﻿Impatiensyingjingensis (Balsaminaceae), a new species from Sichuan, China

﻿Abstract This study describes Impatiensyingjingensis X.Q. Song, B.N. Song & Biao Yang, sp. nov., a new species collected from the Yingjing area of the Giant Panda National Park. This new species is distributed at an altitude of 1400–2100 m, with a plant height of 30–130 cm. The flowers are purple-red or light purple red, with 3–9 flowers on each inflorescence and the dorsal auricle of the lateral united petals is thread-like and about 2 cm long, differing significantly from other species of Impatiens. Furthermore, molecular data, as well as micro-morphological evidence under SEM (of pollens), also support the establishment of the new species.


Introduction
The genus Impatiens L. is the largest genus in the family Balsaminaceae, with more than 1,000 species (Grey-Wilson 1980;Yu et al. 2016;Song et al. 2021;Yuan et al. 2022).It predominantly inhabits the tropical and subtropical mountainous regions of the Eurasian continent and tropical Africa, with a minor presence in temperate zones of Eurasia and North America (Ren 2022).China is one of the five global hotspots for Impatiens diversity (tropical Africa, Madagascar, southern India and Sri Lanka, the eastern Himalayas and southeast Asia), accounting for more than 360 species (Chen et al. 2023).Most species are concentrated in southwest China, specifically in Yunnan, Sichuan, Guangxi, Guizhou and Xizang, showcasing narrow distributions and unique characteristics (Chen 2001;Yuan et al. 2022;Xiang et al. 2023).
In recent years, several new species and new records of this genus, including I. longiaristata, I. tripetala, I. wawuensis, I. longlinensis, I. wutaishanensis, I. longshanensis, I. lihengiana and I. cavaleriei have been discovered in southwest China (Ding et al. 2016;Xue et al. 2020;Liao et al. 2021;Peng et al. 2021;Song et al. 2021;Huang et al. 2023).These findings underscore the presence of numerous potential new species awaiting discovery within this diverse genus.
The Yingjing area of the Giant Panda National Park (GPNP) is located in Yingjing County, Sichuan Province, spanning a total area of 836 km 2 .It is situated in the mountainous area transitioning from the Sichuan Basin to the Qinghai-Tibet Plateau.The unique geographical position, varying altitudes and distinct climate conditions have given rise to a diverse array of flora and fauna, fostering complex ecological communities within the vicinity (Shao et al. 2022).During a survey in the Yingjing area of the GPNP from August to October of 2021, an intriguing Impatiens species was discovered thriving at the forest edge and in valleys at altitudes ranging from 1400 to 2100 m.It was similar to I. lateristachys in overall morphology, but after consulting a large number of specimens and investigating the morphological and micromorphological characteristics, significant differences were observed in flower, inflorescences, dorsal petal, lower petal, lateral united petals and lower sepal.Based on the combination of detailed field observation, morphological, micromorphological and molecular data, we confirmed that it represents a previously undescribed species.The primary objective of this research is to present a detailed description of this newly-discovered species.

Plant sampling and morphological observation
Fresh plant material of the unidentified Impatiens species was collected from the Mount Yunwu area of Yingjing County, within the GPNP.The location was at Shaidianping (29°33.98'N,102°45.00'E,1624 m alt.).The collected specimens were deposited at the Herbarium of Sichuan University (Chengdu, China).Following the guidelines of Flora Reipublicae Popularis Sinica, Balsaminaceae, Tomus 47 (part 2) (Chen 2001) and Balsaminaceae of China (Yu 2012), various morphological characteristics of this species, such as plant height, leaf size and shape, inflorescence type, flower colour, pedicel length, petal width and stamen length, were meticulously observed and measured in the field.A comparative analysis with other Impatiens species was conducted.Additionally, colour photographs of the plants were taken and the inflorescences were dissected indoors.Furthermore, scanning electron microscopy was employed to observe the pollen of the plant.

DNA extraction and sequencing
The fresh adult basal leaves of this species were collected in the field and then dried immediately with silica gel for the next step.Voucher specimens were stored at the Herbarium of Sichuan University (Chengdu, China) under deposition number 202108001.Firstly, we extracted the total genomic DNA from silica-dried leaves with a plant genomic DNA kit (Cwbio Biosciences, Beijing, China) referring to the manufacturer's protocols.Then, the quality and quantity of extracted DNA were tested using 1% agarose gel electrophoresis and high-quality DNA was sequenced on Illumina NovaSeq platform at Personalbio (Shanghai, China) according to the standard Illumina sequencing protocols (Shendure and Ji 2008), with the sequence depth 6G.Paired-end 150 reads were obtained from libraries with an insert size of 300 bp.Finally, the software fastP v.0.15.0 (-n 10 and -q 15) (Chen et al. 2018) was used to filter the low-quality reads and gain high-quality reads.
In addition, total genomic DNA was also employed to amplify the Internal Transcribed Spacer (ITS) regions and the 30 µl amplification system was performed, which included 2 µl extracted total DNA, 10 µl ddH 2 O, 15 µl Taq Master-Mix (CWBio, Beijing, China), 1.5 µl of 10 pmol µl −1 forward primers ITS4 (5'-TCC TCCGCT TAT TGATAT GC-3') and 1.5 µl of 10 pmol µl −1 reverse primers ITS5 (5'-GGA AGTAAA AGTCGT AAC AAG G-3' (White et al. 1990).By executing the following programme: initial denaturation for 3 min at 94 °C, followed by 30 cycles of 45 s at 94 °C, 70 s at 54 °C and 90 s at 72 °C and then a final extension of 10 min at 72 °C, we obtained the amplified PCR products.Finally, we used a 1.5% (w/v) agarose TAE gel to examine all PCR products which were sent to Sangon (Shanghai, China) for sequencing.The software Geneious v.9.0.2 (Kearse et al. 2012) was used to edit the newly-sequenced ITS sequence and consensus sequences were gained.

Plastome assembly and annotation
The plastome of this species was de novo assembled by NOVOPlasty v.2.6.2 (Dierckxsens et al. 2017) with the default parameters and rbcL sequence extracted from the plastome of I. balsamina (MZ902354) as the seed.The Plastid Genome Annotator (PGA) (Qu et al. 2019) was applied to annotate the plastome of this new species, setting the plastome of I. balsamina (MZ902354) as reference.Then, we manually corrected the start and stop codons and intron positions in Geneious v.9.0.2 (Kearse et al. 2012) based on the plastomes of congeneric species.The circular plastome map of this new species was drawn by the online Organellar Genome DRAW tool (OGDRAW) (Lohse et al. 2007).Finally, the newly-generated ITS sequence and plastome of this Impatiens species were submitted to the NCBI under accession numbers OR982404-OR982405 and OR978441, respectively.

Phylogenetic analysis
To investigate the phylogenetic position of this species, 62 complete plastome data and 62 ITS sequences were employed to reconstruct the phylogenetic trees, and Hydrocera triflora was selected as outgroup (Suppl material 1).The plastome data and ITS sequences were straightway aligned with MAFFT v.7.221 (Katoh and Standley 2013) to generate the matrix, respectively.The matrixes were subjected to Maximum Likelihood (ML) and Bayesian Inference (BI) analyses.The ML analyses were inferred by adopting RAxML version 8.2.11 (Stamatakis 2014) with the GTRGAMMA model and 1000 replicates as suggested by the RAxML manual.The BI analyses were performed by using MrBayes v.3.2.7 (Ronquist et al. 2012) with the best-fit substitution model (GTR+I+G) for plastome data and (GTR+G) for ITS sequence determined by ModelTest v.3.7 (Posada and Crandall 1998)  1,000,000 generations, sampling every 500 generations.The run finished when the average standard deviation of split frequencies was below 0.01.The first 25% of samples were discarded as burn-in and the remaining trees were maintained to yield the consensus tree.The phylogenetic trees of two analyses were visualised and edited by Interactive Tree of Life (iTOL) (Letunic and Bork 2019) and the nodes under 50% bootstrap support were collapsed.

Morphological characteristics
We investigated carefully the morphology of this new species and observed that its distinctive morphological features are its flower, inflorescences, dorsal petal, lower petal, lateral united petals and lower sepal, such as it has purple-red or light purple-red flowers with 7-12 pairs of lateral veins.The inflorescences are axillary, slightly shorter than the leaf length or approximately equal to the leaf blade in length, 3-9 flowered arranged in a one-sided raceme on the inflorescence axis.The dorsal petal is orbicular approximately 15 mm in diameter, with a concave apex and obtuse tip, the mid-vein on the back thickening with narrow wings, wings 2-angled.The lower petal is gradually narrowing at the base into a sickle-shaped spur approximately 2 cm long; lateral united petals 2-lobed, auricle linear approximately 2 cm, elongate, inserted into spur (Fig. 1, Table 1).

Micromorphological observations of pollen
In further investigation of this species, we also observed its micromorphology of pollen grains under the scanning electron microscope.The results showed that the pollen grains of this species had a unique micromorphology, characterised by single-grain pollen with a flattened spherical shape.Its polar view was capsule-like, irregular and reticulated (Fig. 2).

The structure and features of plastome
This species exhibited a typical quadripartite structure (Fig. 3), with a length of 151,642 bp, including a large single copy region (LSC: 82,588 bp), a small single copy region (SSC: 17,628 bp) and a pair of inverted repeat regions (IRs: 25,713 bp).The total GC content was 36.80%, with the GC content of the IRs regions being 43.1%, significantly higher than that of the LSC region (34.6%) and the SSC region (29.3%).The genome encoded 114 unique genes, including 80 protein-coding genes, 30 tRNA genes and four ribosomal RNA genes (Table 1).In addition, these unique genes had four categories: Self-replication, Genes for photosynthesis, other genes and Genes of unknown function (Table 2).

Phylogenetic analysis
We employed 62 complete plastomes and 62 ITS sequences to reconstruct the phylogeny of this new species.Although the plastome data and ITS sequence yielded incongruent tree topologies, both strongly supported the fact that this new species clustered with other Impatiens members, belonging to the genus Impatiens.In the plastome-based and ITS-based phylogenetic tree, the results of the Maximum Likelihood (ML) and Bayesian Inference (BI) analyses generated well-resolved topologies and the topologies were highly identical as expected (Fig. 4).It was clearly observed that this new species formed a clade with I. piufanensis in the plastome-based tree (BS = 100, PP = 1.00) (Fig. 4A).However, in the ITS-based phylogenetic tree, this new species was clustered with I. lateristachys (BS = 100, PP = 1.00) (Fig. 4B).In addition, both phylogenetic trees also indicated that this new species was clearly distant from other related Impatiens members that were morphologically similar to it, including I. faberi, I. drepanophora, I. siculifer and I. imbecilla (Fig. 4).

Discussion
China is recognized as a significant hotspot for the distribution of Impatiens.It offers a variety of habitat conditions for the genus Impatiens and breeds a variety of Impatiens resources, including regional endemics and Chinese-specific varieties.Due to the complexities associated with collection and identification, there is a lack of comprehensive and in-depth floristic surveys for the genus Impatiens in China.As a result, the resource status and phylogenetic relationships of the genus Impatiens in China, especially in key and vulnerable areas, are still unclear.Therefore, strengthening the floristic surveys and specimen collection of the Impatiens genus, especially in these critical and vulnerable regions, is an important task for the current research in taxonomy and floristics.Through the investigation and research of the genus Impatiens in the Yingjing area, it has been found that the distribution points and population numbers of I. yingjingensis are relatively small, mainly being found in the valley, forest edge and roadsides at altitudes of 1400-2100 m in the areas of Mount Yunwu.Surveys and protection have not received adequate attention and it is essential to strengthen the investigation of the local resources of I. yingjingensis and conduct systematic research on the species diversity of the genus Impatiens in Yingjing County.
Through field observation and literature review, I. yingjingensis was found to bear the closest morphological resemblance to I. lateristachys, I. drepanophora, I. siculifer, I. imbecilla and I. faberi.However, their distinct differences were noted.
The key features that distinguish I. yingjingensis and I. lateristachys are lower sepal and lateral sepals.Lower sepal in I. yingjingensis is sickle-shaped, the leaves are boat-shaped and the mouth is flat, while the lower sepal in I. lateristachys is angular with an oblique blunt mouth.The lateral sepals in I. yingjingensis are ovate with a pointed apex, while the ones in I. lateristachys are diamond-shaped with a truncated base and three veins, about 2 mm long.The most notable feature that distinguishes I. yingjingensis from I. drepanophora and I. siculifer is flower colour.I. yingjingensis has purple-red or light purplish-red flowers, whereas the flowers in I. drepanophora and I. siculifer are yellow.In addition, I. yingjingensis can be easily distinguished from I. imbecilla by the bracts.Bracts in I. yingjingensis are lanceolate, while I. imbecilla has ovate-lanceolate bracts.Additionally, the dorsal petal between I. yingjingensis and I. imbecilla is also different.,The dorsal petal of I. yingjingensis is 2-angled, orbicular with a concave apex and the midvein on the back thickening with narrow wings, whereas two shallow clefts at the top, the mid-rib on the back thickened and a cockscomb-like projection of dorsal

Translational initiation factor infA
Genes for photosynthesis Subunits of photosystem I psaA, psaB, psaC, psaI,psaJ, ycf3**, ycf4 Subunits of photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT petal are observed in I. imbecilla.I. yingjingensis can be clearly distinguished from I. faberi by their lateral sepals.The ovate lateral sepals with a pointed apex are detected in I. yingjingensis, but green, egg-shaped and a thickened mid-rib of lateral sepals with 3-5 veins are found in I. faberi (Table 1).Furthermore, we also observed the pollen grains of I. yingjingensis and found they have unique micromorphology (single-grain pollen with a flattened spherical shape, polar view capsule-like, irregular and reticulated) (Fig. 2).Previous studies have illustrated that the pollen grains of different species of Impatiens show significant differences in size, equatorial view, polar view and exine thickness and these morphological characteristics serve as natural evidence for the systematic classification of the genus Impatiens (Zhang et al. 2014;Zeng et al. 2016;Zhang et al. 2023).
Therefore, both the morphological and micromorphological features strongly support that I. yingjingensis is very different from other members of Impatiens and should be treated a new member of the genus Impatiens.
Although the explicit systematic position of I. yingjingensis remains undefined, both phylogenetic trees based on plastome data and ITS sequences, strongly supported that I. yingjingensis is nested within the genus Impatiens, indicating its affiliation with the genus.Consistent with Yu et al. (2016), Impatiens can be divided into I. subgen.Clavicarpa and I. subgen.Impatiens.The molecular phylogenetic analysis of Impatiens species, based on complete plastomes and ITS sequences, supported our proposed new species, to cluster into a clade which belongs to I. subgen.Impatiens.To confirm the phylogenetic position of I. yingjingensis within Impatiens, further molecular sequences, such as additional nuclear DNA fragments, are required in future studies.Thus, the molecular evidence further bolsters the argument that I. yingjingensis should be classified as a novel member of Impatiens.In conclusion, considering the evidence obtained from morphology, micromorphology and molecular evidence, the designation of I. yingjingensis as a new species of Impatiens is both logical and compelling.Etymology.The species is named after Yingjing County, Sichuan Province, China, which is the type locality.The Chinese name is given as "荥经凤仙花".

Figure 1 .
Figure 1.Habitat and morphology of I. yingjingensis A habitat B plants C leaf D flower branch E the floral anatomy of I. yingjingensis F, G flower, lateral view H flower, front view I, J capsule K seed.

Figure 2 .
Figure 2. SEM images of pollen grains A polar view B, C partial view.

Figure 3 .
Figure 3. Plastome map of this new species.Genes shown in outside and insides of the circle are transcribed counter-clockwise and clockwise, respectively.The dark grey area of the inner circle denotes the GC content of plastome.

Figure 4 .
Figure 4. Phylogenetic trees constructed by Maximum Likelihood (ML) and Bayesian Inference (BI).The bootstrap values (BS) of ML and posterior probabilities (PP) of BI are listed at each node.(*) represents the node with PP = 1.00/BS = 100."-" means the values < 0.50/50.Red background indicates the newly-sequenced unknown Impatiens species A plastomes-based tree B ITS-based tree.

Figure 6 .
Figure 6.Impatiens yingjingensis A plants B the floral anatomy of Impatiens yingjingensis C capsule D seed E flower, lateral view F flower, front view.(Drawn by Liuqing Zhu)

Table 1 .
Comparative morphological characters of I. yingjingensis and related species.

Table 2 .
Annotated unique genes information of I. yingjingensis.