Eutrema nanum, 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.

Cruciferae , Eutrema nanum , molecular phylogeny, Sichuan, Eutrema sinense

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 C.A.Mey., Thellungiella O.E.Schulz, Neomartinella Pilg., Platycraspedum O.E.Schulz, Chalcanthus Boiss. and Pegaeophyton Hayek & Hand.-Mazz. (Al-Shehbaz and Warwick 2005; 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, 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.

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 (E. scapiflorum (Hook.f. & Thomson) Al-Shehbaz, G.Q.Hao & J.Quan Liu, E. sinense (Hemsl.) G.Q.Hao, J.Quan Liu & Al-Shehbaz, E. hookeri Al-Shehbaz & Warwick, E. fontanum (Maxim.) Al-Shehbaz & Warwick, E. verticillatum (Jeffrey & W.W.Sm.) Al-Shehbaz & Warwick and E. deltoideum (Hook.f. & Thomson) 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).

We extracted the total DNA and amplified and sequenced four DNA markers, the nuclear internal transcribed spacer (ITS) and three chloroplast DNA (cpDNA) regions (psbA-trnH, rbcL, matK), following Hu et al. (2015) and Hao et al. (2017a). The sequences firstly reported here were placed in GenBank under the accession numbers (MH702367, MH793597, MH793598, MH793599). We aligned all sequences using Clustal X (Thompson et al. 1997) and refined them manually. We concatenated three cpDNA sequences into a single matrix for Maximum Parsimony (MP) and Maximum Likelihood (ML) analyses. We coded indels using the simple code method by GapCoder (Young and Healy 2003). We constructed phylogenetic relationships based on two datasets (ITS and cpDNAs) using MP analyses by PAUP* 4.10b (Swofford 2003) and ML analyses using RAxML 7.2.6 (Stamatakis 2006). MP analyses employed a heuristic search with 10,000 replicates and TBR branch swapping and bootstrap values (Felsenstein 1985) were estimated with 1000 replicates and 100 random-addition-sequence replicates per bootstrap replicate. ML analyses were performed with raxmlHPC -f a -s sequence. phy -n boot2 -m GTRGAMMA -x 1234 -# 1000 -n outname. The most suitable GTRGAMMA models were used and bootstrap analyses were estimated with 1000 replicates.

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 and 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.

This research was supported by grants from the National Natural Science Foundation of China (31700164 and 31590821).