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New insights into the taxonomy of tribe Euclidieae (Brassicaceae), evidence from nrITS sequence data
expand article infoHongliang Chen§, Ihsan A. Al-Shehbaz|, Jipei Yue, Hang Sun
‡ Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
§ University of Chinese Academy of Sciences, Beijing, China
| Missouri Botanical Garden, St. Louis, United States of America
Open Access

Abstract

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.

Keywords

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

Introduction

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.

Three different taxonomic treatments and current delimitation of Euclidieae. Number of species included in the study and the total species number of the genus based on current delimitation (BrassiBase) are given in parentheses (sampled/total).

de Candolle (1821) Schulz (1936) Al-Shehbaz (2012) Current delimitation (BrassiBase)
Euclidium W. T. Aiton Anastatica L. Atelanthera Hook. f. & Thomson Anzhengxia Al-Shehbaz & D. A. German (1/1)
Ochthodium DC. Boreava Jaub. & Spach Braya Sternb. & Hoppe Atelanthera Hook. f. & Thomson (1/1)
Pugionium Gaertn. Bunias L. Catenulina Soják Braya Sternb. & Hoppe (13/22)
Euclidium W. T. Aiton Christolea Cambess. Catenulina Soják (1/ 1)
Hymenophysa C. A. Mey. Cryptospora Kar. & Kir. Christolea Cambess. (1 /2)
Lachnoloma Bunge Cymatocarpus O. E. Schulz Cryptospora Kar. & Kir.(/3)
Myagrum L. Dichasianthus Ovcz. & Yunussov Cymatocarpus O. E. Schulz (1/3)
Neslia Desv. Dilophia Thomson Dichasianthus Ovcz. & Yunussov (1 /1)
Ochthodium DC. Euclidium W. T. Aiton Dilophia Thomson (1/2)
Octoceras Bunge Lachnoloma Bunge Euclidium W. T. Aiton (1/1)
Schimpera Hochst. & Steud. ex Schimper Leiospora (C.A.Mey.) Dvořák Lachnoloma Bunge (1/1)
Spirorhynchus Kar. & Kir. Lepidostemon Hook. f. & Thomson Leiospora (C.A.Mey.) Dvořák (5/8)
Tauscheria Fisch. ex DC. Leptaleum DC. Lepidostemon Hook. f. & Thomson (2/6)
Texiera Jaub. & Spach Neotorularia Hedge & J. Léonard Leptaleum DC. (1/2)
Octoceras Bunge Metashangrilaia Al-Shehbaz & D. A. German (1/1)
Pycnoplinthopsis Jafri Neotorularia Hedge & J. Léonard (4/6)
Pycnoplinthus O. E. Schulz Octoceras Bunge (1/1)
Rhammatophyllum O. E. Schulz Pycnoplinthopsis Jafri (1 /1)
Shangrilaia Al-Shehbaz, J. P. Yue & H. Sun Pycnoplinthus O. E. Schulz (1/1)
Sisymbriopsis Botsch. & Tzvelev Rhammatophyllum O. E. Schulz (5/7)
Solms-laubachia Muschl. Rudolf-kamelinia Al-Shehbaz & D. A. German (1/1)
Spryginia Popov Shangrilaia Al-Shehbaz, J. P. Yue & H. Sun (1/1)
Streptoloma Bunge Sisymbriopsis Botsch. & Tzvelev (3/4)
Strigosella Boiss. Solms-laubachia Muschl. (23/33)
Tetracme Bunge Spryginia Popov (2/7)
Streptoloma Bunge (1/2)
Strigosella Boiss. (3/24)
Tetracme Bunge (3/9)

Materials and methods

Plant materials and molecular data

This study comprised 33 genera and 88 species, including 28 genera and 82 species of Euclidieae. Forty-nine ITS sequences of 37 species were newly generated here, and all others were downloaded from GenBank (Table 2 and Appendix 1). Six species of Lineage III (sensu Beilstein et al. 2006), namely Sterigmostemum sulphureum (Banks & Sol.) Bornm. and S. billardieri (DC.) D. A. German (Anchonieae), Bunias erucago L. (Buniadeae), Clausia aprica (Stephan ex Willd.) Korn.-Trotzky (Dontostemoneae), and Hesperis sibirica L. and H. isatidea (Boiss.) D. A. German & Al-Shehbaz (Hesperideae), were used as outgroups.

List of studied taxa including voucher information and GenBank accession numbers.

Taxon Geographical origin Collection number (Herbarium: KUN) GenBank accession No.
Anzhengxia yechengnica (Z. X. An) Al-Shehbaz & D. A. German Pishan, Xinjiang YC-XZ111 MH237681
Yecheng, Xinjiang YC-XZ115 MH237682
Braya parvia (Z. X. An) Al-Shehbaz & D. A. German Aheqi, Xinjiang YC-XZ090 MH237683
Zhada, Xizang YC-XZ150 MH237684
Braya rosea Bunge Aketao, Xinjiang YC-XZ105 MH237685
Kunming, Yunnan SCSY-042 MH237686
Braya scharnhorstii Regel & Schmalh. Aketao, Xinjiang YC-XZ101 MH237687
Christolea crassifolia Cambess. Aketao, Xinjiang YC-XZ103 MH237688
Dilophia salsa Thomson Pishan, Xinjiang YC-XZ128 MH237689
Qumalai, Qinghai ZH645 MH237690
Euclidium syriacum (L.) W. T. Aiton Urumqi, Xinjiang YC-XZ076 MH237691
Eurycarpus lanuginosus (Hook. f. & Thomson) Botsch. Zhada, Xizang YC-XZ152 MH237692
MH237693
MH237694
Leiospora eriocalyx (Regel & Schmalh.) F. Dvořák Yecheng, Xinjiang YC-XZ122 MH237695
Pishan, Xinjiang YC-XZ125 MH237696
Leiospora pamirica (Botsch. & Vved.) Botsch. & Pachom. Aketao, Xinjiang YC-XZ102 MH237697
Aketao, Xinjiang YC-XZ104 MH237698
Lepidostemon rosularis (K. C. Kuan & Z. X. An) Al-Shehbaz Cuona, Xizang ZJW3888 MH237699
Metashangrilaia forrestii (W. W. Sm.) Al-Shehbaz & D. A. German Baqing, Xizang YZC227 MH237700
Pycnoplinthus uniflora (Hook. f. & Thomson) O. E. Schulz Ritu, Xizang YC-XZ134 MH237701
Rudolf-kamelinia korolkowii (Regel & Schmalh.) Al-Shehbaz & D. A. German Aheqi, Xinjiang YC-XZ089 MH237702
Aketao, Xinjiang YC-XZ107 MH237703
Shangrilaia nana Al-Shehbaz, J. P. Yue & H. Sun Shangrila, Yunnan CHY008 MH237704
Sisymbriopsis mollipila (Maxim.) Botsch. Yecheng, Xinjiang YC-XZ119 MH237705
Sisymbriopsis pamirica (Y. C. Lan & Z. X. An) Al-Shehbaz, Z. X. An & G. Yang Aketao, Xinjiang YC-XZ100 MH237706
Sisymbriopsis schugnana Botsch. & Tzvelev Aketao, Xinjiang YC-XZ106 MH237707
Solms-laubachia angustifolia J. P. Yue, Al-Shehbaz & H. Sun Daocheng, Sichuan YZC252 MH237708
Solms-laubachia baiogoinensis (K. C. Kuan & Z. X. An) J. P. Yue, Al-Shehbaz & H. Sun Gongbujiangda, Xizang YZC195 MH237709
Solms-laubachia calcicola J. P. Yue, Al-Shehbaz & H. Sun Leiwuqi, Xizang YZC233 MH237710
Solms-laubachia eurycarpa (Maxim.) Botsch. Basu, Xizang YZC023 MH237711
Solms-laubachia himalayensis (Cambess.) J. P. Yue, Al-Shehbaz & H. Sun Ritu, Xizang YC-XZ130 MH237712
Zhada, Xizang YC-XZ151 MH237713
Solms-laubachia jafrii (Al-Shehbaz) J. P. Yue, Al-Shehbaz & H. Sun Lhasa, Xizang YZC214 MH237714
Jilong, Xizang NZ143 MH237715
Solms-laubachia kashgarica (Botsch.) J. P. Yue, Al-Shehbaz & H. Sun Aheqi, Xinjiang YC-XZ096 MH237716
Solms-laubachia lanata Botsch. Lhasa, Xizang YZC215 MH237717
Solms-laubachia linearlifolia (W. W. Sm.) O. E. Schulz Deqin, Yunnan YZC001 MH237718
Solms-laubachia linearis (N. Busch) J. P. Yue, Al-Shehbaz & H. Sun Pishan, Xinjiang YC-XZ123 MH237719
Solms-laubachia mieheorum (Al-Shehbaz) J. P. Yue, Al-Shehbaz & H. Sun Angren, Xizang YC-XZ160 MH237720
Solms-laubachia platycarpa (Hook. f. & Thomson) Botsch. Dangxiong, Xizang YZC216 MH237721
Solms-laubachia prolifera (Maxim.) J. P. Yue, Al-Shehbaz & H. Sun Mangkang, Xizang YZC019 MH237722
Solms-laubachia pulcherrima Muschl. Lijiang, Yunnan ChenHongliang202 MH237723
Solms-laubachia retropilosa Botsch. Xiangcheng, Sichuan ChenHongliang209 MH237724
Solms-laubachia villosa (Maxim.) J. P. Yue, Al-Shehbaz & H. Sun Yushu, Qinghai YZC239 MH237725
Solms-laubachia xerophyte (W. W. Sm.) H. F .Comber Shangrila, Yunnan YZC277 MH237726
Solms-laubachia zhongdianensis J. P. Yue, Al-Shehbaz & H. Sun Shangrila, Yunnan CHY007 MH237727
Strigosella africana (L.) Botsch. Altay, Xinjiang YC-XZ031 MH237728
Yecheng, Xinjiang YC-XZ117 MH237729

DNA extraction, PCR amplification, and sequencing

Total genomic DNA was extracted from silica gel-dried leaf materials using the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China) following the manufacturer’s protocol. The ITS region was amplified with the primers ITS-18F as modified by Mummenhoff et al. (1997) and ITS4 (White et al. 1990). All polymerase chain reactions (PCR) were performed in a 25 μL volume consisting of 1–2μL sample DNA (approx. 1–10 ng), 12.5μL Premix TaqTM (Takara Biomedical Technology, Beijing, China), 1μL of 10 μM stock of each primer, adjusted to 25 μL with ddH2O. The PCR protocol of the ITS region involved a hot start with 4 min at 94 °C, and 30–32 cycles of amplification (1 min denaturing at 94 °C, 45–60 s annealing at 52–53 °C, 60–80 s extension at 72 °C), and a final elongation step for 10 min at 72 °C. The sequencing primers are the same as amplified primers.

Phylogenetic analyses

Original chromatograms were evaluated with Sequencher 4.1.4 (Gene Codes Corporation 2002) for base confirmation and contiguous sequences editing, and sequences were aligned with MAFFT v7.311 (Katoh et al. 2002, Katoh and Standley 2013) and were manually adjusted with MEGA 7.0.14 (Kumar et al. 2016). The aligned sequences were analyzed with maximum parsimony (MP) and Bayesian inference (BI).

Maximum parsimony analysis were performed by PAUP* 4.0a161 (Swofford 2018) with all characters unweighted. Heuristic parsimony searches were conducted with 100 replicates of random addition of sequences to search for multiple islands of most parsimonious trees (Maddison 1991). Bootstrap analyses (BS) (Felsenstein 1985) to assess the relative support for monophyletic groups were calculated from 1000 replicates using a heuristic search with ten random-addition subreplicates, TBR branch swapping and MULPARS in effect. For Bayesian inference analysis, jModeltest v2.1.7 (Darriba et al. 2012) was used to select the best-fitted model of nucleotide substitution based on the Akaike information criterion (AICc), and the SYM+I+G model was selected for the ITS dataset. Bayesian inference based on the Markov chain Monte Carlo methods (Yang and Rannala 1997) was performed using MrBayes v3.2.6 (Ronquist et al. 2012), four simultaneous Monte Carlo Markov chains (MCMCs) were run for five million generations, and one tree sampled every 1000 generations. The first 1250 trees (25% of total trees) were discarded as burn-in. The remaining trees were summarized in a 50% majority-rule consensus tree, and the posterior probabilities (PP) were calculated.

Results

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.

Discussion

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.

Comparsions on morphological characters of Solms-laubachia, Sisymbriopsis, Rhammatophyllum, and Eurycarpus.

Sisymbriopsis Rhammatophyllum Solms-laubachia Eurycarpus
Habit annual, biennial, or perennial herbs shrubs or subshrubs perennial herbs perennial herbs
Trichomes simple and/or stalked forked or dendritic softly malpighiaceous, submalpighiaceou, rarely subdendritic absent or simple, rarely short-stalked, 2-rayed simple mixed with stalked 1- to 3-forked ones
Basal leaves rosulate or not not rosulate rosulate rosulate
Leaf margin dentate, rarely subentire entire entire or 3- to 9(to 11)-toothed entire
Leaf venation pinnate pinnate pinnate or palmate pinnate
Cauline leaves present present present or absent absent
Flower in racemes, ebracteate or bracteate corymbose in racemes, ebracteate corymbose solitary or in racemes, ebracteate or bracteate corymbose in racemes, ebracteate corymbose
Sepals equal, nonsaccate subequal, nonsaccate equal, nonsaccate equal, nonsaccate
Petal colour white or lavender yellow, creamy white, or rarely purple purple, blue, pink, or rarely white purple
Anther apex obtuse or apiculate apiculate obtuse obtuse
Anther shape ovate or oblong oblong oblong-linear to ovate oblong
Median nectaries present absent or present absent or present present
Fruit shape dehiscent silicles, linear, flattened and latiseptate dehiscent siliques, linear, latiseptate dehiscent silique or silicle, linear, oblong, ovate, lanceolate, or ellipsoid, latiseptate or terete dehiscent silicles, oblong, elliptic, ovate-oblong, or ovate-lanceolate, strongly latiseptate
Fruit valve valves papery, prominently veined, glabrous or pubescent, torulose valves papery, prominently veined, pubescent, torulose valves papery, reticulate veined, glabrous or pubescent, smooth or torulose valves obscurely veined, glabrous, smooth
Septum complete complete complete or rarely perforated or reduced to a rim complete or reduced to a rim
Style obsolete obsolete or distinct obsolete or distinct obsolete
Stigma capitate, entire or 2-lobed, lobes not decurrent capitate, entire or 2-lobed, lobes not decurrent capitate, entire or 2-lobed, lobes not decurrent capitate, entire
Seed uniseriate, wingless or rarely distally with a small wing uniseriate, winged, margined, or wingless uniseriate or biseriate, wingless, seed coat reticulate, rugose, or papillate, not mucilaginous when wetted biseriate, wingless, seed coat minutely reticulate, not mucilaginous when wetted
Cotyledons obliquely accumbent accumbent or rarely incumbent accumbent incumbent or accumbent

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.

Acknowledgements

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).

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Appendix 1

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).