Impatiens wuyiensis (Balsaminaceae), a new species from Fujian of Southeast China, based on morphological and molecular evidences

Southeast Asia, together with tropical Africa, Madagascar, South India and Sri Lanka, and the eastern Himalayas, are the five primary hotspots of species diversity of Impatiens (Balsaminaceae). China is also rich in Impatiens species, especially in the limestone karsts or ‘Danxia’ landforms. With zygomorphic flowers and diverse corolla morphology and color, the species in Impatiens are well-known for their ornamental use, but they are also notorious in taxonomy. During the preparation of revision of Impatiens in Zhejiang and adjacent regions, an unknown species was collected from Mt. Wuyi in Fujian Province, Southeast China. Phylogenetic analyses based on nuclear ITS, chloroplast atpB-rbcL and trnL-F sequences, together with micromorphology of pollen grains and seed coats, strongly supported the close relationship of the new species with Impatiens platysepala Y.L.Chen and I. chloroxantha Y.L.Chen. In turn, both molecular data and morphological characters also were sufficient to distinguish the new species from the other two counterparts. Our detailed morphological observations and molecular phylogenetic analyses support the recognition of Impatiens wuyiensis as a species new to science.


Background
, containing more than 1000 species, is one of the largest genera of seed plants and is a notorious genus for taxonomic difficulty (Hooker 1908;Chen 1978;Yu et al. 2016). The genus Impatiens has zygomorphic flowers with great diversity both in corolla color and morphology, thus it was regarded as 'the dicot counterpart of the orchid' (Yuan et al. 2004;Yu et al. 2016). Some species, e.g. Impatiens balsamina L. ('balsam') and I. walleriana Hook.f. ('busy lizzie'), are well-known for horticultural value and widely cultivated in East to South China. The genus Impatiens differs from its sister group Hydrocera Blume ex Wight & Arn., a monotypic genus, in having lateral petals (sometimes also called wings) united in pairs and valvate and explosive capsules (Chen 2001;Chen et al. 2007). With membranous dried flowers and easily dehiscent fruits on specimens, descriptions of floral and fruit characters on herbarium specimens may therefore be incomplete or ambiguous. Consequently, field investigations are essential for accurately describing the reproductive characters (Chen 1978;Yu 2012).
The genus Impatiens has five recognized diversity hotspots: viz. tropical Africa, Madagascar, South India and Sri Lanka, the eastern Himalayas, and Southeast Asia (Song et al. 2003;Yuan et al. 2004). China is also rich in Impatiens species, including over 250 species which are mainly distributed in south-western regions, especially in Yunnan, Sichuan, Guangxi, Tibet and Guizhou (Chen 1978;Chen et al. 2007;Shui et al. 2011). Recently, molecular phylogenetics has contributed greatly to the understanding of relationships within Impatiens (Yuan et al., 2004;Janssens et al. 2006Janssens et al. , 2007Janssens et al. , 2012Yu et al. 2016). Moreover, micromorphology of pollen grains and seed coats of Impatiens have great taxonomic values (Lu 1991;Lu and Chen 1991;Song et al. 2005;Janssens et al. 2012;Cai et al. 2013), and systematic implications (Jin et al. 2008;Guo et al. 2016;Zeng et al. 2016). Chen (1988Chen ( , 1989Chen ( , 1999 carried out the taxonomic studies and described eight new species from Zhejiang, Anhui and Jiangxi provinces. However, the previous collection of this area was not satisfactory till now. Our field surveys of Impatiens from Zhejiang and adjacent regions in East China have resulted in the discovery of several new species and some infraspecific taxa (Impatiens taishunensis, Chen and Xu 1993;I. jiulongshanica, I. neglacta, I. suichangensis and I. tienmushanica var. longicalculata, Xu and Chen 1999;I. huangyanensis, Jin and Ding 2002;I platysepala var. kuocangshanica, Jin and Zhang 2003;I. yilingiana and I. huangyanensis subsp. attenuata, Jin et al. 2008;I. chekiangensis var. multiflora and var. cangnanensis, Xu et al. 2018). In this study we report an additional distinctive new species, I. wuyiensis, from one of the typical Danxia landforms in northern Fujian Province, China. Danxia, literally meaning 'rosy cloud' , is a type of sandstone landform found in southeastern, southwestern and northwestern China that consist of a red bedrock characterized by steep cliffs, isolated peaks, steep pillars, ravines, mountains and hills that have formed after a long period of erosion by wind and running water (Liu et al. 1999;Peng et al. 2018). The Danxia landform in Fujian and Guangdong Provinces of China shows the block mountains and looks like isolated islands (Peng et al. 2018).
Based on its flower structure, and micromorphology of pollen grains and seeds, Impatiens wuyiensis is similar to I. platysepala and I. chloroxantha, but evidences of molecular data (ITS, trnL-F and atpB-rbcL) and other morphological characters well support the status of this taxon as new.

SEM observations on pollens and seeds
Dried mature pollen grains and seeds of the new species, together with its allied Impatiens platysepala and I. chloroxantha, were directly collected from the field (see further methodological details in Appendix 1). Pollen grains and seeds were mounted on stubs using doublesided adhesive tape, and directly coated with a layer of gold. The coated pollen grains and seeds were observed and photographed under a Hitachi SU8010 SEM. The sizes of pollen grains and seeds were respectively measured on twenty grains per species using light microscopy. Pollen terminology follows Wang and Wang (1983) and Lu (1991), and that of seed follows Liu et al. (2004) and Lu and Chen (1991).

Taxon sampling and DNA sequencing
Seventeen samples representing eight species and one variety of Impateins were collected, and total genomic DNA of these samples were isolated from dried leaf tissue using the TIANGEN Plant Genomic DNA Kit (China). Three DNA regions, ITS (White et al. 1990), atpB-rbcL (Janssens et al. 2006), and trnL-F (Taberlet et al. 1991) were amplified. PCR (the polymerase chain reaction) mixture (25µL) contained 12.5 μL 2 × Reaction Mix, 1.0 μL each primer (10 pmol/µL), 1.0 μL genomic DNA (< 0.5 μg), 0.5 μL Golden DNA Polymerase (2.5 U/ μL) and 9.2 μL ddH2O. The PCR conditions were: 1 cycle of 3 min at 94 °C for denaturation, 35 cycles of 30 s at 94 °C for denaturation (for ITS, 38 cycles), 30 s at 55 °C for annealing and 1 min at 72 °C for extension (for ITS, 45 s), with a final 5 min extension at 72 °C. The successful PCR products were checked using a 1% agarose gel electrophoresis. Sequencing was carried out on an ABI 3730 automated sequencer (Applied Biosystems, USA).
A total of 116 sequences representing 34 taxa (Impatiens species and varieties, plus Hydrocera triflora as outgroup) were used to test the phylogenetic placement of the new species. The sampling representatively covered all the sections (with the exception of sect. Tuberosae) known in the phylogeny of Impatiens ). Fifty-one sequences representing nine taxa being collected in the field were newly generated in this study. The rest of the sequences were obtained from GenBank (Appendix 1).

Phylogenetic analysis
Sequences were assembled and edited using DNAStar Lasergene 7.1 software. Assembled sequences of the same locus were aligned using MAFFT online version (https :// mafft .cbrc.jp/align ment/serve r/) and trimmed manually in Mega 7.0 (Kumar et al. 2016). The best-fit nucleotide substitution models were determined by the Akaike Information Criterion (AIC) using JmodelTest 2 (Darriba et al. 2012), which resulted in ITS for GTR + I + G, atpB-rbcL for GTR + G and trnL-F for GTR + G. Bayesian Inference (BI) was conducted in MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003), running for 10 million generations and sampling one tree every 1000 generations with four chains of Markov chain Monte Carlo (MCMC). A 50% majority-rule consensus tree was calculated after discarding the first 25% trees as burn-in. A Maximum parsimony (MP) analysis was conducted using PAUP v.4.0b10 (Swofford 2003), using a heuristic search algorithm with 1000 random addition replicates and tree bisection-reconnection (TBR) branch-swapping. Node support was assessed using 1000 MP bootstrap (BS) replicates.
Seeds of the new species, Impatiens platysepala and I. chloroxantha were all ovoid, slightly compressed, and surface cells regularly elevated, with granules on cell surfaces. The micromorphology of seed coat was revealed to be closely similar in the three studied species (Fig. 2). The seed size of the new species (length × width) was 2.39 (± 0.12) × 1.29 (± 0.12) mm, and those of I. platysepala and I. chloroxantha were respectively 2.51 (± 0.10) × 1.54 (± 0.11) mm and 2.96 (± 0.20) × 1.63 (± 0.16). Observed seed size of I. chloroxantha was larger than in I. platysepala and the new species (Fig. 2).
The combined dataset of ITS, atpB-rbcL and trnL-F included 2505 aligned characters (ITS: 697 bps, atpB-rbcL: 869 bps and trnL-F: 939 bps). The phylogram with posterior probability values (PP) of Bayesian analysis and bootstrap supports (BS) of Maximum parsimony analysis is respectively depicted in Fig. 3. Five individuals of the new species were shown forming a well-supported clade (PP = 1.00, and BS = 99), which was sister to the clade of two similar species, I. platysepala and I. chloroxantha. Together with the other ten speices (I. chekiangensis, I. noli-tangere, I. tienmushanica, I. davidi etc.), these species are easily distiguished as members in I. sect. Impatiens, which is characterized by inflorescence shortly racemose or umbel-like, ovary 5-capellate, and capsules clavate ).

Conclusion
According to our results, we proceed to formally describe the new species.
Distribution, habitat and ecology: Impatiens wuyiensis seems to be restricted to Mt. Wuyi. It is widely distributed along several scenic spots, such as Dawangfeng, Tianyoufeng, Shuiliandong and Lianhuafeng views. It grows in moist places by roadside or in grasslands, on rock face, under forest or at forest margins, at an elevation between 220 and 430 m ( Fig. 5: a, b).
Phenology: Flowering and fruiting of this new species is from early April to late September.
Etymology: The specific epithet 'wuyiensis' is derived from the type locality, Mt. Wuyi, northern Fujian Province.
Notes: This new species is morphologically similar to Impatiens platysepala and I. chloroxantha in habit and floral structure (Chen 1988(Chen , 2001Zhang et al. 2017), but is easily distinguished from these two species in having flowers golden yellow, lateral sepals densely purplered spotted, upper petals with abaxial midvein clavate or corned at midle, bracts herbaceous, linear or narrowly ovate-lanceolate, 3-4 mm long, ca. 0.5 mm wide. Morphological characters distinguishing the new species from Impatiens platysepala and I. chloroxantha are detailed in Table 1, Figs. 5 and 6.
Micromorphology of pollen grains and seeds of the new species, Impatiens platysepala and I. chloroxantha revealed that these three species are closely related. Phylogenetic analysis detected that the new species is also related to I. platysepala and I. chloroxantha, but considerably different from each another with strong supports.
Additional specimens examined (