Description of a New Species and Lectotypification of Two Names in Impatiens Sect. Racemosae (Balsaminaceae) from China

Impatiens longiaristata (Balsaminaceae), a new species from western Sichuan Province in China, is described and illustrated here based on morphological and molecular data. It is similar to I. longiloba and I. siculifer, but differs in its lower sepal with a long arista at the apex of the mouth, spur curved downward or circinate, and lower petal that is oblong-elliptic and two times longer than the upper petal. Molecular analysis confirmed its placement in sect. Racemosae. Simultaneously, during the inspection of the protologues and type specimens of allied species, it was found that the types of two names from this section were syntypes based on Article 9.6 of the International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code). According to Articles 8.1, 9.3, and Recommendations 9A.1, 9A.2, and 9A.3, the lectotypes of these two names are here designated.


Introduction
Impatiens L. (Balsaminaceae) is one of the most species-rich genera of angiosperms with more than 1000 species [1,2]. It is mainly distributed in tropical and subtropical Africa, Madagascar, southern India and Sri Lanka, eastern Himalayas and Southeast Asia [3,4]. In contrast to the wide distribution of the genus, many Impatiens species are endemic to specific mountains or valleys. Most of the species are forest plants, usually growing in moist semi-shaded places, rarely in semi-arid places [2,5]. Flowers of Impatiens are zygomorphic and have a wide variety of morphology and colors [4,5]. The diversity of species has resulted in an extremely complex interspecific relationship in this genus. Since Hooker and Thomson [6] provided the first infrageneric classification for Impatiens based only on morphological data, several infrageneric classifications of the genus have been proposed in the last century [7][8][9]. However, all of these classifications were based on a few macromorphological characters only. Fortunately, phylogenetic studies using nucleotide sequences have made great contributions to our understanding of the evolutionary relationships of Impatiens in the last two decades [4,5,[10][11][12][13]. Of these studies, the most notable is that in which Yu et al. [5] proposed a new classification based on phylogenetic and morphological evidence in 2016. They divided this genus into two subgenera: I. subgen. Impatiens and I. subgen. Clavicarpa S. X. Yu ex S. X. Yu & Wei Wang. I. subgen. Impatiens was further divided into seven sections, of which I. sect. Racemosae Hook. f. et Thomson was characterized by its inflorescences racemose, many-flowered; lateral sepals 2, rarely 4 with inner 2 reduced; capsule linear; and seed ovoid. China is a hotspot for the distribution of Impatiens and has numerous balsam species, most of which are distributed in the southwest of the country. The Flora of China lists 227 species, of which 187 are endemic [14]. According to Du et al. [15], a total of 69 new taxa of Impatiens have been described in China between 2000 and 2019, and 13 new taxa [16][17][18][19][20][21][22][23][24][25][26][27][28] and three new records [28][29][30] have been published in the last two years, bringing the total species reported for China to nearly 290.
In 2019, we collected an Impatiens species that was morphologically similar to Impatiens siculifer Hook. f. [31] (p. 246) and I. longiloba Craib [32] (p. 161), from Shimian county, Sichuan province. The examination of the morphology and molecular analyses suggested that our Impatiens was significantly different from the two allied species and other known species. Moreover, while examining the protologue of allied species we noted that two names, I. aquatilis Hook. f. [31] (p. 247) and I. clavicuspis Hook. ex W. W. Smith [33] (p. 337), were based on syntypes according to Article 9.6 of the International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code) [34]. Hence, in the present study on Impatiens from China, a new species is described, and lectotypes are designated for two names.

Materials and Methods
In this study, the morphological terminology for species description follows Grey-Wilson [2]. The infrageneric classification of Impatiens uses the recent research result from Yu et al. [5]. Herbaria are indicated with their acronyms, according to Thiers [35].

Identification and Description of New Taxon
During a botanical exploration in western Sichuan in 2019, we noticed an interesting aristate yellow-flowered species of Impatiens growing in the moist area under a broadleaved forest (the arista was up to 1 cm long in the bud stage). Specimens were collected, preserved, and deposited in the Herbarium of Wuhan Botanical Garden, Chinese Academy of Sciences (HIB) for further identification. We then dissected the flower and carefully reviewed related literature [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]36,37] and herbaria specimens in a bid to ascertain the identity of the collected species. However, we were unable to match the new plants with any described species based on morphology. Molecular phylogenetic analyses (described below) resolved the new plants as being closely related to I. longiloba and I. siculifer, both of which differ in morphology. Therefore, we conclude that our plants represent a new species, and we describe it as such below.
The morphological description of new species was based on careful field observation of plants, measurement of herbarium specimens, as well as plant photographs taken in the field. Comparisons between the new species and allied species were based on photographs of herbarium specimens and literature [14,31,32,36,38].

Taxon Sampling and DNA Sequencing
Based on previous studies [4,5,10], 2 DNA sequences (nrITS and plastid DNA atpB-rbcL) of 117 species (containing 3 outgroups) were selected to build the phylogenetic tree. All sequences, except new species, were downloaded from GenBank, and GenBank accession numbers are presented in Table S1. The corresponding sequence of the new species was provided in this study.
Genomic DNA of the new taxon was extracted from silicagel-dehydrated leaf tissue using Mag-MK Plant Genomic DNA Extraction Kits (Sangon Biotech, Shanghai, China). The primer sequences and polymerase chain reaction (PCR) amplified procedure of nrITS and atpB-rbcL sequences followed Yuan et al. [5] and Janssens et al. [10]. PCR products were sequenced by Tsingke Biotechnology Co., Ltd. using sanger sequencing.

Phylogenetic Analyses
All phylogenetic analyses were done under PhyloSuite v1.2.2 [39], an integrated and scalable desktop platform. Sequence data were aligned with MAFFT [40] and concatenated using PhyloSuite. The best evolutionary models for two regions (ITS and atpB-rbcL) were Plants 2021, 10, 1812 3 of 11 selected using ModelFinder [41] according to the AIC criterion. The best-fit models for the Maximum likelihood (ML) analysis are GTR + F + R5 (ITS) and GTR + F + R3 (atpB-rbcL), and for Bayesian inference (BI) they are GTR + F + I + G4 (ITS) and GTR + F + G4 (atpB-rbcL). Maximum likelihood (ML) analysis was inferred using IQ-TREE [42] for 1000 standard bootstraps. Bayesian inference (BI) analysis was inferred using MrBayes 3.2.7a [43], running for 10,000,000 generations, starting from different random trees and sampled every 1000 generations, in which the initial 25% of sampled data were discarded as burn-in.

Lectotypification
The protologue and relevant literature were investigated as references for the nomenclaturally typical element of Impatiens aquatilis and I. clavicuspis. It was evident that more than one collection was cited in the protologue, indicating that those specimens are syntypes in light of Article 9.6 from the International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code) [34]. The designation of lectotypes was necessary following Articles 8.1, 9.3 and recommendations 9A.1, 9A.2, and 9A.3 of the Shenzhen Code. Diagnosis: Impatiens longiaristata is similar to I. longiloba and I. siculifer, but can be distinguished by its lower sepal with a long arista (1 cm) appearance from the bud stage, spur curved downward or circinate, and lower petal that is oblong-elliptic, tip rounded, and two times longer than the upper petal.

Results and Taxonomic Treatment
Type

Distribution and Ecology
Impatiens longiaristata is currently known only from the type location in Shimian County, Sichuan Province, China. It grows in moist places under broad-leaved forests and frequently in association with Pilea sp., Athyrium sp., Salvia sp., and Impatiens oxyanthera.

Etymology
The specific epithet "longiaristata" is derived from the lower sepal with a long arista at the apex of mouths. It is most conspicuous when the flowers are in bud.

Phenology
The new species was observed flowering from August to September and fruiting from September to October.

Distribution and Ecology
Impatiens longiaristata is currently known only from the type location in Shimian County, Sichuan Province, China. It grows in moist places under broad-leaved forests and frequently in association with Pilea sp., Athyrium sp., Salvia sp., and Impatiens oxyanthera. In this study, phylogenetic analyses based on combined datasets of nuclear ITS and plastid atpB-rbcL intergenic spacer DNA sequences confirmed the new species to be in the Impatiens sect. Racemosae and a close relative to I. longiloba and I. siculifer (Figure 2).
Plants 2021, 10, x FOR PEER REVIEW 5 of 11 In this study, phylogenetic analyses based on combined datasets of nuclear ITS and plastid atpB-rbcL intergenic spacer DNA sequences confirmed the new species to be in the Impatiens sect. Racemosae and a close relative to I. longiloba and I. siculifer (Figure 2). Only the ML tree is shown, because its topology is nearly identical to the BI tree. Impatiens longiaristata is highlighted in red.

Conservation Status
The new species is currently only known from a single population with ca. 300 mature individuals at the type locality. We thereby consider that I. longiaristata can be evaluated as vulnerable (D1) based on the International Union for Conservation of Nature (IUCN) Red List Categories and Criteria [44]. Only the ML tree is shown, because its topology is nearly identical to the BI tree. Impatiens longiaristata is highlighted in red.

Conservation Status
The new species is currently only known from a single population with ca. 300 mature individuals at the type locality. We thereby consider that I. longiaristata can be evaluated as vulnerable (D1) based on the International Union for Conservation of Nature (IUCN) Red List Categories and Criteria [44].

Lectotypification
Lectotypification is among the key aspects of plant systematics. During the perusal of literature, the typification of two species named Impatiens aquatilis and I. clavicuspis was considered paramount. This prompted an investigative review of original literature and materials in the author's herbarium during the collection period. We found that three gatherings of herbarium were cited in protologues of I. aquatilis and four gatherings were cited in protologues of I. clavicuspis. The digital images of herbaria cited by these two names, holding in K, P and E were studied. Based on Articles 8. A critical examination of digital images from all collections reveals that the Ducloux, F. 532 digital image registered Henry, A. as the collector on the mounted herbarium sheets in Kew. In addition, this collection was cited at the Museum d'Histoire Naturelle herbarium but could not be traced among online digital images under Ducloux, F. 532 collections in P but was rather found in the Kew digital herbarium. It is not clear whether at one point herbarium specimens were transferred from P to K or the digital images were obtained by the Royal Botanical Gardens, Kew during databasing and digitization sessions of 185,937 specimens at P [45]. This makes Ducloux, F. 532 gathering significant. Notwithstanding this, Ducloux, F. 532 has descriptions from the author's handwriting with a signature of his name and also has clear illustrations with candid footnotes and hand drawings. After a comparison among all syntypes, Ducloux, F. 532 from Yunnan, Mengtsze alt. 5500 ft. in K is selected to serve as the lectotype (Figure 3) for the name I. aquatilis because it is the best representative material. It is also complete, and its descriptions rhyme well with the details of the original publication.
Impatiens clavicuspis Hook. ex W. W. Smith (1915: 337 . All examined collections of digital images had hand drawings of the plant reproductive illustrations (pods and flower morphological characteristics). Furthermore, 1004 and 1006 had accompanying comprehensive handwritten fieldnotes that matched the original description. However, after a critical examination of all these syntypes, we found the specimen displaying the best representative material and locality information (including elevation), numbered 9762, best fitted the original portal description and was preserved in the Kew herbarium. Moreover, this specimen retains the original pigmenta-tion, making the collections more appealing and providing a precise representation of the living plant species. Hence, we designate it here as the lectotype (Figure 4).  that matched the original description. However, after a critical examination of all these syntypes, we found the specimen displaying the best representative material and locality information (including elevation), numbered 9762, best fitted the original portal description and was preserved in the Kew herbarium. Moreover, this specimen retains the original pigmentation, making the collections more appealing and providing a precise representation of the living plant species. Hence, we designate it here as the lectotype ( Figure  4).

Discussion and Conclusions
Impatiens longiaristata is morphologically and phylogenetically allied to I. longiloba and I. siculifer. The information available about the distribution of the latter two species suggests that I. longiloba is endemic to the type locality in Thailand and that I. siculifer is distributed in southwest China [9,10] and Vietnam [46]. Morphologically, all three have a similar type of inflorescences (racemose), the color of flowers (yellow with red spots), and the shape of lower sepals (funnelform). However, I. longiaristata can be easily distinguished from these two allied species by its long arista of the lower sepal, the shape of the spurs, and the lateral united petals (Figure 1). The lower sepal has an arista ca. 1 cm long at the apex of the mouth in I. longiaristata, while it is shortly rostellate or aristate (no more than 5 mm) in I. siculifer and apiculate in I. longiloba. The spur is curved downward or circinate in I. longiaristata, while it is curved upward in I. siculifer and slightly curved at the tip in I. longiloba. The lateral united petals of I. longiaristata are ca. 3 cm long, the upper petal is triangular and semi-circular, and the lower petal is oblong-elliptic, tip rounded, ca. 1.8 cm long, and two times longer than the upper petal, whereas in I. siculifer the lateral united petals are ca. 1.8 cm long, the upper petal is subtriangular, and the lower petal is lorate with an acuminate tip, and in I. longiloba the lateral united petals are ca. 2.5 cm long and the lower petal is lorate, elongate, and four times longer than the upper petal. The lateral veins of leaves are 5-7 pairs in I. longiaristata, but they are 10-12 pairs in I. longiloba and 5-11 pairs in I. siculifer. The lateral sepals are two pairs in I. longiaristata and I. longiloba, but just a pair in I. siculifer. The detailed morphological comparisons between new species and allied species are shown in Table 1. The results of the BI and ML analyses showed that these three species were in a well-supported clade (PP = 0.99 and BP = 85), in which I. longiaristata and I. longiloba formed a highly supported clade (PP = 1.00 and BP = 100) with I. siculifer forming a sister clade. It was also noted that I. longiaristata was morphologically closer to I. longiloba than to I. siculifer (Table 1). Based on detailed morphological studies and phylogenetic analysis, Impatiens longiaristata is a new taxon in science. Detailed descriptions, color plates, phylogenetic analysis, and comparisons with species of similar taxa are provided. The lectotypes of I. aquatilis and I. clavicuspis are also designated.