3urn:lsid:arphahub.com:pub:F7FCE910-8E78-573F-9C77-7788555F8AADPhytoKeysPK1314-20111314-2003Pensoft Publishers10.3897/phytokeys.100.2475624756Research ArticleBrassicaceaeBrassicalesPhylogenyTaxonomyCenozoicAsiaNew insights into the taxonomy of tribe Euclidieae (Brassicaceae), evidence from nrITS sequence dataChenHongliang12Al-ShehbazIhsan A.3YueJipeiyuejipei@mail.kib.ac.cn1SunHanghsun@mail.kib.ac.cn1Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming, Yunnan 650201, ChinaKunming Institute of Botany, Chinese Academy of SciencesKunmingChinaUniversity of Chinese Academy of Sciences, Beijing 100049, ChinaUniversity of Chinese Academy of SciencesBeijingChinaMissouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166-0299, USAMissouri Botanical GardenSt. LouisUnited States of America
Corresponding authors: Hang Sun (hsun@mail.kib.ac.cn); Jipei Yue (yuejipei@mail.kib.ac.cn)
Academic editor: K. Marhold
201821062018100125139FF96FF8E-FFB6-9427-FF99-766DFFAAFF8113000640103201801052018Hongliang Chen, Ihsan A. Al-Shehbaz, Jipei Yue, Hang SunThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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-laubachialanuginosa (Eurycarpuslanuginosus) 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.
Chen H, Al-Shehbaz IA, Yue J, Sun H (2018) New insights into the taxonomy of tribe Euclidieae (Brassicaceae), evidence from nrITS sequence data. PhytoKeys 100: 125–139. https://doi.org/10.3897/phytokeys.100.24756
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 Arabidopsisthaliana (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 methodsPlant 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 Sterigmostemumsulphureum (Banks & Sol.) Bornm. and S.billardieri (DC.) D. A. German (Anchonieae), Buniaserucago L. (Buniadeae), Clausiaaprica (Stephan ex Willd.) Korn.-Trotzky (Dontostemoneae), and Hesperissibirica 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.
Anzhengxiayechengnica (Z. X. An) Al-Shehbaz & D. A. German
Pishan, Xinjiang
YC-XZ111
MH237681
Yecheng, Xinjiang
YC-XZ115
MH237682
Brayaparvia (Z. X. An) Al-Shehbaz & D. A. German
Aheqi, Xinjiang
YC-XZ090
MH237683
Zhada, Xizang
YC-XZ150
MH237684
Brayarosea Bunge
Aketao, Xinjiang
YC-XZ105
MH237685
Kunming, Yunnan
SCSY-042
MH237686
Brayascharnhorstii Regel & Schmalh.
Aketao, Xinjiang
YC-XZ101
MH237687
Christoleacrassifolia Cambess.
Aketao, Xinjiang
YC-XZ103
MH237688
Dilophiasalsa Thomson
Pishan, Xinjiang
YC-XZ128
MH237689
Qumalai, Qinghai
ZH645
MH237690
Euclidiumsyriacum (L.) W. T. Aiton
Urumqi, Xinjiang
YC-XZ076
MH237691
Eurycarpuslanuginosus (Hook. f. & Thomson) Botsch.
Lepidostemonrosularis (K. C. Kuan & Z. X. An) Al-Shehbaz
Cuona, Xizang
ZJW3888
MH237699
Metashangrilaiaforrestii (W. W. Sm.) Al-Shehbaz & D. A. German
Baqing, Xizang
YZC227
MH237700
Pycnoplinthusuniflora (Hook. f. & Thomson) O. E. Schulz
Ritu, Xizang
YC-XZ134
MH237701
Rudolf-kameliniakorolkowii (Regel & Schmalh.) Al-Shehbaz & D. A. German
Aheqi, Xinjiang
YC-XZ089
MH237702
Aketao, Xinjiang
YC-XZ107
MH237703
Shangrilaianana Al-Shehbaz, J. P. Yue & H. Sun
Shangrila, Yunnan
CHY008
MH237704
Sisymbriopsismollipila (Maxim.) Botsch.
Yecheng, Xinjiang
YC-XZ119
MH237705
Sisymbriopsispamirica (Y. C. Lan & Z. X. An) Al-Shehbaz, Z. X. An & G. Yang
Aketao, Xinjiang
YC-XZ100
MH237706
Sisymbriopsisschugnana Botsch. & Tzvelev
Aketao, Xinjiang
YC-XZ106
MH237707
Solms-laubachiaangustifolia J. P. Yue, Al-Shehbaz & H. Sun
Daocheng, Sichuan
YZC252
MH237708
Solms-laubachiabaiogoinensis (K. C. Kuan & Z. X. An) J. P. Yue, Al-Shehbaz & H. Sun
Gongbujiangda, Xizang
YZC195
MH237709
Solms-laubachiacalcicola J. P. Yue, Al-Shehbaz & H. Sun
Leiwuqi, Xizang
YZC233
MH237710
Solms-laubachiaeurycarpa (Maxim.) Botsch.
Basu, Xizang
YZC023
MH237711
Solms-laubachiahimalayensis (Cambess.) J. P. Yue, Al-Shehbaz & H. Sun
Ritu, Xizang
YC-XZ130
MH237712
Zhada, Xizang
YC-XZ151
MH237713
Solms-laubachiajafrii (Al-Shehbaz) J. P. Yue, Al-Shehbaz & H. Sun
Lhasa, Xizang
YZC214
MH237714
Jilong, Xizang
NZ143
MH237715
Solms-laubachiakashgarica (Botsch.) J. P. Yue, Al-Shehbaz & H. Sun
Aheqi, Xinjiang
YC-XZ096
MH237716
Solms-laubachialanata Botsch.
Lhasa, Xizang
YZC215
MH237717
Solms-laubachialinearlifolia (W. W. Sm.) O. E. Schulz
Deqin, Yunnan
YZC001
MH237718
Solms-laubachialinearis (N. Busch) J. P. Yue, Al-Shehbaz & H. Sun
Pishan, Xinjiang
YC-XZ123
MH237719
Solms-laubachiamieheorum (Al-Shehbaz) J. P. Yue, Al-Shehbaz & H. Sun
Angren, Xizang
YC-XZ160
MH237720
Solms-laubachiaplatycarpa (Hook. f. & Thomson) Botsch.
Dangxiong, Xizang
YZC216
MH237721
Solms-laubachiaprolifera (Maxim.) J. P. Yue, Al-Shehbaz & H. Sun
Mangkang, Xizang
YZC019
MH237722
Solms-laubachiapulcherrima Muschl.
Lijiang, Yunnan
ChenHongliang202
MH237723
Solms-laubachiaretropilosa Botsch.
Xiangcheng, Sichuan
ChenHongliang209
MH237724
Solms-laubachiavillosa (Maxim.) J. P. Yue, Al-Shehbaz & H. Sun
Yushu, Qinghai
YZC239
MH237725
Solms-laubachiaxerophyte (W. W. Sm.) H. F .Comber
Shangrila, Yunnan
YZC277
MH237726
Solms-laubachiazhongdianensis J. P. Yue, Al-Shehbaz & H. Sun
Shangrila, Yunnan
CHY007
MH237727
Strigosellaafricana (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 Octoceraslehmannianum 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 Eurycarpuslanuginosus (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 Buniaserucago (PP / BS = 0.59 / 76; Figure 1), indicating that S.lanuginosa is neither a member of genus Solms-laubachia nor of the tribe Euclidieae.
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.
https://binary.pensoft.net/fig/211555Discussion
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.
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-laubachialanuginosa, its three accessions formed a clade clustered with Buniaserucago (Buniadeae), Hesperissibirica, 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 Streptolomadesertorum 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 Octoceraslehmannianum (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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Taxa and accession numbers downloaded from GenBank for the ITS sequences used in the study.
Outgroups: Tribe Anchonieae: Sterigmostemumbillardierei (DC.) D. A. German (DQ357513), S.sulphureum (Banks & Sol.) Bornm. (KJ663764). Tribe Buniadeae: Buniaserucago L. (GQ497885). Tribe Dontostemoneae: Clausiaaprica (Stephan ex Willd.) Korn.-Trotzky (LK021257). Tribe Hesperideae: Hesperisisatidea (Boiss.) D. A. German & Al-Shehbaz (GQ497882); Hesperissibirica L. (DQ357549). Ingroups: Tribe Euclidieae: Atelantheraperpusilla Hook. f. & Thomson (FM164518, FM164519); Brayaalpina 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); Catenulinahedysaroides (Botsch.) Soják (GQ424607); Cryptosporafalcata Kar. & Kir. (DQ357532); Cymatocarpuspilosissimus (Trautv.) O. E. Schulz (GQ497858); Dichasianthussubtilissimus (Popov) Ovcz. & Junussov (AY353169); Lachnolomalehmannii Bunge (GQ497889); Leiosporabeketovii (Krasn.) D.A. German & Al-Shehbaz (FN821579); L.exscapa (Ledeb.) F. Dvořák (FN821615), L.saposhnikovii (A.N. Vassiljeva) D.A. German & Al-Shehbaz (FN821554); Lepidostemonglaricola (H.Hara) Al-Shehbaz (GQ424542); Leptaleumfilifolium (Willd.) DC. (KJ623485); Neotorulariacontortuplicata (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); Octoceraslehmannianum Bunge (GQ424609); Pycnoplinthopsisbhutanica (H. Hara) Jafri (GQ497878); Rhammatophyllumafghanicum (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); Spryginiafalcata Botsch (FN821518), S.winkleri (Regel) Popov (GQ424563); Streptolomadesertorum Bunge (FM164618, FM164619); Strigosellabrevipes (Bunge) Botsch. (DQ357558), S.scorpioides (Bunge) Botsch. (KJ623536); Tetracmecontorta Boiss. (DQ357600), T.quadricornis (Stephan ex Willd.) Bunge (DQ357602), T.secunda Boiss. (DQ357604).