Over 30 Years of Misidentification: A New Nothospecies Lycoris × jinzheniae (Amaryllidaceae) in Eastern China, Based on Molecular, Morphological, and Karyotypic Evidence

Based on the complete chloroplast genome, morphology, and karyotype evidence, we identified a new nothospecies, Lycoris × jinzheniae S.Y. Zhang, P.C. Zhang & J.W. Shao, in eastern China. This new nothospecies has been inappropriately named Lycoris × albiflora in the previous literature for more than 30 years. However, the new nothospecies resulted from the hybridization of L. sprengeri and L. chinensis and had the following characteristics: the karyotype was 2n = 19 = 3V + 16I, the leaves emerged in the spring, the ratio of filament to corolla length was approximately 1.2, tepals were slightly undulated and curved, and it was distributed throughout eastern China. These characteristics are quite different from those of L. × albiflora; thus, in this study, we named it and provided a detailed morphological description and diagnosis.


Introduction
Lycoris Herb. (Amaryllidaceae), first described as L. aurea (L'Her.) Herb., is only naturally distributed in East Asia [1]. Possessing large, beautiful, and colorful flowers, this genus of plants has great ornamental value and potential for horticultural applications. Possibly due to their highly consistent living habitats, some different species of Lycoris may overlap in distribution, resulting in extensive interspecific hybridization events. At present, there are 27 legitimate species names in this genus, but only 9 entities are considered original fertile diploid species, i.e., L. chinensis, L. sprengeri, L. radiata, L. longituba, L. aurea, L. traubii, L. sanguinea, L. wulingensis, and L. tsinlingensis. The remaining species have been proven to be or possibly to be nothospecies [1][2][3][4][5][6][7][8][9][10]. Although most F1 Lycoris hybrids have little ability to reproduce sexually due to disorders of their chromosome pairing during meiosis, they can vegetatively reproduce via bulb segments and can form a considerable number of clonal clusters in the wild [1, 8,[11][12][13][14]. Moreover, these nothospecies have a greater variety of floral colors, so they usually have higher horticultural utilization value. Therefore, accurate and legitimate naming is helpful for follow-up in-depth research and application [1, 2,6,8,15].
In recent years, in the process of collecting germplasm resources and performing systematic research on the genus Lycoris, we found that L. sprengeri (leaves emerging in the spring, pink-blue flowers, and endemic to China) and L. chinensis (leaves emerging in the spring, yellow flowers, and distributed in China and South Korea) sometimes overlap in distribution, and a special type of infertile species with white flowers and leaves emerging in the spring can be found in their overlapping populations (Figure 1). These plants were formerly recognized as Lycoris × albiflora Koidz. in Chinese taxonomic and horticultural studies, and subsequent molecular systematic studies adhered to this taxonomic opinion [4,5,16]. However, through a literature review and specimen examination, we found that L. × albiflora was named after a type collected in Japan with white tepals and leaves emerging in the autumn [1,17]. After researching the karyotype and molecular phylogeny, L. × albiflora is now considered to be the nothospecies descendant of L. traubii (leaves emerging in the autumn, yellow flowers, and distributed in Japan and the Taiwan Province of China) and L. radiata (leaves emerging in the autumn, red flowers, and widely distributed in East Asia), and is unlikely to be distributed in mainland China [1,3,11,12]. Therefore, the previous identification of such special species (with white flowers and leaves emerging in the spring) may be incorrect. In the 1980s, Ms. Lin Jin-zhen, a pioneer in the cross-breeding of Lycoris in China , is different from L. × albiflora in the origin of hybridization. Furthermore, there were significant differences between the two hybrids in terms of leaf emergence stage, leaf shape, tepal shape, and filament length (Table 1). Therefore, these special infertile species are a new nothospecies that has not been officially described. In honor of Ms. Lin's contribution to the hybrid breeding of Lycoris, we hereby name the nothospecies as Lycoris × jinzheniae S.Y. Zhang, P.C. Zhang & J.W. Shao and describe it.

Morphological Characteristics and Fertility
Principal component analysis of five vegetative characters, five propagule characters, and their combination showed that there was obvious morphological differentiation between L. chinensis, L. sprengeri and L. × jinzheniae (including 30 artificial hybridization plants), and L. × jinzheniae morphologically fell between the first two. The wild populations and the artificial hybrid population were consistent based on the 95% confidence interval, suggesting that they are morphologically similar (Figure 4a). In six quantitative characteristics, there was no obvious difference in morphology among three wild populations and one artificially hybridized population of L. × jinzheniae ( Figure 4b). However, the morphological differences between L. × jinzheniae, L. chinensis, and L. sprengeri were conspicuous and significant. In general, the morphological indicators of L. × jinzheniae were almost between the latter two species (Figure 4b).
To compare fertility more intuitively, we cross-sectioned the capsules of the three Lycoris species at the same time and found that both L. sprengeri and L. chinensis had many developing seeds, but L. × jinzheniae could not bear any filled seeds ( Figure 5G). This was not an accidental phenomenon but occurred in all populations. Diagnosis. Lycoris × jinzheniae is similar to L. × albiflora in the color of flowers, but it can be distinguished from the latter by leaves emerging in spring, leaf apex blunt, a ratio of filament to corolla length of 1.2:1, and slightly undulate and recurved tepal. There are also very obvious morphological differences between L. × jinzheniae and its parents, as shown in Table 1 and Figure 5.  Table 2, and the circles represent the 95% confidence intervals. (b) Comparison of and variation in 6 morphological characters. The codes of the abscissa correspond to the populations in Table 2. In the boxplot, the horizontal line shows the median, while the bottom and top of the box show the first and third quartiles. Boxplots marked with different letters differ significantly (post hoc test, p < 0.05). Description. Perennial herb. Bulbs nearly oval or fusiform, 3-4 cm in diameter, and epidermis brown with fine lines. Leaves linear, often 6-10, blunt apex, 40-55 cm-long, 1.2-2 mm-wide, covered with little white powder; upper surface dark green, midvein slightly pale; bottom surface light green with a raised midrib. Inflorescence scapose, 40-60 cm-high, green or reddish-brown; two bracts, lanceolate, 3-4.5 cm-long, 1-1.5 mm-wide; flowers 5-8 per umbels, pedicels 3-5 cm-long, diameter 4-7 mm; buds rose-red; flowers usually white, sometimes pale pink or yellowish; tepals oblanceolate, 5-6.5 cm-long, approximately 8-12 mm-wide, apex slightly reversed and slightly undulated; floral tube 1-2 cm-long. Filament 7-9 cm-long, white, slightly longer than tepals, anther yellow, length of 5 mm; pistil length 7.5-8.5 cm, apex purplish-red, lower part white. Ovary 6-8 mm in diameter, spherical, and green. Capsules three-lobed, green, infertile.
Phenology. Flowering from August to early September. Leaves emerge in mid-February and wither in late May. Distribution and habitat. Lycoris × jinzheniae is currently found in the Anhui, Jiangsu, and Zhejiang provinces of China and grows in moist hillside forests ( Figure 6). It always grows in the overlapping distribution area of L. chinensis and L. sprengeri. Occasionally, residents living around its range dig and cultivate them near their homes as horticultural plants.
Vernacular name. 秀丽石蒜 (xiù lì shí suàn). Reproduction. It can only reproduce asexually by bulb splitting. Typically, one mature bulb can turn into three mature bulbs after 2 years.
Conservation status. Although the number of Lycoris × jinzheniae in the wild is very small, as an F1 hybrid that cannot reproduce sexually, it has no protective significance, and a large number of individual plants can be obtained through artificial hybridization. We propose classifying its conservation status as least concern (LC) according to the IUCN Red List criteria [19].

Discussion
In angiosperms, the chloroplast genome is normally maternally inherited [1, 4,5]. In the phylogenetic tree that we constructed based on the chloroplast genome, Lycoris× albiflora clustered with L. radiata and constituted Clade 2, with a robust bootstrap (BS = 100, PP = 1.00, Figure 3), which is consistent with previous results [3]. This supports the previous viewpoint that L. radiata is the maternal parent of L. × albiflora, which was concluded from morphological characteristics and distribution patterns [1]. However, the wild plants of L. × jinzheniae were nested with the offspring produced by artificial hybridization between L. sprengeri (female parent) and L. chinensis (male parent), forming Clade 3 together with L. sprengeri. This was a sister group with L. sanguinea (Clade 4) (Figure 2), suggesting that the new hybrid was relatively distant from L. × albiflora in phylogenetic relationship and that L. sprengeri is the possible maternal parent of L. × jinzheniae.
Through the observation of multiple populations and individuals, we found that the karyotype of the new hybrid (including wild populations and artificial hybrid populations) was an odd 19 and was stable (2n = 19 = 3V + 16I), which is quite different from that of L. × albiflora (2n = 17/18 = 5V + 12I/6V + 12I) [1,12]. In the field, L. × jinzheniae concomitantly occur with L. sprengeri and L. chinensis, especially when the populations of these two species are less than 100 m apart; they can always be found within or near the L. sprengeri populations. Therefore, combining the distribution, karyotype, and molecular evidence, we can deduce that L. sprengeri (2n = 22 = 22I) is the maternal parent of the new nothospecies, and the anther accompanying plant L. chinensis (2n = 16 = 6V + 10I) is its male parent. These two wild diploid parents each provide a set of chromosomes that can constitute the karyotype of the new hybrid. We also note that the karyotypes of L. longituba and L. aurea are (or can be) 2n = 6V + 10I [1, 11,12]. The karyotype of L. aurea is highly variable, and the 6V+10I type is only found in Southwest China and Hainan Province [1]. The wild population of L. longituba is scarce and is now only distributed in the hills along the Yangtze River in parts of the Jiangsu and Anhui provinces. Thus, we have not found that L. longituba or L. aurea are distributed near or overlapped with L. sprengeri thus far, which means it is very unlikely that these two species participated in the hybridization of L. × jinzheniae.
Interestingly, all the evidence we found thus far indicates that L. × jinzheniae is produced by L. sprengeri was relatively high (approximately 40%), and a L. × jinzheniae germplasm nursery was successfully cultivated. Although some seeds were obtained by reverse hybridization, subsequent descriptions did not mention that these seeds can successfully grow seedlings. Under natural conditions, we found overlapping areas (nearly less than 100 m) of both L. chinensis and L. sprengeri in seven areas ( Figure 6). L. × jinzheniae appeared in every such area, ranging from several clusters to more than 50 clusters, and they were all, without exception, closer to or on the edge of L. sprengeri populations. No individual plants closer to populations of L. chinensis have been found thus far. Furthermore, some other studies have shown that L. sprengeri, as a female parent, participates and forms several hybrid offsprings, but a hybrid formed from a male L. sprengeri parent has not yet been reported [1,4,5,20]. At present, there are similar reports of unidirectional hybridization in Ligularia, Sonneratia, Rhizophora, and other taxa, and the asymmetry of bidirectional hybridization is also prevalent in angiosperms [21][22][23].
Research suggests that parental rarity in the wild or postmating isolation may be some of the factors contributing to hybrid asymmetry [22]. The specific hybridization participation mechanism by which L. sprengeri can only be used as a female parent but not as a male parent is still unclear.
Morphologically, the 30 individual plants from artificial hybridization and the 90 individual plants from three wild populations were the same in terms of vegetative and propagule characteristics (Figures 5 and 6). This suggests that their morphological characteristics were relatively similar and consistent, although L. × jinzheniae can be produced by multiple independent crosses (including artificial crosses). However, this new nothospecies can be distinguished from L.× albiflora by leaf emerging time (spring vs. autumn), leaf apex shape (blunt vs. acuminate), ratio of filament to corolla length (1.2:1 vs. 2:1), and tepal undulation and recurve degree (slight vs. strong).
From the above, L.× jinzheniae was quite different from L.× albiflora in origin, morphology, karyotype, and natural distribution (Figures 2-6, Table 1). Here, we named it and provided a detailed morphological description and diagnosis.

Plant Material
From 2016 to 2021, materials were collected in the Eastern China field and Hangzhou Botanical Garden (Table 2 and Figure 6). These bulbs were cultivated in a homogenous garden located in Fengyang County, Chuzhou City, Anhui Province, for morphological and karyotype observation.
Based on pre-experiments and previous studies, we selected L. × jinzheniae and its putative parents (L. sprengeri and L. chinensis), L. × albiflora and its putative parents, L. radiata, and L. sanguinea (infertile original diploid and the sister groups of L. sprengeri) to construct a phylogenetic tree. Information on the newly generated complete chloroplast genome of Lycoris is shown in Table 3. The chloroplast genome sequences of some other species were downloaded from NCBI ( Figure 2). Table 3. Information for newly generated complete chloroplast genome of Lycoris.

Chloroplast Genome Acquisition and Phylogenetic Analysis
Sequencing samples were obtained from leaves, which were dried in silica gel and collected from wild populations or homogenous garden. DNA extraction used a modified cetyltrime thylammonium bromide (CTAB) extraction protocol [24] with Qubit TM dsDNA HS Assay Kit (Invitrogen, Waltham, MA, USA)-mediated cleaning [25]. After polymerase chain reaction (PCR) and agarose gel electrophoresis, DNA quality control was ensured using a NanoDrop 1000 Spectrophotometer. Next-generation sequencing (NGS), which used Illumina HiSeq 6000 and FastQC, was outsourced to The Germplasm Bank of Wild Species in Southwest China (China, Kunming). We employed Getorganelle v1.7.1 and 3G raw data from the previous step to complete the assembly of the chloroplast genome [26]. The assembled chloroplast genome was annotated by PGA [27]. This study was performed on the 12 newly reported complete chloroplast genomes and 7 complete chloroplast genomes from NCBI. Narcissus poeticus was selected as the outgroup (Table 2, Figure 2) [20,[28][29][30][31].
The sequences of 20 chloroplast genes shared by all plastomes were aligned using MACSE v2 in PhyloSuite [32,33]. The phylogenetic relationship, which included maximum likelihood (ML) and Bayesian inference (BI) methods, was implemented in IQtree, and MrBayes with the best-fit model of DNA substitution estimated by ModelFinder [34][35][36]. ML analysis was conducted using the GTR+G+I model with 1000 bootstrap replicates, and Bayesian analysis was constructed using MrBayes with 8 independent chains for 1,000,000 generations and sampling every 1000 generations [37].

Karyotype Observation
The actively dividing root tips were obtained by burying the bulb in moist sand. Root tips were treated with 0.1% colchicine for 12 h and then treated with fixative solution (glacial acetic acid: absolute ethanol ratio = 1:3) for 24 h in the dark. The samples were washed with water, soaked in 1 mol/L hydrochloric acid, and placed in a water bath at 60 • C for 15 min [8,[10][11][12]38]. After washing with water, the root tips were stained with modified phenol fuchsin for 15 min, and the karyotypes were observed by pressing temporary mounts. Twenty bulbs of each species in each population were selected to verify the accuracy, photographed with an electronic eyepiece, and processed using Photoshop.
Some scholars divide the chromosomes of Lycoris into three categories based on shape, M, T, and A, but based on our karyotype observation findings, the A-type and T-type chromosomes are indistinguishable, and there is also morphological variation or centering between them [11,12,19,39]. Therefore, we only distinguish between V-shaped (meta or submetacentric) and I-shaped (acrocentric) karyotypes [19,39].

Morphological Statistical Analysis
Based on the materials in Table 1, 5 vegetative characters (leaf length, leaf width, bulb diameter, bulb weight, and leaf twist angle) and 5 propagule characters (tepal length, floral tube length, filament length, symmetry degree of flower, and undulate degree of tepal) of the species were used as units (artificial hybridization of Lycoris × jinzheniae is listed separately). SPSS ver. 19.0 was used to standardize and extract the principal components and test the cumulative contribution rate of the first two principal components. Then, the devtools and ggbiplot software packages of R v3.6.0 were used. Principal component analysis was performed on all traits, vegetative traits, and propagule traits [37].
Three vegetative characters (leaf length, leaf width, and bulb diameter) and three propagule characters (tepal length, floral tube length, and filament length) were selected, and SPSS ver. 19.0 was used to make boxplots to compare morphological differences and test for significance [10].

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The molecular data that support the findings of this study are openly available in GenBank (see Table 3).