Eutremananum (Brassicaceae), a new species from Chola Shan, Southwest China

Abstract Eutremananum, a new high-elevation (4500–4600 m) species from Chola Shan, Sichuan (Southwest China), is described and illustrated. It is similar morphologically to E.nepalense but is readily distinguished by having oblong to elliptic or obovate to spatulate (vs. suborbicular to broadly ovate) leaves, glabrous (vs. puberulent) sepals and ovate to oblong fruit 4–7 × 2–3 mm with flattened valves (vs. ovoid to subglobose fruit 2–3 × 1.8–2 mm with rounded valves). The genetic differences amongst E.nanum, E.nepalense and other close relatives are further confirmed by phylogenetic analyses using ITS and cpDNA sequence variations. The new combination E.sinense is proposed.


Introduction
The boundaries of Eutrema R.Br. (Brassicaceae or Cruciferae) have recently been expanded to include 38 species, several of which were previously placed in the six genera Taphrospermum Hao et al. 2017a). The taxonomic knowledge of this genus is still incomplete because numerous collections from the high-elevation regions in Southwest China were often overlooked and many areas remain poorly explored. We reported two new species during recent field investigations and molecular analyses (Hao et al. 2016(Hao et al. , 2017b. Here we report the third one, Eutrema nanum, found in Chola Shan at a high elevation of 4500-4600 m in Sichuan Province, Southwest China. This new species is morphlogically similar to E. nepalense (Al-Shehbaz, Kats Arai & H.Ohba) Al-Shehbaz, G.Q.Hao & J.Quan Liu but, as shown below, it is readily distinguished by several aspects of leaves and fruit. The phylogenetic studies on both species and their other relatives were also conducted herein and the results support the recognition of this novelty. In addition, one of six species which were used to determine the systematic position of E. nanum was found to need a taxonomic combination and a new name Eutrema sinense (Hemsl.) G.Q.Hao, J.Quan Liu & Al-Shehbaz is therefore proposed herein.

Material and methods
We examined morphological traits of Eutrema nanum and several relative species. We followed Hu et al. (2015) and Hao et al. (2017a) in examining the genetic differences between this novelty (two accessions) and the morphologically similar E. nepalense (one accession). In order to determine the systematic position of E. nanum, we futher included six species ( O.E.Schulz) in our analyses. All six species were shown to be close relatives to E. nepalense in our previous study (Hao et al. 2017a) and two (E. scapiflorum and E. sinense) were previously placed in the genus Pegaeophyton. The related E. integrifolium Bunge (see Hao et al. 2017a) was selected as the outgroup. The collection information of the sampled species is listed in Table 1 and Figure 3 and the voucher specimens were deposited in the Sichuan University Herbarium (SZ).
Eutrema nanum is morpholgically most similar to E. nepalense, from which it is readily distinguished by having oblong, elliptic, obovate to spatulate leaves, glabrous sepals and ovate to oblong larger fruit 4-7 × 2-3 mm with flattened, glabrous valves. In contrast, E. nepalense (https://www.gbif.org) has suborbicular to broadly ovate leaves, puberulent sepals and ovoid to subglobose smaller fruit 2-3 × 1.8-2 mm with rounded, puberulent valves. Eutrema nanum was only found with around 100 individuals along a stream in a valley about 2 kilometres from the Chola Shan peak, whereas E. nepalense occurs across Himalyas Mountains in Bhtan, China, Nepal and India.
Phenology. Flowering: June-August. Fruiting: August-September. Distribution and habitat. Eutrema nanum is currently known only from Chola Shan, part of Hengduan Mountains in West Sichuan, China (Fig. 3). It grows under rocks by streams close to glaciers, damp or gravelly scree, wet sand at a very high elevation of 4500-4600 m.
Paratype The earliest available epithet of this taxon at the species rank is "sinensis" and it should have been been transferred to Eutrema by Hao et al. (2017a) instead of using the varietal epithet "robustum."

Genetic differences between Eutrema nanum, E. nepalense and other relatives
Sequence data from Eutrema nanum and E. nepalense reveals that one nucleotide substitution in ITS, two in rbcL, 18 in matK and eight substitutions and three indels in psbA-trnH distinguish them very well (Table 2).
Based on sequence variations of ITS and cpDNAs (Table 3), phylogenetic analyses suggested that Eutrema nanum is mostly related to E. nepalense, E. sinense and E. scapiflorum. However, phylogenetic relationships of these four species are incongurent between ITS and plastid DNA tree (Fig. 4). In the ML analyses of ITS sequence data, E. nanum and E. nepalense formed a single cluster sister to E. sinense and together are sister to E. scapiflorum with high support values (>80%) (Fig. 4A). By contrast, in the ML analyses of cpDNAs sequences, the phylogenetic relationships were maintined between E. nanum and sister E. sinense and together as sister to E. scapiflorum, but E. nepalense fell outside that relationship and was separated from them by E. hookeri with medium support (>50%) (Fig. 4B). MP analyses produced almost the same tree topologies with similar bootstrap support values.  603 633 638 657 28 40 48 92 114 115 138-212 228 235 Eutrema nanum

Discussion
Both Eutrema nanum and E. nepalense are small plants similar in flower traits and seed size. However, as discussed above, they are quite different morphologically. In addtion, phylogenetic analyses of cpDNAs variations suggested these two species did not comprise a monophyletic clade. Furthermore, the Himalayan E. nepalense is disjunctly separated by a distance of at least 1200 air kilometres from the Chola Shan (Sichuan, SW China), where E. nanum is endemic (Fig. 3). Eutrema nanum is also closely related to E. sinense in the phylogenetic analyses of the cpDNA sequence variations, but both are easily distinguished from each other. Eutrema nanum is a small and weak herb with entire leaves (0.5-1.5 cm long) and small flowers (petals 2-3 mm long), whereas E. sinense is obviously stout with entire or toothed leaves (1.5-8 cm long) and distinctly larger flowers (petals 8-15 mm long) and fruit (10-20 mm long). Pylogenetic relationships amongst E. nanum, E. nepalense and E. sinense are incongruent between ITS and cpDNA trees. This incongruence may suggest possible hybridisations or incomplete lineage sorting during the rapid and recent species diversifications Soltis 2000, 2009). However, it is not possible at present to determine which of these two factors had caused the incongruent phylogenies observed here. More analyses and molecular data, especially based on more individuals and genomic evidence, are needed to solve these phylogenetic inconsistences.