Sisymbriumlinifolium and Sisymbriopsisschugnana (Brassicaceae), two new records from Xinjiang, China

Abstract Sisymbriumlinifolium and Sisymbriopsisschugnana, previously confined to western North America and Tajikistan, respectively, were discovered in Xinjiang during a recent field trip to this autonomous region of China. The identity of these two species was subsequently confirmed by extensive morphological and molecular studies. The biogeographical significance of these new floristic records is briefly addressed.

Two of the authors (H.L.C and J.P.Y) have conducted a botanical expedition to Xinji-

Plant materials and molecular data
Collected specimens were deposited in KUN, and species identification was based on the floras of China (Zhou et al. 2001), Pan-Himalaya (Al-Shehbaz 2015d), and North America ) and studies on Sisymbrium L. (Al-Shehbaz 1988, 2006, Warwick and Al-Shehbaz 2003 and Sisymbriopsis (Al-Shehbaz et al. 1999).
The nrITS sequence of Sisymbriopsis schugnana was included in our previous study on the phylogeny of the tribe Euclidieae (Chen et al. 2018b), while nrITS sequences of four individuals of Sisymbrium linifolium were generated and analysed in this study. An additional 48 sequences, representing 19 Sisymbrium species (mostly from Warwick et al. 2002) Mutlu and Karakuş (2015), N. torulosa and C. bursa-pastoris were used as outgroups.

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 ITS-4 (White et al. 1990). All polymerase chain reactions (PCR) were performed in a 25 μl volume consisting of 1μl sample DNA (approx. 5-10 ng), 12.5μl Premix Taq (Takara Biomedical Technology, Beijing, China), 1μl of 10 μM stock of each primer, adjusted to 25 μl with ddH 2 O. The PCR protocol of the ITS region involved a hot start with 4 min at 94 °C, and 32 cycles of amplification (1 min denaturing at 94 °C, 45 s annealing at 53 °C, 60 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 analysed with maximum parsimony (MP) and Bayesian Inference (BI).
Parsimony analysis was performed with heuristic searches of 1000 replicates with random stepwise addition using tree bisection reconnection (TBR) branch swapping as implemented in PAUP* 4.0a161 (Swofford 2018). All characters were weighted equally, and gaps were treated as missing data. 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 (AIC), 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 v3.2.5 (Ronquist et al. 2012), four simultaneous Monte Carlo Markov chains (MCMCs) were run for 3 million generations, and one tree sampled every 1000 generations. The first 750 trees (25% of total trees) were discarded as burn-in. The remaining trees were summarised in a 50% majority-rule consensus tree, and the posterior probabilities (PP) were calculated.

Results
The aligned ITS dataset comprised 24 species (59 accessions) with 584 characters, of which 192 were variable and 152 (26.03%) were parsimony-informative. Four individuals of the newly collected Sisymbrium from Xinjiang have exact sequences, and sequence divergence between them and S. linifolium ranged from 0-0.2%, which was lower than that of 1.5% compared with S. polymorphum (Murray) Roth.
The generated MP trees had a very similar topology to the Bayesian tree, thus only the BI topology, which is almost as same as the result of Mutlu and Karakuş (2015), is shown. The four Xinjiang Sisymbrium clustered with S. linifolium (PP/BS = 1/97), and then clustered with S. polymorphum (PP/BS = 0.65/63) and S. loeselii L. (PP/BS = 0.78/54) (Figure 4b). Furthermore, sequences alignment revealed that the Xinjiang plants and North American S. linifolium shared several specific nucleotide residues that are different from S. polymorphum (Figure 4a), which further their identity as S. linifolium.

Sisymbrium linifolium (Nutt.) Nutt
The generic placement of Sisymbrium linifolium has long been in dispute. It was originally placed in Nasturtium W.T.Aiton (Nuttall, 1834), and then transferred to Sisymbrium (Nuttall in Torrey and Gray, 1838) and Schoenocrambe Greene (Greene, 1896). Though several authors claimed that, on aspects of habit, leaves and flowers morphology, this species is very similar to the Eurasian S. polymorphum and retained it in Sisymbrium (Payson 1922, Schulz 1924, A1-Shehbaz 1973, while others kept it in Schoenocrambe (Rollins 1982(Rollins , 1993. Molecular phylogenetic study on Sisymbrium, using ITS sequence data, revealed that S. linifolium is most closely related to S. polymorphum within the Old World Sisymbrium clade of tribe Sisymbrieae, while all other New World Sisymbrium were placed in various genera of the tribe Thelypodieae (Warwick et al. 2002). These results prompted Warwick and Al-Shehbaz (2003) to propose nomenclatural adjustments for some Sisymbrium species and further delimit Sisymbrium to include only 40 Old World species, plus North American S. linifolium, instead of the 96 species previously assigned to it (Al-Shehbaz, 2006).
Based on morphology, the Xinjiang Sisymbrium material we collected could be identified as S. polymorphum, but both phylogenetic analyses and sequence alignments supported its placement in S. linifolium (Figure 4). This conclusion makes the distribution range of S. linifolium extended from North America into north-western China, with a large range disjunction (Figure 3). One possible explanation for such distribution is a recent introduction of seeds of S. linifolium from North America to China by unintentional human activities. Many weeds of the mustard family (e.g., Capsella bursa-pastoris, Thlaspi arvense L., and Sisymbrium orientale) are invasive in both continents under preferable habitats (Zhou et al. 2007) such as farmlands, construction sites and ruins, waste places, disturbed sites, and roadsides. The Xinjiang S. linifolium was collected from a rocky hillside near the provincial road S229 in Jeminay County ( Figure 1C). This locality is far from any villages or towns and, therefore, the possibility that its occurrence was the result of human activity is less likely. However, introduction with road construction material cannot be excluded as well.
Another possible explanation is that Sisymbrium linifolium actually has both North American and Central Asian distribution, and most, if not all, of its Asian populations  were misidentified as the very similar species, S. polymorphum. Further molecular phylogenetic studies and crossing experiments on more populations from both continents are needed to determine whether a single species or two are in fact involved. If it turned out that the species grows on both continents, then the name for the combined species should be the earlier-published one, S. polymorphum.

Sisymbriopsis schugnana Botsch. & Tzvelev
Sisymbriopsis Botsch. & Tzvelev was originally recognised as a monospecific genus including S. schugnana as its type (Botschantsev and Tzvelev 1961). A second species, S. mollipila (Maxim.) Botsch., was transferred from Sisymbrium by Botschantsev (1966), andAl-Shehbaz et al. (1999) recognised three other species. Of the five species currently assigned to the genus, S. schugnana is endemic to Tajikistan, S. mollipila occurs in China, Kyrgyzstan, and Tajikistan, and the other three species are endemic to China. However, in a molecular phylogenetic study by Warwick et al. (2004), S. mollipila and S. yechengensis (C.H.An) Al-Shehbaz, C.H.An & G.Yang were found unrelated to each other, and the former was close to some Neotorularia species, whereas the position of S. yechengensis was unresolved. In a later phylogenetic study (Warwick et al. 2007), S. mollipila was found nested within a clade containing species of the genera Desideria, Rhammatophyllum O.E.Schulz, and Solms-laubachia, whereas    The material studied here was collected from alluvium of the Muztagata (also Mugtag Ata) Glacial Public Park in Tashkurgan County, Xinjiang, an area close to the borders of Tajikistan. The plant has decumbent stems, dentate and palmately veined leaves, linear and latiseptate secund fruit, and white to pink flowers (Figure 2). Our initial morphological studies failed to identify the plant using Zhou et al. (2001), but subsequent molecular sequence comparison narrowed its identity to Sisymbriopsis, and its unique secund fruits led to its recognition as S. schugnana and a new record from China. In addition, species of S. pamirica (Y.C.Lan & C.H.An) Al-Shehbaz, C.H.An & G.Yang, S. mollipila, and Anzhengxia yechengnica (C.H.An) Al-Shehbaz & D.A. German were recently included in a phylogenetic study on the tribe Euclidieae (Chen et al. 2018b). Three Sisymbriopsis species formed a monophyletic subclade embedded in the Solms-laubachia s.l. clade, and A. yechengnica was close to Pycnoplinthus uniflora (Hook.f. & Thomson) O.E.Schulz., these findings suggesting that the real identity of Sisymbriopsis is still awaiting further studies (Chen et al. 2018b).
Sisymbriopsis schugnana is narrowly distributed in the eastern Pamir (Figure 3), a dry and cold desert plateau currently subjected to severe desertification caused by extensive exploitation of dwarf shrub resources, a phenomenon termed "Teresken Syndrome" (Kraudzun et al. 2014). Discovery of the first population of S. schugnana within the poorly explored Chinese mountains bordering Tajikistan should promote further botanical explorations in similar areas of adjacent neighbouring countries.