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.

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.
The xeromorphic adaptations of Asparagus species are beneficial to the establishment of "Xeroscaping" [58][59][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][62][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][66][67] and the USA [60,64].
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].

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.
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.
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][88][89], is important in studying phylogeny and resolving taxonomical problems [89][90][91][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.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.

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).
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 Nova-Seq 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.

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.

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.
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.
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 proteincoding (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

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.

Comparative genome analysis
The IR boundaries of the seven genomes were relatively conserved, with some minor variations (contractions and deletions) (Fig 8).
B Located in the region 9167-9994 bp; for NC 047458, trnG-UCC, at 36924-36994 bp, had no intron.In the LSC/IR B 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 IR B also exhibited variation, with lengths  The ycf1 pseudogenes was retained in the border IR B /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 IR B /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/IR A border for all species except A. officinalis, in which an IR A 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/IR A junction, unlike in the other species.
At the IR A /LSC border, rps19 (137-279 bp long) in IR A 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.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

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.
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].

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.

Fig 1 .
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.https://doi.org/10.1371/journal.pone.0266376.g001 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 (Fig5), with most (ca.two-thirds) found in LSC regions and one-fifth and one-tenth, respectively, found in SSC and IR regions.

Fig 2 .
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.https://doi.org/10.1371/journal.pone.0266376.g002

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.C, pseudogene.Not to scale.https://doi.org/10.1371/journal.pone.0266376.g008