Wrightieae R. Br.

2.4. Wrightieae Wrightieae are sister to the rest of the APSA clade and include three genera (Livshultz et al., 2007). Potential PAs were detected in leaves of 5 of 11 species of Wrightia R.Br. and in seeds of Wrightia tinctoria (Table 2) but none of the three species of Pleioceras Baill. and Stephanostema K. Schum sampled (Table S2). The most frequent candidate ions had m/z 432.3–432.5, containing both m/z 120 and 138 fragments. These compounds have a similar mass to a modified glycoside, 2-aminobenzoyl O-β- D-apiofuranosyl-(1 → 6)-β- D-glucopyranoside (MW = 431.4), from W. antidysenterica, that is also predicted to yield m/z 120 and 138 fragments (Fig. 5) (Srinroch et al., 2019). Thus we consider it unlikely that these compounds are PAs. In W. tinctoria samples, there is coelution with m/z 270.2, 300.2 and 328.3 compounds that appeared with lower abundances (counts per second). These compounds are identified as PAs with moderate confidence based on their fragmentation patterns (m/z 120, 138). Alterations to the chromatography method associated with these scans can increase the resolution of these compounds currently co-eluting within the chromatography, thereby increasing the sensitivity to these ions by diminishing ion suppression. One of these candidate PAs (m/z 300.2) could be a breakdown product of the putative aminobenzoyl glycoside (m/z 432.4) (Fig. 5). The D-apiofuranosyl residue, joined by a beta-1,6 linkage to the D-glucopyranoside, could be lost via insource fragmentation resulting in the appearance of the precursor ion, m/z 432, in addition to a m/z 300 ion in PREC 120 and 138 scans at the same retention time (Fig. 5). A standard of 2-aminobenzoyl O -β -D-apiofuranosyl-(1 → 6)- β -D-glucopyranoside would need to be analyzed on the devised precursor scans to determine if insource fragmentation is a potential cause of the m/z 300 ion in W. tinctoria samples. The benzoxazinoid glycoside, blepharin (MW = 327.3), reported from W. religiosa (Sahakitpichan et al., 2018), has a similar mass to another candidate PA from W. tinctoria (m/z 328.3). Blepharin is not predicted to readily yield m/z 120 or 138 compounds under our experimental conditions, but a mass spectrum for the compound, obtained in negative electrospray ionization (ESI-) mode, has low abundance ions at m/z 118 and 136 (Horai et al., 2010; Tsugawa et al., 2019). Multiple other PREC 120 only ions appeared in these samples including m/z 210.2, 352.1, and 460.0. We suggest with low confidence these molecular ions may be associated with PAs. Table 2 Samples analyzed on all PREC scans (m/z 120, 138, 156). Samples determined as N: no candidate ions detected, Y (low): low confidence, candidate ions detected only in PREC 120, Y (mod):moderate confidence, candidate ions detected in PREC 120 and PREC 138; Y (low): only candidate ion with mass 432.4–432.5 detected in PREC 120 and PREC 138; Y (high): high confidence, candidate ions detected in PREC 120, 138, and 156. ND: not detected. Vouchers in Table S2. (continued on next page) Table 2 (continued) Tribe Species Author sample organ Determination PREC PREC PREC Uncorrected Relative # 120 138 156 Retention Time (min) (continued on next page) a Aminobenzoyl glycoside of similar mass and predicted fragmentation pattern previously reported in Wrightia antidysenterica (Srinroch et al., 2019) (Fig. 5). Previously reported alkaloids from Wrightia species include steroidal (pregnane) alkaloids from Wrightia pubescens subsp. laniti (Blanco) Ngan (syn. Wrightia javanica A.DC.) (Kawamoto et al., 2003). This species is sampled here (Table S2) and determined as PA negative. Three species, W. tinctoria, W. dubia Spreng., and W. arborea (Dennst.) Mabb. (syn. W. tomentosa Roem. & Schult.), are used as sources of indigo (Ngan, 1965), and the indole-containing compounds indigotin and indirubin are known from W. tinctoria, as well as the quinolizidine, tryptanthrin (Khyade, 2014), the later also reported from W. pubescens R. Br. (Traxler et al., 2021). Recent studies of W. religiosa, W. antidysenterica, and W. pubescens have identified benzoxazinoids, megastigmane glycosides, indoxyl glycosides, simple aromatic glycosides, flavonoid glycosides, and lignans (Sahakitpichan et al., 2018; Srinroch et al., 2019; Traxler et al., 2021), but only one compound, the aforementioned aminobenzoyl glycoside (Srinroch et al., 2019), is predicted to yield m/z 120 and 138 fragments (Fig. 5).Published as part of Barny, Lea A., Tasca, Julia A., Sanchez, Hugo A., Smith, Chelsea R., Koptur, Suzanne, Livshultz, Tatyana & Minbiole, Kevin P. C., 2021, Chemotaxonomic investigation of Apocynaceae for retronecine-type pyrrolizidine alkaloids using HPLC-MS / MS, pp. 1-15 in Phytochemistry (112662) (112662) 185 on pages 5-8, DOI: 10.1016/j.phytochem.2021.112662, http://zenodo.org/record/825966