replace "&" with "and" here and elsewhere in the text)

Reply: Agreed. All the sign “&” were placed by the word “and” in the main text and title.

Comment 4: The exceptions might be L178-179 (cites the original text), L197-200 (cites the original text), L218-220 (only for mentioning Asparagus myersii, since a binomial name has been announced), L222-223 (cites the original text), Table 1, L614-616 (a full name has been announced), L647-648 (as it complies with the ICN rules), L649-651 (but being the common name, "asparagus” should stand in lowercase).

Reply: Thank you so much for your consideration for these exception. However, two more exceptions we would like to request:

L165-167: In 1890, Regel published the name Asparagus sprengeri based on the cultivated plants growing in Natal.

Reason: Cites the original text

L187-188: Both names Aspararagopsis aethiopica (and later, Asparagus aethiopicus) and Asparagopsis densiflora were adopted in parallel for 116 years, from 1850 to 1965.

Reason: Please noticed that there are two genera involved: Asparagopsis and Asparagus. Both the abbreviation of these two genera are “A.” which would be confusing. Moreover, please noticed that Asparagopsis aethiopica and Asparagopsis densiflora were the name published by Kunth in 1850 (Please refer to the S2 Table).

Comment 5: Moreover, vernacular expressions such as L101 (circumscription of Asparagus), L117 (relationships within Asparagus), L125 (only one native Asparagus), L126 (Other common exotic Asparagus), L232 (7 Asparagus were examined) (and in many other places) were not clarified as required in the previous review round.

Reply: Agreed. Please see the amendment in the manuscript.

Comment 6: 102-107: The terms "genus" and "subgenus" should stand in lowercase.

Reply: Agreed. Please see the amendment in the manuscript.

Comment 7: L127-129 and elsewhere in the text: I must repeat: common names should be written in lowercase, i.e., "sprengeri asparagus" (however, I'm not aware of this Latinized common name, but "Sprenger's asparagus" might be acceptable, after Carl Ludwig Sprenger), "lace fern", etc.

Reply: Agreed. The common name “Sprenger’s asparagus” is adopted.

Comment 8: The authors are urged to meticulously check the text once again for proper English usage while following the comments stated above. I still strongly encourage the authors to have the MS proofread by a native English speaker or professional editing agency.

Reply: Agreed. We employed a professional editing agency to proofread the manuscript.

Thank you very much for your kindly reviews and consideration.

Yours sincerely,

Dr. David TW LAU

Curator of the Shiu-ying Hu Herbarium

School of Life Sciences

The Chinese University of Hong Kong

Submitted filename: Rebuttal Letter.docx

Click here for additional data file.

21 Mar 2022

Complete chloroplast genomes of Asparagus aethiopicus L., A. densiflorus (Kunth) Jessop ‘Myers’, and A. cochinchinensis (Lour.) Merr.: Comparative and phylogenetic analysis with congenerics

PONE-D-21-33675R2

Dear Dr. LAU,

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Academic Editor

PLOS ONE

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Reviewers' comments:

31 Mar 2022

PONE-D-21-33675R2

Complete chloroplast genomes of Asparagus aethiopicus L., A. densiflorus (Kunth) Jessop ‘Myers’, and A. cochinchinensis (Lour.) Merr.: Comparative and phylogenetic analysis with congenerics

Dear Dr. LAU:

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