Two New Species of Impatiens from China, and Taxonomic Insights into the Longifilamenta Group, Which Is Endemic to China

Impatiens longshanensis (The LSID for the name Impatiens longshanensis is: 77219154-1) sp. nov. and I. lihengiana (The LSID for the name I. lihengiana is: 77219153-1) sp. nov., from Hunan, China, are described and illustrated here. The molecular phylogenetic study suggests that I. longshanensis and I. lihengiana should be placed in the I. sect. Impatiens. A detailed description, diagnostic characters between the two new species and allied species, pollen and seed morphology, and color photographs are provided. In addition, based on wide sampling, we found that the longifilamenta group, an endemic group to China, whose members have basal lobes of lateral united petals with long filamentous hairs, shows significant morphological variability. In this paper, we discuss the taxonomic significance of morphological characteristics within this group. Based on a literature review and observation of living materials in the field, an updated identification key for this group is also proposed.


Introduction
Balsaminaceae comprises two genera, the monotypic Hydrocera Blume ex Wight & Arnott [1] and Impatiens Linnaeus [2]. Impatiens is one of the most species-rich genera of angiosperms, having more than 1000 species. These are mainly distributed in the mountainous regions of the tropics and subtropics, and have tropical Africa, Madagascar, South India and Sri Lanka, Sino-Himalaya, and South-East Asia as their centers of diversity [3,4]. The genus is distinguished by zygomorphic flowers with tremendous diversity in corolla color and morphology, and lateral petals always united in pairs. The fruit is a fleshy, explosive capsule, and seeds are often dispersed elastically from valves when ripe [5,6]. Impatiens species occur in diverse habitats, such as in forest understories, roadside ditches, valleys, abandoned fields, along streams and in seepages, usually in mesic or wet conditions, although some species can tolerate drier habitats [6]. Due to their beautiful appearance and long flowering period, many species of Impatiens are grown all over the world as ornamental plants [7]. Many new species of Impatiens have been discovered and described in recent years [8][9][10][11][12][13][14][15]. There are more than 280 species of Impatiens distributed in China [5,16,17]. Most of these are found in southwestern China, which is a key diversity area of the genus.
As is well known, Impatiens is notoriously difficult to classify [3,18]. Morphologically, Impatiens are usually fleshy plants, with fine and fragile flowers, usually folded and co-alesced in dried specimens, hence losing their original shape and therefore difficult to reconstruct. The botanist Hooker called them "a terror to botanists" and "deceitful above all plants, and desperately wicked". Yu et al. [6] divided the genus Impatiens into two subgenera, I. subgen. Clavicarpa and subgen. Impatiens [15], based on both morphological and molecular evidence, and the latter was further subdivided into seven sections: Semeiocardium, Racemosae, Fasciculatae, Impatiens, Tuberosae, Scorpioidae, and Uniflorae.
The longifilamenta group in Impatiens was once treated as I. sect. longifilamenta [17], then merged into I. sect. Impatiens [6]. This group is endemic to China, and markedly characterized by its 1-or 2-flowered racemose inflorescences, pedicel with 2 bracts, lower sepal saccate or funnelform, with striate, basal lobes of lateral united petals with a filamentous long hair. However, this group has not attracted much attention, and there are few studies related to it. In this paper, we discuss the morphological characteristics with taxonomic significance in this group, and provide its updated identification key.
During the botanical explorations from 2012 to 2020 in Longshan County, Hunan province, the authors encountered two interesting species of Impatiens growing on wet shady habitats under an evergreen forest. Specimens were collected carefully, and the flowers and fruits were preserved in formalin-acetic-alcohol (FAA) solution for further identification. After careful examination of the relevant specimens and literature [5,16,17], the authors confirm the two as new species, and describe them here.

Etymology
The specific epithet "longshanensis" refers to the locality of the type specimen, Longshan County, Hunan, China.
Phenology: Flowering and fruiting were observed in the field from September to November.

Habitat and Distribution
Impatiens longshanensis is currently known only from the type locality in Longshan County, Hunan Province, China ( Figure 4). It was found growing in shaded moist places, along creek sides, at altitudes of between 1000 and 1300 m.

Etymology
The specific epithet "lihengiana" is given in honor of Prof. Heng Li, a taxonomist at Kunming Institute of Botany, Chinese Academy of Sciences, who has made significant contributions to plant taxonomy.
Phenology: Flowering and fruiting were observed in the field from September to November.

Habitat and Distribution
Impatiens lihengiana is currently known only from the type locality in Longshan County, Hunan Province, China ( Figure 4). It grows in shaded moist places, along streams, between 450 and 650 m. The surface of the pollen grains under SEM of the two species appears to be similar.

Molecular Phylogenetic Analysis
Consistent with Yu et al. [6], Impatiens can be divided into I. subgen. Clavicarpa and I. subgen. Impatiens. Within I. subgen. Impatiens, several sections can be recognized. The molecular phylogenetic analysis of Impatiens based on ITS and atpB-rbcL supported ten species, namely, I. soulieana, I. lecomtei, I. platychlaena, I. bullatisepala, I. davidii, I. dicentra, I. fissicornis, I. tayemonii, and our two proposed new species, to cluster into a clade which belongs to I. sect. Impatiens (Figure 7). This result is also consistent with the morphological classification in which these species are all characterized by lateral united petals with a long filamentous appendage.

Taxonomic Insights
The results of our phylogenetic reconstruction show that the longifilamenta group in Impatiens is a phylogenetically and morphologically cohesive group (Figure 7). As is currently known, this group consists of 25 species growing as annual herbs, characterized by 1-3 flowered racemose inflorescence, and basal lobes of lateral united petals having an apex with a filamentous appendage [5,13,14,[19][20][21][22]. The morphological characteristics of lateral sepals, lower sepals, and lateral united petals have a taxonomic significance within these species, crucial for their identification. They are endemic to China, distributed from Taiwan to Qinghai-Tibet Plateau.
The molecular data supported that Impatiens soulieana is a separate subclade (Figure 7). Morphologically, the apex of distal lobes in Impatiens soulieana is retuse (a distinctive characteristic that defines this species), whereas the apex of distal lobes in other species in this clade is entire, and the apex is constricted into filamentous hair. Thus, the phylogenetic hypothesis is congruent with the observed morphological distinctiveness. Interestingly, the apex of distal lobes in Impatiens oblongipetala is also retuse, but more phylogenetic evidence is needed to clearly understand its taxonomic status.
Based on morphological characteristics, there are significant variations in the structure of the flower within this group ( Figure 8A-F). The distinctive characteristic of this group is that the apex of distal lobes of lateral united petals has a filamentous appendage, with few exceptions, and the filamentous appendages are also diverse ( Figure 8G-O). Previous descriptions of this group were mostly based on dried herbarium specimens. However, the filamentous appendages of distal lobes also need to be observed with a stereomicroscope and recorded in the field, because their complex morphological characters are often poorly preserved in herbarium materials and, hence, the filamentous appendages may be ignored. Based on many years of field observations, in addition to literature consultation, the following identification key is proposed, and hence the description herein.

Discussion
Based on morphological evidence and molecular phylogenetic study, the two new species should be placed in Impatiens sect. Impatiens. I. longshanensis is similar to I. dicentra in its single-flowered inflorescence, yellow flowers, and saccate lower sepal. However, I. longshanensis can easily be distinguished from similar species by its green equilateral lateral sepals, reniform dorsal petal, oblong basal lobe, and dolabriform distal lobes. A more detailed morphological comparison between I. longshanensis and I. dicentra is provided in Table 1. I. lihengiana is superficially similar to I. davidii in having singleflowered inflorescence, dolabriform distal lobes, and 4-colpate pollen grains, but differs by its yellow-green lateral sepals, purple spotted, 1-veined, funnelform lower sepal, not clawed lateral united petals, and ovate-lanceolate basal lobe. A detailed comparison of similar species is given in Table 2. Overall, the evidence from the combinations of morphology and phylogeny shows that the longifilamenta group is a cohesive group. For this group, an apex with a filamentous appendage in basal lobes of lateral united petals is the most noticeable feature distinguishing it from other species of Impatiens. In addition, this group is endemic to China, and mainly distributed in western Sichuan, Hubei, southern Henan, Guizhou, northwest Guangxi, and Jiangxi, Taiwan, and it favors montane elevations (1000-3000 m).
Based on the material examined in the present study, the morphological characters of this particular group show a significant variability. The morphology of the lower sepal and lateral sepals shows the diversity of flower structures. The shape of the lower sepal is variable, ranging from navicular, infundibular to saccate ( Figure 8P-V). The margin of the lateral sepals is dentate or entire. The abaxial midvein of the lateral sepals ranges from slightly thickened, carinate to a spinelike appendage ( Figure 8W-Z,Aa-Gg). Furthermore, the color of flowers of some populations displays a significant variability and instability within intraspecies in these Impatiens taxa. For example, in the previous literature [7], lateral sepals of Impatiens platychlaena are purple, but in this study, the color changes gradually, ranging from purple, yellow with purple spots to pure yellow, based on years of field observation (Figure 8Dd-Gg). Similarly, the flower of Impatiens toxophora is multicolor with the flower color varying from white, yellow to purple within the same population (Figure 8Hh-Ll). These variabilities may blur the lines between species to some extent, hence posing serious challenges to species identification and classification. Therefore, their taxonomic status needs future investigation and more phylogenetic evidence to enhance the understanding of the species relationships within this particular group.

Gross Traits
Specimens of the two new species were collected from Longshan County and quality color photographs were taken in the field. The morphological characteristics of the two species reported in this paper are mostly based on measurements and observations of living plants, specimens, and color photographs.

SEM Observations
Mature whole pollen grains and seeds were collected from flowers and fruits, respectively, in the natural habitats of the plants, and later observed directly under an anatomical macroscope (Olympus SZX10). Dried pollen grains and seeds were carefully mounted on circular metal stubs using double-sided adhesive tape, sputter coated with gold using the JEC-3200 Auto Fine Coater, and then examined and photographed using the JSM-IT500 SEM. Pollen characters were described according to the literature [23,24], and seed characters were described according to the literature [25][26][27].

DNA Sequencing and Phylogenetic Analyses
Genomic DNA of the two new species was extracted from silica gel-dried leaves (Yi-Yan Cong 35443, Yan Xiao LS-794 at HNNU, CSH) using Mag-MK Plant Genomic DNA extraction kits (Sangon Biotech, Shanghai, China). PCR product sequencing was carried out using TSINGKE Biological Technology. Thirty-three representative species from Impatiens were chosen to construct a phylogenetic tree with Hydrocera trifloral as outgroups. DNA sequences of these 34 species were downloaded from GenBank, with the exception of the two new species, I. dicentra, I. platychlaena, and I. longialata. Species names and GenBank accession numbers are provided in Supplementary Materials Table S1.