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 View Fig ) ( 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 View Fig ). 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 View Fig ). 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.

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Table 2 ( continued)

Tribe Species Author sample organ Determination PREC PREC PREC Uncorrected Relative

# 120 138 156 Retention Time (min)

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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 View Fig ).