Amana hejiaqingii (Liliaceae), a New Species from the Dabie Mountains, China

: In this paper, a new species from Central China, Amana hejiaqingii (Liliaceae), is described and illustrated. It resembles A. anhuiensis and A. tianmuensis but differs from A. anhuiensis as it has one white vein on its lower leaf and yellow anthers. It also differs from A. tianmuensis by possessing solitary pink ﬂowers with longer and wider tepals. The principal coordinates analysis separated the three species based on morphological data. Cytological observation showed that A. hejiaqingii is diploid (2 n = 2 x = 24). Molecular phylogenetic analyses further supported its species delimitation.


Introduction
The genus Amana Honda (1935: 20; Liliaceae) is characterized by 2−3(-4) opposite or verticillate bracts at the upper part of flowering stem and a distinct beak of the fruit, which separates it from Tulipa L. (Tan et al., 2005 [1]). Amana consists of nine perennial herbaceous species and is endemic to East Asia (Ohwi and Kitagawa 1992 [2]; Chen and Mordak 2000 [3]; Shen 2001 [4]; Tan et al., 2007 [5]; Tan et al., 2008 [6]; Han et al., 2014 [7]; Li et al., 2017 [8]; Wang et al., 2019 [9]; Wang et al., 2022 [10]). Amana belongs to spring ephemerals, blooming in early spring. The above-ground tissues die after bearing fruit and become dormant underground (Struik, 1965 [11]). This genus mainly occurs in temperate deciduous or subtropical evergreen broadleaved/mixed forests. Amana is diploid, and its basic chromosome number is x = 12, except for two ploidy levels in A. edulis: diploid in northern populations and tetraploid in southern populations (Wang et al., 2022 [10]; Wu et al., 2022 [12]). This genus is confirmed to be in a monophyletic group and sister clade with Erythronium (Li et al., 2017 [8]). A suspected new species was found during fieldwork from 2016 to 2022 in the Dabie Mountains, China. Our morphological examination and phylogenetic analyses further verified that it is a new species. Therefore, we describe and illustrate it here.

Sampling, Sequencing and Assembly
The new species was investigated in eight localities in the Dabie Mountains bordering the Henan and Hubei provinces from 2016 to 2022 ( Figure 1). Genomic DNA was extracted from silica-gel-dried leaves with modified CTAB reagent (Plant DNAzol, Shanghai, China), according to the manufacturer's protocol. Libraries were pooled and sequenced with 150 bp paired-end reads using the Illumina HiSeq X10 platform at China National GeneBank (CNGB, Shenzhen, China). Raw reads were assembled into plastome sequences by GetOrganelle (Jin et al., 2020 [13]) with default settings. The newly generated sequences can be accessed on GenBank (Table 1). Voucher specimens were deposited at the Herbarium of Zhejiang University (HZU, acronyms according to Thiers et al., 2016 [14]).

Cytological Analysis
Actively growing droppers were collected in the field for chromosome counting. The dropper tip materials were pretreated in 0.1% colchicine for 4.5 h and then fixed in Carnoy's Fluid (3 absolute alcohol: 1 glacial acetic acid, v/v) for 12-24 h. Afterwards, they were converted into anhydrous ethanol and stored at −20 • C in the refrigerator for further treatment. The fixed dropper tips were bathed in 37 • C enzyme solution (a mixture of 2% cellulase and pectinase (2:1)) for 1 h. After hydrolysis, the materials were rinsed with distilled water several times. Finally, stained tips with Carbol fuchsin and chromosome compression tablets were made for observation. Photos were captured by a SOPTOP DMCX40 microscope (SOPTOP, Ningbo, China). Karyotype formula was based on the measurement of mitotic metaphase chromosomes photos. The degree of karyotype asymmetry (As.K%, Arano 1963), karyotypic symmetry division category (KA Type, Stebbins 1971 [16]), mean centromeric asymmetry index (MCA), coefficient of variation of chromosome length (CVCL), coefficient of variation (CVCI), and total haploid karyotype length (THL) were evaluated (Paszko 2006 [26]) with GTR + I + G substitution model. The Markov chain Monte Carlo (MCMC) algorithm was run with two independent chains and default priors for 10,000,000 generations. Trees were sampled every 1000 generations.

Morphological Comparisons
Amana hejiaqingii (Figures 2 and 3) is similar to A. anhuiensis (Figure 4) and A. tianmuensis (Wang et al., 2022 [10]) in bulbs, leaves and flowers. However, it differs from A. anhuiensis as it has lower leaves with a grayish-white midvein (vs. with more than one white vein), and yellow anthers (vs. light-purple) at maturity. Additionally, the widest part of the lower leaf of A. hejiaqingii is usually in the 2/8-3/8 position (upper part) of the leaf, while A. anhuiensis is usually at 1/8 position (top part) of the leaf. Meanwhile, it differs from A. tianmuensis as it has a solitary pink flower (vs. mostly solitary, sometimes two, white). Moreover, the outer and inner tepals of A. hejiaqingii were mostly longer and wider than A. tianmuensis (Table 2).

Principal Coordinates Analysis
A principal coordinates analysis showed three separated clusters ( Figure 5). The first principal coordinates (PCoA1) accounted for 31.77%, and the second principal coordinates (PCoA2) accounted for 13.34% of the total variance.

Principal Coordinates Analysis
A principal coordinates analysis showed three separated clusters ( Figure 5). The first principal coordinates (PCoA1) accounted for 31.77%, and the second principal coordinates (PCoA2) accounted for 13.34% of the total variance.

Cytology Observation
A total of six populations were observed, and the numbers of metaphase chromosomes were 2n = 2x = 24 = 3 m + 11 sm + 10 st. The length of chromosome varied from 5.75 to 10.05 μm. The ratio of the longest to shortest chromosome was 2.13. The total haploid length of the chromosome set (THL) was 99.10. The coefficient of variation of centromeric index (CVCI) was 18.83. The coefficient of variation of chromosome length (CVCL) was 11.60. The mean centromeric asymmetry (MCA) was 50.42. As.K% was 75.50. KA belonged to Stebbins's-3B ( Figure 6).

Molecular Phylogeny
The complete plastome sequence length of Amana hejiaqingii was 151,513-151,516 bp (large single copy (LSC) 81,862-81,864 bp, small single copy (SSC) 17,107-17,112 bp and inverted repeat (IR) 26,271 bp with 36.7%, 34.6%, 30.1% and 42% GC content; Figure S1). The total alignment was 73,033 bp with 3065 variable sites. The phylogenetic tree based on plastid genes (Figure 7) revealed that A. hejiaqingii was distantly related to A. tianmuensis with robust supports (BIPP = 1.0, MLBS = 100). This finding is congruent with our ongoing phylotranscriptomic study on the genus Amana.  Distribution and habitat: Up to now, Amana hejiaqingii is found in eight localities across 233.7 km in the Dabie Mountains bordering Henan and Hubei provinces. It grows in moist deciduous broad-leaf forests on mountain slopes at elevations of 70-530 m. In future investigations, we think that more populations will be found in the area in and around these localities.

Amana hejiaqingii
Etymology: The specific epithet is named in memory of Professor Jia-qing He, a Chinese botanist who was dedicated to plant investigation in the Dabie Mountains. He walked about 12,684 km and collected nearly 10,000 specimens during a 255-day fieldtrip in the Dabie Mountains, becoming the first person ever to make a comprehensive wild plant investigation of the Dabie Mountains.
Conservation Status: Amana hejiaqingii is distributed in several places in Henan and Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/taxonomy2030022/s1, Figure S1: The complete plastome circle diagram of Amana hejiaqingii; Table S1: Morphological data of Amana anhuiensis, A. hejiaqingii and A. tianmuensis.