Eutrema giganteum (Brassicaceae), a new species from Hengduan Mountains in Sichuan Province, southwest China, is described, and its relationships to the closely related E. yunnanense is discussed based on morphological, cytological, and molecular data. It is similar morphologically to E. yunnanense but is readily distinguished by having robust (vs. slender), erect (vs. decumbent), and branched (vs. mostly simple), and rather tall stems (60–110 cm vs. 20–60 cm); curved (vs. straight), smooth (vs. torulose), and shorter fruit (5–8 mm vs. 8–15 mm); and fewer ovules per ovary (1–4 vs. 6–10). All examined individuals from different populations of E. giganteum clustered into a single clade sister to E. yunnanense in phylogenetic analyses using the combined nuclear ITS and plastid DNA datasets. Our cytological studies revealed that the chromosome number of E. giganteum is 2n = 44, with a genome size of 1160 (±8) Mb, while that of E. yunnanense is 2n = 28, with a genome size of 718 (±15) Mb. Multiple lines of evidence support the recognition of E. giganteum as a distinct species well differentiated from E. yunnanense.

Brassicaceae , Cruciferae , Eutrema giganteum , new species, Eutrema yunnanense, molecular phylogeny

Eutrema R.Br. (Brassicaceae) is an important genus that includes the model plant for salt-tolerance E. salsugineum (Pall.) Al-Shehbaz & Warwick and the economic wasabi plant E. japonicum (Miq.) Koidz. This genus was expanded to comprise 26 species (Al-Shehbaz and Warwick 2005; Warwick et al. 2006) with 16 transferred from four previously genera, Taphrospermum C.A.Mey., Thellungiella O.E.Schulz, Neomartinella Pilger, and Platycraspedum O.E.Schulz (Al-Shehbaz and Warwick 2005; Warwick et al. 2006). Since then, several new species were described (Ning et al. 2005; Al-Shehbaz 2007; Gan and Li 2014; Xiao et al. 2015; Hao et al. 2015, 2016).

During botanical expeditions to Hengduan Mountains in southwest China from 2013 to 2016, we discovered three populations of Eutrema that were strikingly unusual in having large size, big cordate leaves, and stout, erect and branched stems. Only E. yunnanense Franch. has such similar big cordate leaves (10–20×10–16 cm), but its stems are slender, decumbent, and rarely branched. Therefore, it was immediately suspected these populations may represent an undescribed new species. In order to further test this hypothesis, morphological, molecular, and cytological studies are presented here on those two species and two related species E. japonicum and E. thibeticum Franch. were conducted with herein.

For morphological comparisons and taxonomical treatments, we examined more than ten living individuals from each population of Eutrema giganteum (three populations) and E. yunnanense (two populations), and photos of all herbarium specimens of E. yunnanense preserved in the Chinese Virtual Herbarium (http://www.cvh.org.cn/). We followed Hu et al. (2015) and Hao et al. (2015, 2016) in examining genetic differences between two morphological groups, and three individuals were studied from each population. In order to determine the systematic position of E. giganteum, we further sampled two populations each for E. japonicum and E. thibeticum because they were phylogenetically related to E. yunnanense. We sampled one individual each of E. schulzii Al-Shehbaz & Warwick, E. heterophyllum (W.W. Sm,) H. Hara, E. verticillatum (Jeffrey & W.W. Sm.) Al-Shehbaz & Warwick, E. integrifolium (DC.) Bunge, E. altaicum (C.A. Mey.) Al-Shehbaz & Warwick, and E. salsugineum as ingroups. We chose one sample of Chalcanthus renifolius (Boiss. & Hohen.) Boiss. as the outgroup. The distribution of sampled populations listed in Table 1. Voucher specimens were deposited in the Sichuan University Herbarium (SZ).

We extracted total DNA from silica gel-dried leaves using the modified CTAB method (Doyle and Doyle 1990). The internal transcribed spacer (ITS) and four chloroplast DNA regions (trnL-F, psbA-trnH, rbcL, matK) were amplified for phylogenetic analyses. The five pairs of primers used for amplifying and sequencing trnL-F, psbA-trnH, rbcL, matK and nuclear nrITS were the same as those used by Hu et al. (2015). PCR amplification and sequencing approaches followed Hu et al. (2015) and Hao et al. (2015). For those ITS sequences with double peaks in those possible hybrids, we further cloned them using vector pGEM-T (Promega, Madison, Wisconsin). We selected ten positive clones for sequencing with primers “sp6” and “t7”. We deposited all new sequences in GenBank under the accession numbers KY969594–KY969625. We aligned DNA sequences using Clustal X (Thompson et al. 1997) and MEGA 5.10 (Tamura et al. 2011) and refined them manually. We concatenated sequences from all four cpDNA fragments into a single matrix for Maximum parsimony (MP) and Maximum likelihood (ML) analyses because of their common inheritance without obvious recombination. To evaluate the congruence of the plastid and nuclear datasets, we employed the incongruence length difference (ILD) test (Farris et al. 1995). The ILD test was carried out using the PAUP* 4.10b (Swofford 2003) with the following settings: 1000 replications, each using a heuristic search with 100 random-addition-sequence replicates and TBR branch swapping. We performed ILD tests between each pair of the cpDNA dataset, and between the nrITS dataset and the combined cpDNA dataset. The P-values smaller than 0.01 were considered to be significant incongruent (Cunningham 1997). We reconstructed phylogenetic relationships based on three datasets (nrITS, cpDNAs and combined nrITS+cpDNAs) respectively using MP analyses by PAUP* 4.10b (Swofford 2003), employing a heuristic search with 10,000 replicates and TBR branch swapping. We estimated bootstrap values (Felsenstein 1985) with 1000 replicates and 100 random-addition-sequence replicates per bootstrap replicate. Because indels may contain potential phylogenetic information (Simmons et al. 2001), we coded them using the simple code method by GapCoder (Young and Healy 2003) for phylogenetic analyses. We performed ML analyses using RAxML 7.2.6 (Stamatakis 2006) with the order: raxmlHPC -f a -s sequence. phy -n boot2 -m GTRGAMMA -x 1234 -# 1000 -n outname. We selected the GTRGAMMA model and estimated ML bootstrap analyses with 1000 replicates. We followed Hao et al. (2015) to carry out chromosome number count and genome-size determination.

Different species concepts emphasize the different criteria to define a new species (Wheeler and Meier 2000). An integrative practice using multiple criteria to circumscribe species boundaries and define a new species will produce relatively objective and operational taxonomy (Su et al. 2015; Hu et al. 2015; Liu 2016). Based on morphological, cytological, and molecular data, the new species E. giganteum is described here as a new species distinct from the closely related E. yunnanense. First, our observations of herbarium specimens and living plants suggested that E. giganteum is most similar to E. yunnanense, but could be distinguished by distinct morphological traits, including stem, fruit, ovule number, and individual size. Second, a species should be delimited as an evolutionarily distinct lineage (de Queiroz 1998, 2007; Stockman and Bond 2007; Fujita et al. 2012; Hendrixson et al. 2013; Mckay et al. 2013). Our molecular phylogenetic analyses of combined nrITS and cpDNA datasets, all examined individuals of E. giganteum clustered into a single lineage, sister to Eutrema yunnanense. Third, chromosome number count and genome-size measure revealed the obviously cytological discrepancy between E. giganteum and E. yunnanense. The chromosome number of E. giganteum is 2n = 44, whereas E. yunnanense has the number of 2n = 28. Consistent with the difference of the chromosome number, we also found that the genome size of E. giganteum is almost 1.5 times larger than that of E. yunnanense. These chromosomal and genomic differences are likely to lead to the obvious reproductive isolations between E. giganteum and E. yunnanense. In addition, our unpublished data suggested that some populations of E. thibeticum have the chromosome number of 2n = 16. Both E. giganteum and E. thibeticum are co-distributed in the Hengduan Mountains in western Sichuan where the former occurs at the high elevation while the latter at the low elevation. It seems likely that E. giganteum (2n = 44) originated from a hybridization between E. thibeticum (2n = 16) and E. yunnanense (2n = 28) although further molecular evidence and experimental hybridization are needed. Overall, all available lines of evidence suggest that E. giganteum should be recognized as a distinct new species.

This research was supported by grants from National Natural Science Foundation of China (31590821) and Sichuan Province Youth Science and Technology Innovation Team (2014TD003).