As currently delimitated, the species-rich mustard tribe Euclidieae DC. (Brassicaceae) comprises 28 genera and 152 species distributed primarily in Asia. To date, no tribe-wide comprehensive phylogenetic analysis has been conducted. In this study, sequence data from the nuclear ribosomal internal transcribed spacer (nrITS) region of 82 species in all 28 genera of Euclidieae were used to test its monophyly and infer inter- and intra-generic relationships within. Phylogenetic analyses revealed that Rhammatophyllum and Sisymbriopsis are embedded within Solms-laubachia s.l., and Solms-laubachia lanuginosa (Eurycarpus lanuginosus) fell outside the tribe. Therefore, Solms-laubachia s.l. as currently recognized is not monophyletic and its generic delimitation needed further study. Besides, our results suggest that the genera Lepidostemon, Neotorularia, and Tetracme are polyphyletic.

Euclidieae , Brassicaceae , Cruciferae , Solms-laubachia , Phylogeny, nrITS

The Brassicaceae (Cruciferae) includes 52 tribes, 341 genera, and 3,997 species (BrassiBase: https://brassibase.cos.uni-heidelberg.de/, accessed 5 February 2018, Koch et al. 2012, Kiefer et al. 2014) distributed worldwide, primarily in the temperate regions (Al-Shehbaz 1984, Al-Shehbaz et al. 2006). The family is academically and economically important (Franzke et al. 2011, Al-Shehbaz 2012, Huang et al. 2016), as it contains the well-known model plant species Arabidopsis thaliana (L.) Heynh. and many crops (e.g., cabbage, cauliflower, turnip, rape, canola, radish, wasabi) and ornamentals (e.g., species of Lobularia Desv., Iberis L., Hesperis L., Matthiola W. T. Aiton). Although the family is easily recognized morphologically, it is often difficult to assign an individual plant to a given genus, and there are tremendous controversies on generic delimitations and tribal assignments (Al-Shehbaz et al. 2006, Al-Shehbaz 2012). The total number of tribes and genera they include varied among different systems. For example, Schulz (1936) and Janchen (1942) recognized 19 and 15 tribes, respectively. By contrast, others (e.g., Zhou et al. 2001, Appel and Al-Shehbaz 2003) adopted an alphabetical arrangement of genera. The use of molecular sequences to infer phylogenetic relationships during the past two decades have greatly improved our understanding of the evolution within the Brassicaceae. That led Al-Shehbaz et al. (2006) to propose the first phylogenetic tribal classification system based on prior molecular studies, especially the chloroplast ndhF sequences (Beilstein et al. 2006), and had since been expanded to include 52 tribes (Al-Shehbaz 2012, German and Friesen 2014, Chen et al. 2016). Generic boundaries had also been redefined, and studies on Solms-laubachia s.l. (Yue et al. 2006, 2008), Eutrema R. Br. (Al-Shehbaz and Warwick 2005), Microthlaspi F. K. Mey. (Ali et al. 2016), to name a few, demonstrate that trend.

The tribe Euclidieae DC. was established ca. 200 years ago (de Candolle 1821), and it has been accepted in subsequent tribal classifications, though the delimitation of its component genera remained controversial. Of the 14 genera recognized by Schulz (1936) in the tribe, only Euclidium W. T. Aiton and Lachnoloma Bunge were maintained by Al-Shehbaz (2012) (Table 1). As currently delimited (Warwick et al. 2007, Al-Shehbaz and German 2016, BrassiBase) (Table 1), the tribe comprises 28 genera and 152 species, including the species-rich Solms-laubachia Muschl. (33 spp.), Strigosella Boiss. (24 spp.), and Braya Sternb. & Hoppe (22 spp.), as well as 12 monospecific genera.

Because taxa sampling varied in previous studies, the interrelationships among genera of Euclidieae varied a great deal. In order to gain knowledge of phylogenetic relationship of the tribe, we conducted the first comprehensive study that included representatives of all genera.

The aligned ITS dataset comprised 88 species (100 accessions) with 609 characters, of which 256 were variable and 187 (30.7%) were parsimony-informative.

The resolution of MP analysis was relatively weaker than the outcome of BI analysis. Only the topologies of Bayesian phylogenetic analysis were shown (Figure 1). The result clearly showed that all 28 genera of Euclidieae formed a moderately to strongly supported clade (PP / BS = 0.99 / 61; Figure 1). Dilophia Thomson, Lachnoloma, and Spryginia Popov formed the early branching lineage of the tribe in BI analysis (Figure 1). Five species of Rhammatophyllum, three of Sisymbriopsis, and 23 of Solms-laubachia formed a well-supported subclade within Euclidieae (PP / BS = 0.95 / 68; Figure 1), and then clustered with Anzhengxia Al-Shehbaz & D.A.German and Pycnoplinthus O.E.Schulz (PP / BS = 0.99 / 55; Figure 1).

All Braya species formed a subclade (PP / BS = 1 / 89; Figure 1) sister to Shangrilaia Al-Shehbaz, J. P. Yue & H. Sun, Metashangrilaia Al-Shehbaz & D. A. German, Lepidostemon Hook. f. & Thomson, and Pycnoplinthopsis Jafri (Figure 1). Species of Neotorularia Hedge & J. Léonard, Streptoloma Bunge, Octoceras Bunge, Tetracme Bunge, Cymatocarpus O. E. Schulz, Cryptospora Kar. & Kir., Atelanthera Hook. f. & Thomson, and Catenulina Soják clustered into one clade in BI analysis (PP = 1; Figure 1), whereas both Neotorularia and Tetracme were found to be polyphyletic. As for the four species of Neotorularia, N. contortuplicata (Stephan ex Willd.) Hedge & J. Léonard and N. torulosa (Desf.) Hedge & J. Léonard formed one clade (PP / BS = 1 / 99; Figure 1), while N. tetracmoides (Boiss. & Hausskn.) Hedge & J. Léonard and N. dentata (Freyn & Sint.) Hedge & J. Léonard each formed a solitary branch. The three species of Tetracme formed two independent subclades in BI analysis, one of which comprised of T. quadricornis (Stephan ex Willd.) Bunge and T. contorta Boiss. (PP = 0.94; Figure 1), and the other consisted of T. secunda Boiss. and Octoceras lehmannianum Bunge (PP = 0.61; Figure 1).

In addition to the above clades, species of Leiospora (C. A. Mey.) Dvořák and Strigosella formed two well supported clades, suggesting that both are monophyletic. However, Solms-laubachia lanuginosa (Hook. f. & Thomson) D. A. German & Al-Shehbaz (formerly Eurycarpus lanuginosus (Hook. f. & Thomson) Botsch.) did not fall within the Solms-laubachia-Rhammatophyllum-Sisymbriopsis clade. Instead, three accessions of this species formed a clade with outgroup taxa Bunias erucago (PP / BS = 0.59 / 76; Figure 1), indicating that S. lanuginosa is neither a member of genus Solms-laubachia nor of the tribe Euclidieae.

Figure 1. 

Phylogenetic tree resulting from Bayesian analysis of the ITS sequences of the 88 Brassicaceae species, of which 28 genera and 82 species in Euclidieae. Posterior probabilities are indicated above branches. Bootstrap support values (>50%) are noted below branches.

Our results suggested that Solms-laubachia s.l. is not monophyletic, within which both Rhammatophyllum and Sisymbriopsis are embedded. Besides, S. lanuginosa fell outside of the clade. The closeness of Solms-laubachia, Rhammatophyllum, and Sisymbriopsis was revealed in previous studies (e.g., Belstein et al. 2006, 2008, Warwick et al. 2007, German et al. 2009). However, these studies only sampled one or two representative species of each genus and therefore did not reach a convincible conclusion on their generic status. By contrast, this study sampled 23 of 33 species of Solms-laubachia, five of seven of Rhammatophyllum, and three of four of Sisymbriopsis, representing thus far the most complete taxa sampling on these three genera.

Solms-laubachia had recently been subjected to a series of studies, including taxonomy (Lan and Zhou 1981, Al-Shehbaz and Yang 2000), cytology (Yue et al. 2003, 2004), molecular phylogeny (Yue et al. 2006, 2008), and biogeography (Yue et al. 2009). As traditionally circumscribed, this genus contained nine to thirteen species distributed from Southwest China to East-Himalayan. However, molecular phylogenetic studies demonstrated that Desideria Pamp. and Phaeonychium O. E. Schulz should be included in it, and that led to greatly expanding of the morphological and geographic boundaries of Solms-laubachia. For example, previously delimited Solms-laubachia species have entire, pinnately veined leaves and latiseptate fruit, whereas the expanded Solms-laubachia also has palmately veined leaves, and terete fruit. The geographic distribution of Solms-laubachia s.l. is also expanded westward into Central Asia.

Rhammatophyllum consists of shrubs or subshrubs with soft malpighiaceous, submalpighiaceous, or rarely subdendritic trichomes, filiform to linear or lanceolate, entire cauline leaves, and dehiscent fruit with torulose valves. Its seven species are distributed from Turkmenistan and W Kazakhstan into W Mongolia (Botschantzev 1987, Al-Shehbaz and Appel 2002, Kamelin 2002, German et al. 2006, Moazzeni et al. 2014). By contrast, Sisymbriopsis includes annual, biennial or perennial herbs primarily with stalked and 1- or 2-forked to dendritic trichomes, pinnately lobed to coarsely dentate or rarely subentire basal and cauline leaves, and linear, flattened and latiseptate fruit with torulose valves and complete septum. Its four species are distributed in Afghanistan, China (Qinghai, Xinjiang, and Xizang), Kyrgyzstan, and Tajikistan (Al-Shehbaz et al. 1999, Al-Shehbaz and German 2016).

Although our results suggest combining Solms-laubachia, Rhammatophyllum, and Sisymbriopsis into one monophyletic genus, merging these three genera into one will make it vastly heterogeneous morphologically (Table 3).The combined genus would be highly variable by encompassing nearly all habit types in the family, nearly all petals colors, and almost all inflorescence types, and would be almost impossible to delimit morphologically. Alternatively, one could keep both Rhammatophyllum and Sisymbriopsis as separate monophyletic genera (Figure 1), and split Solms-laubachia s.l. into several well-delimited smaller genera depending on how different the species cluster together. Because our phylogenetic analyses was based on single ITS sequence fragments, infra-generic relationships can be satisfactorily resolved only by further studies dealing with cpDNA and other single-copy nuclear markers.

As for the outlier Solms-laubachia lanuginosa, its three accessions formed a clade clustered with Bunias erucago (Buniadeae), Hesperis sibirica, and H. isatidea (Hesperideae). Because it fell out of Solms-laubachia and the remainder of Euclidieae, we suggest restoring its previous status in the genus Eurycarpus Botsch. The incongruence between taxonomic treatments based strictly on morphology call for the need to draw generic limits and relationships after conducting adequate molecular phylogenetic analyses. Identifying the tribal position of Eurycarpus is beyond the scope of this paper, and it will be conducted in the near future with nuclear and chloroplast sequences data.

The monospecific genus Metashangrilaia was established based on M. forrestii (W.W.Sm.) Al-Shehbaz & D.A.German, a species used to be put in Braya. Previous molecular analyses revealed that it had very distinct ITS sequences and formed a well-supported clade sister to the rest of Braya (Warwick et al. 2004). Besides, it showed great morphological divergences from other Braya species (Al-Shehbaz et al. 2004, Al-Shehbaz and German 2016). All these led Al-Shehbaz and German (2016) to separate it from Braya and accommodate it in the newly established Metashangrilaia. This study provides further evidence on a strong sister taxon relationship between Metashangrilaia and Shangrilaia (Figure 1), supporting the decision by Al-Shehbaz and German (2016).

Our results also suggest that Neotorularia, Tetracme, and Lepidostemon are not monophyletic. Of the four species sampled from Neotorularia, the generic type N. torulosa clustered with N. contortuplicata, and they were sister to Streptoloma desertorum Bunge, while N. tetracmoides and N. dentata each formed an independent clade (Figure 1). The three sampled Tetracme formed two separate clades, one of which was T. contorta and T. quadricornis, whereas the other was T. secunda and Octoceras lehmannianum (Figure 1). The non-monophyly of both genera is congruent with previous studies (Warwick et al. 2004, 2007).

Finally, Lepidostemon used to be a monospecific genus, the type species is L. pedunculosus Hook. f. & Thomson. It was expanded by Al-Shehbaz (2000, 2002), to include six species endemic to the Mid-western Himalaya (Al-Shehbaz 2015). The ITS sequence of L. glaricola (H. Hara) Al-Shehbaz (Couvreur et al. 2010) did not fall with our newly sequenced L. rosularis (K. C. Kuan & Z. X. An) Al-Shehbaz in one clade. However, due to limited data and low solution of ITS sequences, further studies with extensive sampling and more molecular markers are needed to clarify the taxonomic circumscription of the non-monophyletic genera – Neotorularia, Tetracme, and Lepidostemon.

We are grateful to Prof. Dunyan Tan for help in field work, and to Minshu Song for assistance on molecular studies. Thanks are due to Dr Dmitry A. German for generously giving advice and helpful discussions. This study was supported by the National Natural Science Foundation of China (31590823, 31170181) and the National Key R&D Program of China (2017YFC0505200).

Taxa and accession numbers downloaded from GenBank for the ITS sequences used in the study.

Outgroups: Tribe Anchonieae: Sterigmostemum billardierei (DC.) D. A. German (DQ357513), S. sulphureum (Banks & Sol.) Bornm. (KJ663764). Tribe Buniadeae: Bunias erucago L. (GQ497885). Tribe Dontostemoneae: Clausia aprica (Stephan ex Willd.) Korn.-Trotzky (LK021257). Tribe Hesperideae: Hesperis isatidea (Boiss.) D. A. German & Al-Shehbaz (GQ497882); Hesperis sibirica L. (DQ357549). Ingroups: Tribe Euclidieae: Atelanthera perpusilla Hook. f. & Thomson (FM164518, FM164519); Braya alpina Sternb. & Hoppe (AY353096), B. fernaldii Abbe (AY353152), B. gamosepala (Hedge) Al-Shehbaz & S. I. Warwick (AF137565), B. glabella Richardson (AF137578), B. humilis (C. A. Mey.) B. L. Rob. (AY237325), B. linearis Rouy (AY353102), B. longii Fernald (AY237310), B. pilosa Hook. (KT727927), B. siliquosa Bunge (AY353105), B. thorild-wulffii Ostenf. (AY353098); Catenulina hedysaroides (Botsch.) Soják (GQ424607); Cryptospora falcata Kar. & Kir. (DQ357532); Cymatocarpus pilosissimus (Trautv.) O. E. Schulz (GQ497858); Dichasianthus subtilissimus (Popov) Ovcz. & Junussov (AY353169); Lachnoloma lehmannii Bunge (GQ497889); Leiospora beketovii (Krasn.) D.A. German & Al-Shehbaz (FN821579); L. exscapa (Ledeb.) F. Dvořák (FN821615), L. saposhnikovii (A.N. Vassiljeva) D.A. German & Al-Shehbaz (FN821554); Lepidostemon glaricola (H.Hara) Al-Shehbaz (GQ424542); Leptaleum filifolium (Willd.) DC. (KJ623485); Neotorularia contortuplicata (Stephan ex Willd.) Hedge & J. Léonard (AY353165), N. dentata (Freyn & Sint.) Hedge & J. Léonard (AY353160), N. tetracmoides (Boiss. & Hausskn.) Hedge & J. Léonard (AY353162), N. torulosa (Desf.) Hedge & J. Léonard (AY353167); Octoceras lehmannianum Bunge (GQ424609); Pycnoplinthopsis bhutanica (H. Hara) Jafri (GQ497878); Rhammatophyllum afghanicum (Rech. f.) Al-Shehbaz & O. Appel (DQ357583), R. erysimoides (Kar. & Kir.) Al-Shehbaz & O. Appel (DQ357587), R. gaudanense (Litv.) Al-Shehbaz & O. Appel (DQ357585), R. ghoranum (Rech. f.) Al-Shehbaz & O. Appel (DQ357586), R. pachyrhizum (Kar. & Kir.) O. E. Schulz (DQ357588); Solms-laubachia flabellata (Regel) J. P. Yue, Al-Shehbaz & H. Sun (GQ497886), S. grandiflora J.P. Yue, Al-Shehbaz & H. Sun (DQ523419), S. minor Hand.-Mazz. (DQ523418), S. stewartii (T.Anderson) J. P. Yue, Al-Shehbaz & H. Sun (FN821609), S. sunhangiana J.P. Yue & Al-Shehbaz (EU186027); Spryginia falcata Botsch (FN821518), S. winkleri (Regel) Popov (GQ424563); Streptoloma desertorum Bunge (FM164618, FM164619); Strigosella brevipes (Bunge) Botsch. (DQ357558), S. scorpioides (Bunge) Botsch. (KJ623536); Tetracme contorta Boiss. (DQ357600), T. quadricornis (Stephan ex Willd.) Bunge (DQ357602), T. secunda Boiss. (DQ357604).