taxonID	type	description	language	source
06510721387DFFC203649C3851C6FAB0.taxon	discussion	Apocynaceae is the tenth largest angiosperm plant family, with ca. 5300 species classified in 378 genera (Endress et al., 2018). The family is of particular importance in natural products research because of the occurrence of multiple medicinally important species and compounds including monoterpenoid indole alkaloids, exemplified by the chemotherapy drugs vincristine and vinblastine from Catharanthus roseus (L.) G. Don (Aslam et al., 2010). The expense and difficulty of deriving these valuable molecules from natural sources has motivated the complete biochemical elucidation of their biosynthetic pathway as a steppingstone to its genetic engineering (Caputi et al., 2018; Qu et al., 2019). The genus Asclepias L. and its specialized herbivores are a model system in chemical ecology and evolution of reciprocal adaptations between plants and herbivores, with a particular focus on cardenolides (Agrawal et al., 2012). Pyrrolizidine alkaloids (PAs) are also implicated in co-evolution between Apocynaceae and one of their specialized herbivore lineages, Lepidoptera subfamily Danainae (milkweed and clearwing butterflies). Evolution of the first gene of the PA biosynthetic pathway, homospermidine synthase (hss), has been elucidated (Livshultz et al., 2018 a). Researchers have detected phylogenetic signals in the distribution of all of these compounds and others, including steroidal and phenanthroindolizidine alkaloids and steroidal glycosides, in Apocynaceae (Endress et al., 2018), but knowledge of their taxonomic distribution still lags progress on the phylogeny of the family (Fishbein et al., 2018).	en	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. Phytochemistry (112662) 185: 1-15, DOI: 10.1016/j.phytochem.2021.112662, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112662
065107213878FFC400329B2E5008FB65.taxon	description	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 S 2). 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 S 2. (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) 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 S 2) 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).	en	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. Phytochemistry (112662) 185: 1-15, DOI: 10.1016/j.phytochem.2021.112662, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112662
06510721387BFFC403649B41545BFC52.taxon	description	Eleven species from nine of 13 genera of Malouetieae were sampled. PAs were detected with moderate confidence in Eucorymbia alba Stapf (two samples) and Galactophora schomburgkiana Woodson (one sample) (Table 2). This is the first report of specialized metabolites from these genera. No PAs were detected in the single sample of Galactophora crassifolia (Müll. Arg.) Woodson analyzed (Table S 2), a species that was previously reported as lacking alkaloids (daRocha et al., 1982). Evenly massed ions were detected in the PREC 120 (with MCA) scan in one of two samples of Holarrhena curtisii King & Gamble (m / z 284.3, 300.1) (Table S 2). Kibatalia macrophylla (Pierre) Woodson (one of two samples) contained three possible PAs in its PREC 120 (with MCA) mass spectrum, m / z 284.3, 302.4, and 314.0 (Table S 2). The only previous report of PAs in Malouetieae used the Mattocks’ test (Mattocks, 1967), a colorimetric assay with absorption at 565 nm, to detect unsaturated PAs in the seeds of Holarrhena pubescens (Arseculeratne et al., 1981). In our study, crude extracts of H. pubescens leaves were evaluated for PAs via LC-MS / MS, with no strong evidence of PAs in the PREC scans. In seeds of H. pubescens, two even-massed ions were identified in the PREC 120 scan only, m / z 476.4 and 498.4 (Table 2). These are considered candidate PAs with low confidence. The specialized metabolites of H. pubescens (syn. H. antidysenterica (L.) Wall. ex A. DC., H. floribunda T. Durand & Schinz) are well studied, with steroidal alkaloids and glycosides of primary interest for their potential bioactivity (Kumar et al., 2007; Sinha et al., 2013). Three steroidal amino-glycosides previously reported from H. pubescens (holantosine B, D, and F, MW = 475.3 Da) correspond in mass to one of the ions detected in PREC 120 (Afendi et al., 2011; Janot et al., 1970). Analysis of standards and / or alternative analytical techniques would be required for absolute identification of the compounds detected here.	en	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. Phytochemistry (112662) 185: 1-15, DOI: 10.1016/j.phytochem.2021.112662, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112662
06510721387BFFCB00329D9C53BDFD61.taxon	description	These three tribes form a well-supported monophyletic clade of predominantly New World genera (Fishbein et al., 2018), but the boundaries among them have been redrawn several times (Morales et al., 2017; Sim ˜ oes et al., 2004). Morales et al. (2017) proposed that presence of PAs is a useful chemotaxonomic character for delimiting Echiteae from Odontadenieae and identified their absence in Pentalinon Voigt as supportive of its transfer to Odontadenieae. We sampled eight of fourteen Echiteae, five of nine Odontadenieae, and three of six Mesechiteae genera. PAs were detected in seven of eight sampled Echiteae genera. Only the sampled Prestonia species (P. robusta Rusby, P. tomentosa R. Br.) had no evidence of PAs in the PREC 120 (MCA on) scans (Table S 2), although PAs have been previously reported from P. amabilis J. F. Morales, P. quinquangularis Spreng. [syn. P. acutifolia (Benth. ex Müll. Arg) K. Schum], and P. portobellensis Woodson (Burzynski et al., 2015). PAs in Echites and Parsonsia have been previously reported (Burzynski et al., 2015), but PAs in Bahiella J. F. Morales, Macropharynx (syn. Peltastes), Temnadenia, Rhodocalyx Müll. Arg., and Laubertia A. DC. are here reported for the first time, although Brown (1987) had previously suggested their presence in Macropharynx and Temnadenia based on indirect evidence. Exemplars of these genera had molecular ions appearing at consistent retention times in PREC 120, 138, and 156 scans (Table 2), fragmentation patterns characteristic of the cyclic triesters known from the positive control, Parsonsia alboflavescens (Table 1). Among the 22 species sampled from the other two tribes (Table S 2), only one, Mandevilla boliviensis (J. J. Veitch) Woodson (Mesechiteae), yielded an ion detectable in the PREC 120 scan, but not in PREC 138 or 156 (Table 2). We have low confidence in the identification of this compound as a PA, but this result requires further investigation. Overall, this pattern supports Morales et al. (2017) hypothesis of the chemotaxonomic utility of PA presence for circumscription of Echiteae. PAs are present in all Echiteae genera studied, but presence of detectable PAs varies both among samples of congeneric species and of conspecific individuals (Table S 2, S 3, Table 2). The diversity of PAs within a sample also varies greatly from 18 candidate compounds in Parsonsia alboflavescens (Table 1) to one in Temnadenia odorifera (Vell.) J. F. Morales (Table 2). We have evidence that some of this variation is due to plasticity (see Section 2.11), but the role of genetics remains uncertain. Extensive sampling of species and individuals within Echiteae genera will be necessary to determine if there are any species-specific patterns useful for delimiting sub-tribal or infra-generic taxa.	en	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. Phytochemistry (112662) 185: 1-15, DOI: 10.1016/j.phytochem.2021.112662, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112662
065107213874FFCB03649D7D51EBF952.taxon	description	We sampled 36 species from 17 of 21 genera of Apocyneae. In this tribe, PAs had been previously reported from Anodendron affine Druce (Sasaki and Hirata, 1970) and Amphineurion marginatum (Roxb.) D. J. Middleton (Colegate et al., 2016). These species belong to two early diverging lineages of Apocyneae (subtribes Papuechitinae and Amphineurinae, respectively) and Livshultz et al. (2018 b) suggested that PAs were potentially a chemotaxonomic character for delimiting these early diverging lineages from the rest of the radiation. No PAs were detected with high confidence in any sample of Apocyneae. Two of three accessions of Amphineurion marginatum leaves possessed a molecular ion of m / z 320 which appeared only in the PREC 120 scan with associated chromatography at a late retention time of ~ 15 min (> 3 to 1 S / N) (Table 2). Fragmentation of a PA with an m / z 320 via product ion scan (PIS) by Colegate et al. (2016) was consistent with our PREC 120 findings, as that compound was found to only result in m / z 120 fragment with no m / z 138 or 156 fragments. HRMS was utilized to propose a molecular formula for the m / z 320 ion, C 18 H 26 NO 4, and therefore it was tentatively identified as an open-chain diester (Colegate et al., 2016). Colegate et al. (2016) reported that leaves of Amphineurion marginatum contained the lowest percentage by dry weight of PAs (0.02 %) compared to roots (0.13 %) and stems (0.09 %). This may account for the lack of PA structural diversity and abundance observed in our leaf samples but other factors, environmentally induced plasticity, genetic variation, cannot be ruled out in the present study. A product scan and / or HRMS would need to be completed on the m / z 320 molecular ion in our Amphineurion marginatum leaf samples to understand fully the fragmentation pattern and identity of this compound. The unusual PAs previously identified in Anodendron affine have a platynecine core (Sasaki and Hirata, 1970) that would not be detectable with our strategy. Thus, the occurrence and distribution of platynecine-derived PAs in other Apocyneae species also remains an open question.	en	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. Phytochemistry (112662) 185: 1-15, DOI: 10.1016/j.phytochem.2021.112662, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112662
065107213874FFCB0364988C55D4FD44.taxon	description	We sampled 26 species from 10 of 27 genera of tribe Marsdenieae. Compounds fragmenting to m / z 120 and 138 were present in samples of Marsdenia tinctoria R. Br. and Marsdenia glabra Constantin (Table 2). However, three of these four ions have a molecular mass of 432.5, raising the possibility that these are structurally similar to the aminobenzoyl glycoside previously identified in Wrightia antidysenterica (Srinroch et al., 2019) (Fig. 5) rather than PAs. HRMS would need to be used to determine if the m / z 432 ions observed in Marsdenia and Wrightia species are the same compound. Thus we have moderate confidence in the presence of PAs only in the one sample of M. glabra, based on the presence of an m / z 388.2 ion. The current taxonomic concept of Marsdenia R. Br. is highly polyphyletic (Rodda et al., 2020), and the segregation of monophyletic genera from Marsdenia sensu lato is ongoing (Espírito Santo et al., 2019). Marsdenia tinctoria (the type species) and M. glabra belong to the small group of species segregated as Marsdenia sensu stricto (Bullock, 1956; Forster, 1995). The most frequently reported natural products from species of Marsdenia sensu lato and other genera of Marsdenieae are steroids, steroidal alkaloids, and steroidal glycosides including the antisweet gymnemic acids from the medicinal plant Gymnema sylvestre (Retz.) R. Br. ex Sm. (Liu et al., 1992). Phenanthroindolizidine alkaloids are rare, reported only from Telosma pallida (Roxb.) Craib (Mulchandani and Venkatachalam, 1976). All three common classes of compounds have been previously reported from M. tinctoria (Chowdhury et al., 1994; Gao et al., 2009). Marsdenia tinctoria is also a source of indigo (Mohd Nasuha and Choo, 2016), although the chemical origin of the stain has not been identified in this species.	en	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. Phytochemistry (112662) 185: 1-15, DOI: 10.1016/j.phytochem.2021.112662, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112662
