Wen Xu

Jianhua Wang

Baozhao Ju
email: jubaozhao@163.com

Xiujuan Lan
email: xiujuanlan@139.com

Xixiang Ying
email: yingxixiang@163.com

Dr Didier Stien
email: didier.stien@cnrs.fr

Prof Ying Xixiang

Seven compounds from Portulaca oleracea L. and their anticholinesterase activities

Keywords: Portulaca oleracea L., lignan, isolation, anticholinesterase activity

A novel lignan, identified as 4- (3,4-dihydroxyphenyl)-6,7-dimethoxy-3a,4-dihydronaphtho[2,3-c]furan-1(3H)-one, named oleralignan A (1), together with six known compounds, loliolide (2), isololiolide (3), dehydrololiolide (4), daphnetin (5), esculetin (6), and trans-coumaric acid methyl ester (7) was obtained from Portulaca oleracea L., while compounds 3, 4, and 6-7 were isolated from the plant for the first time. Their structures were elucidated using spectroscopic methods, including one-and two-dimensional NMR and high-resolution electrospray ionization time-of-flight mass spectrometry. In addition, the results of activity assay demonstrated that compounds 1-7 have anticholinesterase activities.

Introduction

Portulaca oleracea L., an annual green herb, belonging to the family of Portulacaceae, with branched and succulent stems, is edible and has a slightly acidic and salty taste similar to spinach [START_REF] Lim | Antioxidant properties of different cultivars of Portulaca oleracea[END_REF]. It is consumed as a vegetable and has been reported to provide better nutrition than the major cultivated vegetables [START_REF] Yazici | Salinity tolerance of purslane (Portulaca oleracea L.) is achieved by enhanced antioxidative system, lower level of lipid peroxidation and proline accumulation[END_REF]. It is widely distributed in Africa, China, India, Australia, Middle East, Europe and America (Rashed et al. 2003). It has been listed by the World Health Organization as one of the most used medi p m 'G P ' [START_REF] Zheng | Antihepatocarcinoma effect of Portulaca oleracea L. in mice by PI3K/Akt/mTOR and Nrf2/HO-1/NF-κB pathway[END_REF]. As a traditional Chinese medicine, it is well-known as diuretic, febrifuge, antiseptic, antispasmodic and vermifuge [START_REF] Shen | Purification and characterization of an antitumor polysaccharide from Portulaca oleracea L[END_REF]. At the same time, P. oleracea L. has been reported to possess a wide variety of pharmacological effects, such as anti-inflammatory [START_REF] Meng | The anti-inflammation and pharmacokinetics of a novel alkaloid from Portulaca oleracea L[END_REF], antibacterial [START_REF] Nayaka | Antibacterial attributes of apigenin, isolated from Portulaca oleracea L[END_REF], antiulcerogenic [START_REF] Karimi | Evaluation of the gastric antiulcerogenic effects of Portulaca oleracea L. extracts in mice[END_REF], antitumor [START_REF] Shen | Purification and characterization of an antitumor polysaccharide from Portulaca oleracea L[END_REF], antioxidant [START_REF] Yang | A new homoisoflavone from Portulaca oleracea L. and its antioxidant activity[END_REF], antidiabetic [START_REF] Bai | Anti-diabetic effect of Portulaca oleracea L. polysaccharideandits mechanism in diabetic rats[END_REF], or anticholinesterase [START_REF] Xiu | A new alkaloid from Portulaca oleracea L. and its antiacetylcholinesterase activity[END_REF], among others. These activities have been related

to its chemical constituents including alkaloids [START_REF] Zhao | Two new alkaloids from Portulaca oleracea L. and their bioactivities[END_REF], flavonoids [START_REF] Yang | New flavonoids from Portulaca oleracea L. and their activities[END_REF], lignans [START_REF] Ma | Four lignans from Portulaca oleracea L. and its antioxidant activities[END_REF], fatty acids [START_REF] Teixeira | Omega-3 fatty acid desaturase genes isolated from purslane (Portulaca oleracea L.): expression in different tissues and response to cold and wound stress[END_REF], or minerals [START_REF] Uddin | Evaluation of antioxidant properties and mineral composition of purslane (Portulaca oleracea L.) at different growth stages[END_REF]). In the present work, we isolated 7 intersting compounds from P.

oleracea L. including 1 new arylnaphthalene lignan containing 2 methoxy groups. At the same time, some lignans have been reported to have anticholinesterase activity, and the methoxy group in lignans can effectively enhance its anticholinesterase effect [START_REF] Salleh | Anticholinesterase and anti-inflammatory constituents from Beilschmiedia pulverulenta kosterm[END_REF]). However, study on the anticholinesterase activity of arylnaphthalene lignans has not been found. Therefore, we undertook to evaluate their f w m f E m ' m .

Results and discussion

Structure elucidation

Figure 1

With regard to the structural determination itself, it was possible to establish the presence of a 1,2,4,5-tetrasubstituted benzene ring based on the signals of protons H-5

(δ H 6.16) and H-8 (δ H 7.16) together with their respective carbons at δ C 114.9 and 113.9 and their HMBC correlations with non-protonated carbons of the benzene ring (Table S1 in supplementary material, Figure 2). Since protons H-5 and H-8 correlated with both C-6 and C-7, the assignment of both methoxy groups was based on the COSY and ROESY correlations of MeO-C7 with H-8, but also the ROESY correlation of MeO-C6 with H-2'. The COSY and ROESY spectra also provided key information regarding the substituents in positions 4a and 8a. Thanks to these data, it was possible to position H-4 close to H-5 and H-9 close to H-8. This was confirmed by the HMBC correlations of H-4 with C-4a and C-8a and of H-9 with C-4a, C-8a and C-8. The COSY spectrum defined a proton sequence H-4/H-3a/H-3, and the proton and carbon chemical shifts in position 3 (δ H 3.97/4.33, δ C 71.5) demonstrated that C-3 was link to an oxygen. The HMBC correlations H-9/C-3a, H-9/C-1, and H-3-y/C-1 completed the 3a,4-dihydroisobenzofuran-1-one subunit.

Figure 2

Additional correlations of H-4 with C-1', C-2' and C-6' demonstrated that an aromatic ring was linked to C-4 and that both ortho positions were protonated. The fact that the signals are very wide and are split does not prevent the structural determination because protons H-2' and H-6' also correlated in ROESY with H-3a, H-4 and for H-2', MeO-C6. As the lateral aromatic ring could not be symmetrical, two hydroxy groups had to be positioned on the C-3' and C-4' carbons. The hydroxy protons are visible in 1 H NMR at δ H 8.97and 9.45. The remaining methine was assigned to position 5'. The relative configuration of compound 1 was ascertained based on the 8.6 Hz coupling between H-3a and H-4 along with the ROESY correlations H-4/H-3-x and H-3a/H-3-y which were consistent with an anti-periplanar configuration of protons H-3a and H-4.

Coupling constants of 8-10 Hz are found in analogous compounds described in the literature, including conidendrin [START_REF] Barton | Preparation and structure of dimethyl-α-conidendrin-8-sulfonamide[END_REF][START_REF] Davies | Intermolecular C-H activation at benzylic positions: synthesis of (+)-imperanene and (-)-α-conidendrin[END_REF]. Eventually, compound 1 was identified as a conidendrin-related lignan and was given the trivial name oleralignan A.

By comparison of the 1 H and 13 C NMR spectral data with literature, the 6 known compounds (2-7) were identified, respectively, as loliolide (2) [START_REF] Mori | Carotenoids and degraded carotenoids, VIII: Synthesis of (+)-dihydroactinidiolide, (+)-and (-)-actinidiolide, (+)-and (-)-loliolide as well as (+)-and (-)-epiloliolide[END_REF], isololiolide (3) [START_REF] Zheng | γ-lactone derivatives and terpenoids from selaginella uncinata and their protective effect against anoxia[END_REF], dehydrololiolide (4) [START_REF] Zheng | γ-lactone derivatives and terpenoids from selaginella uncinata and their protective effect against anoxia[END_REF], daphnetin (5) [START_REF] Xu | Studies on the chemical constituents of Daphne pedunculata[END_REF], esculetin (6) [START_REF] Zhou | A new dicoumarin and anticoagulant activity from Viola yedoensis Makino[END_REF]), trans-coumaric acid methyl ester (7) [START_REF] Wan | Antioxidant and alpha-glucosidase inhibitory phenolics isolated from highbush blueberry flowers[END_REF].

Anticholinesterase activity

Alzheimer's disease (AD) is the most common neurodegenerative disorder of aging, accounting approximately for two-third of all cases of senile dementia [START_REF] Tandon | Molecular genetics of alzheimer's disease: the role of β-amyloid and the presenilins[END_REF]. AD is neurochemically characterised by a consistent deficit in cholinergic neurotransmission, particularly affecting cholinergic neurons in the basal forebrain.

Symptoms of AD and other forms of dementia can be treated by the use of agents which restore the level of acetylcholine through inhibition of both major cholinesterases: acetylcholinesterase and butyrylcholinesterase [START_REF] Loizzo | Chemical analysis, antioxidant, antiinflammatory and anticholinesterase activities of Origanum ehrenbergii Boiss and Origanum syriacum L. essential oils[END_REF].

Studies have shown that some lignans exhibit neuroprotective effects, increasing choline acetyltransferase activity, inhibiting acetylcholinesterase (AChE) activity in vitro, and increasing hippocampal acetylcholine release in vivo [START_REF] Howes | Plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function[END_REF]. Consequently, the anticholinesterase activities of the 7 compounds were evaluated using a microplate assay. Oleralignan A (1) inhibited enzyme activity at 58.31 ± 0.23 μΜ. Compounds 2-7 activity ranged from 71.50 ± 0.39 μΜ 93.88 ± 0.39 μΜ. Overall, oleralignan A exhibited higher anticholinesterase activity than compounds 2-7(Table S2, in supplementary material).

Experimental

General experimental procedures

The NMR spectra were obtained using an AVANCE 600 MHz instrument (Bruker Corporation, Switzerland). HRMS was determined using a 6520

quadrupole-time-of-flight mass spectrometer (Agilent, Palo Alto, CA). The UV data was obtained from a HITACHI U-3010 spectrophotometer (Hitachi Ltd, Tokyo, Japan), the IR spectrum and data obtained from IR200 spectrophotometer (Thermo Electron Corporation, Waltham, MA). In the separation process, the GF 254 (Qingdao Marine Chemical Co., Qingdao, China) was being as TLC filling, the silica gel

Plant materials and chemicals

The whole herbs of P. oleracea L. were collected in Shijiazhuang (Hebei, China)

Isolation and identification

The dried whole herb of P. oleracea L. (250 kg) were extracted twice with 10 volumes of water for 2 hours each time, and then the water extract was concentrated to obtain a crude extract (21 kg), which was subjected to a 100-200 mesh silica-gel column chromatography (61 × 55 cm, approximately, 70 kg) with a 2 volumes of ethyl acetate 3 times as the isocratic elution, affording the fraction evaporated (550 g).

Then, the fraction was subjected to the 80-100 mesh polyamide resin column chromatography (120 × 8 cm, approximately, 1 kg), eluting with water, 30%, 50%, 70% and 100% ethanol to obtain five fractions (4 L each). The fraction of 50% extracting (121 g) was condensed then subjected to a 200-300 mesh silica-gel column (120 × 8 cm, approximately, 1 kg) for further purification with ethyl acetate, ethyl acetate and methanol (5:1, 2:1, v/v) as the gradient eluant, obtaining three fractions as the gradient eluant under medium pressure, acquiring five fractions (A1-A5, 500 mL each). Among that, A1 was purified on a Sephadex LH-20 m 100 φ 2 × 150 cm) using methanol solvent to obtain seven fractions B1-B7, and then these fractions were further prepared by UHPLC, using MeOH-0.1% formic acid as the mobile phase, with flow rate of 1.0 mL/min, and obtained compound

Anticholinesterase activity assay

Acetylcholinesterase activities of compounds were y m f E m 's method [START_REF] Ellman | A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF]. In a 96-well plate, 140 μ PBS (0.1 M, pH = 8.0,

Conclusion

In the study, a novel lignan named oleralignan A and six known compounds were isolated from the water extract of P. oleracea L. The results of anticholinesterase activities assay showed that oleralignan A presented a higher anticholinesterase activity than others, with IC 50 value of 58.31 ± 0.23 μΜ.

( 100 -

 100  Qingdao Marine Chemical Co., Qingdao, China), polyamide resin (80-100 mesh, Taizhou Luqiao Sijia Biochemical Plastic Factory, Zhejiang, China) and ODS (20-40 μm GE Healthcare, Marlborough, MA) were used as column chromatographic filling. The 96-well microplate reader (HBS-1096A) was from Nanjing Detie Experimental Equipment Co., Ltd. (Nanjing, China).

(

   4 L each). The fraction of ethyl acetate (69 g) was subjected to the 20-40 μm octadecylsilyl (ODS) column chromatography (25 × 3 cm, approximately, 150 g, Ultimate XB-C18) with methanol and water(60:40, 70:30, 80:20, 90:10, 100:0, v/v)

  at 20°C for 10 minutes. The absorbance was measured at 405 nm.Eserine and methanol were used as positive and blank controls, respectively. The inhibition rate (%) of each sample AChE was calculated according to the following formula[START_REF] Aktumsek | Antioxidant potentials and anticholinesterase activities of methanolic and aqueous extracts of three endemic Centaurea L. species[END_REF]): Inhibition activity (%) = [(A blank -A sample )/A blank ] × 100%, in which, A sample and A blank were respectively the absorbance of the test compound and the blank control. The anticholinesterase activity was evaluated by the value of IC 50 (means ± SD, n = 3).

  This phenyl ring appears in 1 H NMR in the form of a very broad signal between 6.70 and 6.94 ppm integrating for 3 protons. The signals of the methoxy group in C-6 (δ H 3.73, δ C 55.6) and the methine 3a (δ H 3.55, δ C 39.7) are also very broad due to the proximity of the rotating phenyl ring.

	ring (1'-6').
	Compound 1, a yellowish powder, has the molecular formula C 20 H 18 O 6 , as deduced
	from the HR-ESI-TOF-MS deprotonated molecular ion at m/z 353.1021 [M-H] -
	(calcd. for C 20 H 17 O 6 -, 353.1031), corresponding to 12 degrees of unsaturation. The
	examination of the 1 H, 13 C, and HSQC spectra of 1 demonstrated that several signals
	were very broad in 1 H and 13 C NMR spectra. Some signals were also duplicated. This
	eventually turned out to originate from the slow rotation of the unsymmetrical phenyl

  in June 2017, and identified by Prof. Xixiang Ying. The voucher specimens (No. 20171001) were deposited at School of Pharmacy, Liaoning University of Traditional

	Chinese Medicine. Acetylthiocholine iodide (ATCI) (pur y ≥ 99%	A E
	y ≥200	/m p		were from Dalian Meilun Biotechnology Co., Ltd.
	(Dalian, China), 5, 5-dithiobis-2-	z	DT	p y ≥ 99% was from
	Shanghai Jinsui Bio-Technology Co., Ltd. (Shanghai, China) and eserine (p y ≥
	98%) was from Shanghai Hanxiang Biotechnology Co., Ltd. (Shanghai, China). The
	HPLC grade formic acid, acetonitrile and methanol were purchased from Damao
	m	P	T j		p y ≥ 99.9% . A	w
	analytical grade reagents supplied by Jinfeng Chemical Factory (Tianjin, China), and
	the purified water was from WAHAHA Company (Shenyang, China).

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant number 82073990).

Supplementary material

Supporting information can be found in the online version of this article.

Disclosure statement

The authors have no conflicts of interest to disclose.


Anurag A Agrawal
email: agrawal@cornell.edu

Katalin Böröczky

Meena Haribal

Amy P Hastings

Ron A White

Ren-Wang Jiang

Christophe Duplais

Cardenolides, toxicity, and the costs of sequestration in the coevolutionary interaction

Keywords: Chemical ecology, coevolution, monarch butterfly (Danaus plexippus), milkweeds Asclepias, plant defense theory, plant-insect interactions, toxin sequestration

come

experiments on isolated toxins and the monarch's neural sodium-potassium pump enzymes, we show that tropical milkweed produces a burdensome cardenolide toxin and monarchs convert it to less toxic compounds, the latter sequestered for their own benefit.

Introduction

Although coevolutionary interactions are often portrayed as simplified arms races of reciprocal defense and offense evolution, the dynamics are decidedly more complex. For example, how do plants respond to highly specialized herbivores, and are such adapted consumers immune to plant defenses? On average, specialists are less impacted by particular plant defense compounds than generalists [START_REF] Cornell | Herbivore responses to plant secondary compounds: A test of phytochemical coevolution theory[END_REF][START_REF] Rothwell | Phytochemical defences and performance of specialist and generalist herbivores: a meta-analysis[END_REF], but does this mean that further coevolution is not possible? Even highly specialized herbivores must contend with plant defenses if coevolutionary interactions are proceeding [START_REF] Berenbaum | Postgenomic chemical ecology: from genetic code to ecological interactions[END_REF]. For any herbivorous insect, larval feeding, protection from enemies, and adult oviposition are each key points in the life-cycle where plant chemistry plays a role in the outcome. Thus, the typical cornucopia of chemical compounds in an individual plant presents opportunities for both plant resistance and cooption of this defense by specialist herbivores [START_REF] Renwick | Isothiocyanates stimulating oviposition by the diamondback moth, Plutella xylostella[END_REF][START_REF] Nieminen | Effect of iridoid glycoside content on oviposition host plant choice and parasitism in a specialist herbivore[END_REF][START_REF] Heisswolf | Selection of large host plants for oviposition by a monophagous leaf beetle: nutritional quality or enemy-free space?[END_REF].

It is thus far unclear how often coevolutionary interactions reach equilibrium or "stalemate" as it were [START_REF] Berenbaum | Constraints on chemical coevolution: wild parsnips and the parsnip webworm[END_REF]. Nonetheless, several conditions are predicted to slow or suppress the endless arms race. First, the more specialized an interaction, the greater the investments required and potential challenges to innovation. Second, when different life-stages of herbivores are subject to distinct selection pressures [START_REF] Scheirs | Optimization of adult performance determines host choice in a grass miner[END_REF][START_REF] Jones | Beyond preference and performance: host plant selection by monarch butterflies, Danaus plexippus[END_REF][START_REF] Gripenberg | A meta-analysis of preferenceperformance relationships in phytophagous insects[END_REF], continued coevolution may be restricted because of conflicting selection. And finally, when aspects of the population biology of the species involved reduce local adaptation, such as gene flow and the presence of alternate hosts, asymmetry may emerge in the coevolutionary match between plants and herbivores [START_REF] Thompson | The Geographic Mosaic of Coevolution[END_REF][START_REF] Zangerl | Phenotype matching in wild parsnip and parsnip webworms: Causes and consequences[END_REF]. In the interaction between milkweed plants and monarch butterflies, cardenolides have played a central role in our understanding of coevolutionary specialization, larval feeding, sequestration, and to a lesser extent, oviposition [START_REF] Agrawal | Monarchs and milkweed: A migrating butterfly, a poisonous plant, and their remarkable story of coevolution[END_REF]. Although monarchs are abundant across a broad geographical range, substantial phenotypic and genetic analyses have failed to reveal population differentiation [START_REF] Zhan | The genetics of monarch butterfly migration and warning colouration[END_REF][START_REF] Freedman | Host plant adaptation during contemporary range expansion in the monarch butterfly[END_REF]. A lack of local adaptation is likely due to the four-generation annual cycle, where butterflies feed on diverse milkweed species and yet intermix during migration and overwintering [START_REF] Agrawal | Monarchs and milkweed: A migrating butterfly, a poisonous plant, and their remarkable story of coevolution[END_REF].

There is some evidence that cardenolides can be a burden for monarch caterpillars [START_REF] Brower | Palatability dynamics of cardenolides in monarch butterfly[END_REF][START_REF] Agrawal | Natural selection on common milkweed (Asclepias syriaca) by a community of specialized insect herbivores[END_REF][START_REF] Zalucki | Detrimental effects of latex and cardiac glycosides on survival and growth of first-instar monarch butterfly larvae Danaus plexippus feeding on the sandhill milkweed Asclepias humistrata[END_REF][START_REF] Rasmann | Induced responses to herbivory and jasmonate in three milkweed species[END_REF][START_REF] Seiber | Pharmacodynamics of some individual milkweed cardenolides fed to larvae of the monarch butterfly (Danaus plexippus L.)[END_REF], although costs of sequestration have not been demonstrated. Nonetheless, many assays, even across >10-fold concentrations of cardenolides, fail to show negative effects of cardenolides on monarchs [START_REF] Agrawal | Macroevolutionary trends in the defense of milkweeds against monarchs: latex, cardenolides, and tolerance of herbivory. Monarchs in a Changing World: Biology and Conservation of an Iconic Insect[END_REF]. More mechanistic in vitro work with the monarch's highly resistant sodium-potassium pump (Na + /K + -ATPase), the cellular target of cardenolides, demonstrated that some milkweed cardenolides are strong inhibitors of monarch neural physiology [START_REF] Petschenka | Relative selectivity of plant cardenolides for Na+/K+-ATPases from the monarch butterfly and non-resistant insects[END_REF]. Thus, work with specific compounds that are variable in plants is needed to pinpoint agents of resistance. For sequestration of cardenolides, a model proposed by Nelson [START_REF] Nelson | A model for cardenolide and cardenolide glycoside storage by the monarch butterfly[END_REF] and supported in a review of early work [START_REF] Malcolm | Milkweeds, monarch butterflies and the ecological significance of cardenolides[END_REF] and new research [START_REF] Jones | Cardenolide intake, sequestration, and excretion by the monarch butterfly along gradients of plant toxicity and larval ontogeny[END_REF] suggests that monarchs selectively sequester more polar cardenolides, some compounds are metabolized (modification or detoxification), and others are transported via carriers [START_REF] Seiber | Pharmacodynamics of some individual milkweed cardenolides fed to larvae of the monarch butterfly (Danaus plexippus L.)[END_REF][START_REF] Frick | Uptake and sequestration of ouabain and other cardiacglycosides in Danaus plexippus (Lepidoptera, Danaidae) -evidence for a carriermediated process[END_REF][START_REF] Marty | Metabolism of uscharidin, a milkweed cardenolide, by tissue homogenates of monarch butterfly larvae, Danaus plexippus L[END_REF][START_REF] Seiber | Cardiac glycosides (cardenolides) in species of Asclepias (Asclepiadaceae)[END_REF]. Finally, observational work indicated that monarchs tend to oviposit on intermediate cardenolide concentration plants [START_REF] Oyeyele | Cardiac glycosides and oviposition by Danaus plexippus on Asclepias fruticosa in south-east Queensland (Australia), with notes on the effect of plant nitrogen content[END_REF][START_REF] Zalucki | Oviposition by Danaus plexippus in relation to cardenolide content of three Asclepias species in the southeastern USA[END_REF], suggesting the hypothesis that adult butterflies minimize toxic exposure to larvae while optimizing sequestration of plant poisons.

Asclepias curassavica is surprisingly understudied in its interactions with monarch butterflies, despite being a critical hostplant worldwide (second only to A. syriaca) [START_REF] Agrawal | Monarchs and milkweed: A migrating butterfly, a poisonous plant, and their remarkable story of coevolution[END_REF]. Attack of A. curassavica by monarchs can be strong, and therefore a likely source of selection for plant defense. The species is weedy throughout the tropics and has a plethora of cardenolides, including relatively uncommon compounds, some of which may be detrimental to monarch performance [START_REF] Seiber | Pharmacodynamics of some individual milkweed cardenolides fed to larvae of the monarch butterfly (Danaus plexippus L.)[END_REF][START_REF] Seiber | Cardiac glycosides (cardenolides) in species of Asclepias (Asclepiadaceae)[END_REF][START_REF] Malcolm | Chemical defence in chewing and sucking insect herbivores: plantderived cardenolides in the monarch butterfly and oleander aphid[END_REF][START_REF] Zhang | Structures, chemotaxonomic significance, cytotoxic and Na+, K+-ATPase inhibitory activities of new cardenolides from Asclepias curassavica[END_REF][START_REF] Roeske | Milkweed cardenolides and their comparative processing by monarch butterflies (Danaus plexippus L.)[END_REF][START_REF] Faldyn | Climate change and an invasive, tropical milkweed: An ecological trap for monarch butterflies[END_REF]. In particular, voruscharin is a long-known cardenolide containing a thiazolidine heterocycle (having both nitrogen and sulfur, Fig. 1), yet its previous study was hampered by solubility issues and the inability of thin layer chromatography to separate it from related compounds [START_REF] Seiber | Pharmacodynamics of some individual milkweed cardenolides fed to larvae of the monarch butterfly (Danaus plexippus L.)[END_REF][START_REF] Seiber | Cardiac glycosides (cardenolides) in species of Asclepias (Asclepiadaceae)[END_REF][START_REF] Malcolm | Chemical defence in chewing and sucking insect herbivores: plantderived cardenolides in the monarch butterfly and oleander aphid[END_REF][START_REF] Hesse | African arrow poisons XIV. Voruscharin, a second sulfurcontaining heart poison from Calotropis procera L[END_REF]. In terms of sequestration, it was demonstrated decades ago that monarchs preferentially sequester two cardenolides, calotropin and calactin, especially when feeding on A. curassavica [START_REF] Seiber | Pharmacodynamics of some individual milkweed cardenolides fed to larvae of the monarch butterfly (Danaus plexippus L.)[END_REF][START_REF] Malcolm | Chemical defence in chewing and sucking insect herbivores: plantderived cardenolides in the monarch butterfly and oleander aphid[END_REF][START_REF] Roeske | Milkweed cardenolides and their comparative processing by monarch butterflies (Danaus plexippus L.)[END_REF][START_REF] Reichstein | Heart poisons in the monarch butterfly[END_REF]. For oviposition, two flavonol glycosides were isolated from A. curassavica leaves that stimulate egg laying [START_REF] Haribal | Oviposition stimulants for the monarch butterfly: Flavonol glycosides from Asclepias curassavica[END_REF]. Nonetheless, the relative importance of quercetin glycosides versus cardenolides in oviposition is unknown, and specific cardenolides that impact larval growth and sequestration have not been well-studied. If A. curassavica defends itself against this specialized herbivore in a coevolutionary interaction mediated by plant chemistry, connecting specific toxins to their target site in the context of sequestration and oviposition is critical. In particular, we hypothesized that specific cardenolides modulate a trade-off between benefits of specialization and costs of coping with toxicity.

Here we identify cardenolides produced by A. curassavica and address which compounds are sequestered by monarchs, followed by asking four questions: 1) Do specific cardenolides reduce monarch larval growth or does sequestration impose a burden for larvae? 2) Using in vivo and in vitro assays, do monarchs detoxify or convert particular cardenolides to less toxic forms? 3) What is the relative toxicity (measured as in vitro inhibition of the cellular target, the monarch's Na + /K + -ATPase) of non-sequestered cardenolides, those sequestered intact, and those modified during sequestration? And finally, 4) do monarch oviposition decisions minimize toxicity and optimize sequestration of cardenolides, or are oviposition stimulants (flavonol glycosides) drivers of oviposition?

Results

Plant cardenolides, sequestration, and conversion

Among the >200 A. curassavica plants grown in a common environment, the range of total leaf cardenolides varied 8-fold (0.96 -8.16 mg/g dry mass). Of the nine cardenolides quantified, one apolar compound, thiazolidine ring-containing voruscharin, identified by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS/MS) was, on average, 40% of the total leaf cardenolides. Three other major peaks represented about 10% each of the total (two unidentified compounds at retention times 14.7 and 17.9 minutes, and uscharin). Of the 36 pairwise phenotypic correlations between the nine cardenolides (n=212), all but four were highly significant and positive, and the four exceptions all involved voruscharin (Table S1). A principal components analysis (PCA) yielded two orthogonal PC axes explaining 85% of the variation in the concentrations of the nine cardenolides, with PC1 having loadings of over 0.5 for all peaks except voruscharin, while voruscharin dominated PC2 (Table S2). Thus, voruscharin production appears to be regulated somewhat independently from other A. curassavica cardenolides.

Monarch caterpillars were grown individually on these intact plants and were collected in their fourth instar, weighed, freeze dried, and had their guts removed. Chemical analysis by UV and mass spectrometry revealed an absence of voruscharin from caterpillar bodies (Figures 1A,S1,S2). Three compounds frugoside, calotropin and calactin accounted for 77% of the total sequestered cardenolides (Table S2). While frugoside occurred in both plant and insect tissues, calactin was very low in leaves, and calotropin, if present, was too low to quantify in leaves. The six other compounds sequestered were minor (each <6.5% of the total) and were not found in the plant.

To test if monarchs convert voruscharin to calactin and calotropin, we purified voruscharin from A. curassavica, painted it on leaves of A. tuberosa (a milkweed that is eaten by monarchs in nature but lacks cardenolides), and fed it to monarch caterpillars. In particular, we compared monarchs fed painted versus unpainted A. tuberosa, in addition to their frass and positive controls fed A. curassavica. First, an untargeted MS-based metabolomics analysis showed that the chemical compositions of the groups assessed by PERMANOVA were different (P<0.05), although not all pairwise comparisons were significant (Figure 1A, Table S3). Second, in parallel, cardenolide sequestration was assessed by measuring absolute concentrations based on UV detection (Fig. 1B) and the relative concentrations based on LC-HRMS detection (Fig. S2). Voruscharin was only recovered on painted leaves, and in small quantities in caterpillar frass. Monarchs fed control A. tuberosa leaves lacked cardenolides and those fed painted leaves only contained calactin and calotropin. Finally, as expected, our positive control of monarchs fed A. curassavica foliage sequestered calactin and calotropin, in addition to several polar cardenolides, but lacked voruscharin.

To connect the dots of voruscharin synthesis and degradation, we hypothesized a biochemical model (Fig. S3) and examined transformation dynamics of voruscharin in vivo and in vitro. In particular, we hypothesized that voruscharin first degrades to uscharidin, which is secondarily converted to calactin and calotropin. We detected low levels of uscharidin in monarchs on A. curassavica, but uscharidin was absent in monarchs fed A. tuberosa painted with voruscharin and was found in traces in their frass (Fig. S4). To understand the degradation pathway, we conducted an assay in which pure voruscharin was subjected to different basic pH values and temperatures (Fig S5). Voruscharin is transformed to uscharidin under various alkaline conditions (pH 7-10) and the rate of conversion was enhanced at warmer temperatures (Fig. S5).

The burden of cardenolide consumption and sequestration

Although both plant cardenolide PC axes were associated with total plant cardenolide concentrations (Table S2), only PC2 (as well as the single compound voruscharin) predicted total cardenolides sequestered by monarchs (PC1 F 1,68 =0.296, p=0.588, PC2 F 1,68 =5.501, p=0.022, voruscharin F 1,68 =10.107, p=0.002, Figure 2a). To address the relative importance of voruscharin (as a plant toxin) or sequestered cardenolides (as a burden, i.e., the combined effect of converting cardenolides and storing the preferred compounds), we used these two predictors in multiple regression to predict caterpillar growth (the two variables are only modestly correlated, r=0.36, variance inflation factor = 1.15). Here, monarch growth was strongly negatively predicted by total sequestered cardenolides (R 2 =0.61), but not leaf voruscharin concentration (Figure 2b, sequestered cardenolide F 1,67 =85.660, p<0.001, voruscharin F 1,67 =1.254, p=0.267, Fig. S6). This analysis was robust to different configurations of using plant and sequestered cardenolides as predictors of monarch growth (i.e., using plant PC2 or total plant cardenolides and insect PC1 in multiple regressions, Fig. S6).

To address the inhibitory capacity of different cardenolides on their physiological target, the monarch Na + /K + -ATPase, we isolated and purified frugoside (polar, sequestered intact), uscharin (apolar, 3-thiazoline ring-containing, minor leaf component, not sequestered), voruscharin (apolar, thiazolidine ring-containing, dominant leaf component, not sequestered), calotropin (intermediate polarity, minimal in the plant but sequestered), and calactin (intermediate polarity, minimal in the plant but sequestered) (Figs. S7-9). The inhibitory capacity of these compounds was referenced to two standards (ouabain and digitoxin) and all were compared on the sensitive porcine Na + /K + -ATPase versus the highly resistant enzyme isolated from monarch neural tissues. The two non-sequestered cardenolides were the most potent inhibitors of the monarch Na + /K + -ATPase and yet were among the least inhibitive of the sensitive porcine Na + /K + -ATPase (Fig. 3). In particular, the monarch's Na + /K + -ATPase is typically 50-100 times more resistant to cardenolides than the porcine Na + /K + -ATPase [START_REF] Petschenka | Relative selectivity of plant cardenolides for Na+/K+-ATPases from the monarch butterfly and non-resistant insects[END_REF][START_REF] Vaughan | Insensitivity of lepidopteran tissues to ouabainphysiological mechanisms for protection from cardiac glycosides[END_REF], and this was borne out by the average of 86-fold higher IC 50 for five compounds tested, including 3 sequestered cardenolides and the two standards (Table S4; Figure 3). Strikingly, for nonsequestered uscharin and voruscharin, the monarch was less than 2-fold more resistant than the sensitive Na + /K + -ATPase.

Monarch oviposition

When we tested free-flying monarchs for their oviposition preferences, egg laying was best predicted by intermediate concentrations of total leaf cardenolides and plant height (Figure 4, Poisson GLM: total cardenolides, L-R χ 2 = 5.638, p = 0.018, total cardenolides squared L-R χ 2 = 4.827, p= 0.028, and height L-R χ 2 = 15.395, p <0.001; alternative models including the significant predictive effect of cardenolide PC1 and individual compounds are given in Table S5). Neither leaf voruscharin nor plant PC2 predicted oviposition. Because egg laying was also not predicted by foliar concentrations of two quercetin glycosides in leaf tissues (a quercetin-rhamno-di-hexoside and quercetin-rhamno-hexoside, Table S6, Fig. S10), we tested their stimulatory effect on oviposition by isolating and purifying the compounds. The latter compound was indeed found to be stimulatory in oviposition trails, but only at unrealistically high concentrations (Table S6). Thus, overall, oviposition on whole plants was best predicted by plant height and the set of more polar cardenolides which showed correlated expression, but neither the most potent cardenolides nor the low levels of quercetin glycosides in leaves.

Discussion

It has long-been conventional wisdom that specialist insect herbivores are less impacted by plant defensive chemistry than generalists; and reciprocally, specialist pests that have been persistent over millions of years are potent forces of natural selection for specific plant defenses [START_REF] Cornell | Herbivore responses to plant secondary compounds: A test of phytochemical coevolution theory[END_REF][START_REF] Rothwell | Phytochemical defences and performance of specialist and generalist herbivores: a meta-analysis[END_REF]. Nonetheless, as Berenbaum et al. [START_REF] Berenbaum | Constraints on chemical coevolution: wild parsnips and the parsnip webworm[END_REF] pointed out over 30 years ago, there is potential for stalemate in the metaphorical coevolutionary arms race, where herbivores and plants are well-adapted to each other, with little room for further escalation. In the case of monarchs on milkweed, despite all the various barriers to feeding that milkweeds possess (trichomes, latex, and cardenolides), monarchs seem to prevail by shaving leaf trichomes and trenching latex canals as caterpillars, and possessing a highly cardenolide-insensitive sodiumpotassium pump [START_REF] Agrawal | Monarchs and milkweed: A migrating butterfly, a poisonous plant, and their remarkable story of coevolution[END_REF].

Here we have reported that the dominant cardenolide in one of the most important milkweed host plants for monarchs, the tropical milkweed A. curassavica, is the unusual thiazolidine ring-containing compound voruscharin. The fact that most cardenolides lack nitrogen and sulfur, and that nitrogen is typically limiting for plant growth, suggests that such compounds may be evolutionarily derived and highly potent [START_REF] Seiber | Cardiac glycosides (cardenolides) in species of Asclepias (Asclepiadaceae)[END_REF]. Although voruscharin has been known from A. curassavica and two other species in the Apocynaceae (Gomphocarpus fruticosus and Calotropis procera) for decades, and it was known not to be sequestered by monarchs, its potency, conversion, and impacts on sequestration and oviposition were largely unknown [START_REF] Seiber | Pharmacodynamics of some individual milkweed cardenolides fed to larvae of the monarch butterfly (Danaus plexippus L.)[END_REF][START_REF] Seiber | Cardiac glycosides (cardenolides) in species of Asclepias (Asclepiadaceae)[END_REF][START_REF] Malcolm | Chemical defence in chewing and sucking insect herbivores: plantderived cardenolides in the monarch butterfly and oleander aphid[END_REF][START_REF] Roeske | Milkweed cardenolides and their comparative processing by monarch butterflies (Danaus plexippus L.)[END_REF][START_REF] Hesse | African arrow poisons XIV. Voruscharin, a second sulfurcontaining heart poison from Calotropis procera L[END_REF]. We have found that voruscharin is among the most potent milkweed cardenolides known to inhibit the monarch Na + /K + -ATPase [START_REF] Petschenka | Relative selectivity of plant cardenolides for Na+/K+-ATPases from the monarch butterfly and non-resistant insects[END_REF], but it has rather poor inhibition of the highly sensitive porcine Na + /K + -ATPase, suggesting that this compound and its high expression in A. curassavica may function specifically against the monarch butterfly. In related work, we will show that natural populations of A. curassavica harbor substantial genetic variation in voruscharin production.

In perhaps the best known system of plant-herbivore chemical coevolution, Berenbaum and colleagues (3) revealed how toxic furanocoumarins evolved in plants of the Apiaceae and the means by which lepidopteran herbivores detoxify these compounds. In particular, diverse cytochrome P450 enzymes in insects appear to have specifically evolved to detoxify the compounds. Among swallowtail butterflies, although linear furanocoumarins are metabolized easily, the biochemically novel angular furanocoumarins are much more challenging and have imposed selection to diversify cytochrome P450 enzymes. In the monarch -milkweed system, selective sequestration of more polar compounds is well-known, but metabolic detoxification was thought to be far less important, in part because butterflies store cardenolides for their own defense [START_REF] Seiber | Pharmacodynamics of some individual milkweed cardenolides fed to larvae of the monarch butterfly (Danaus plexippus L.)[END_REF][START_REF] Malcolm | Milkweeds, monarch butterflies and the ecological significance of cardenolides[END_REF][START_REF] Jones | Cardenolide intake, sequestration, and excretion by the monarch butterfly along gradients of plant toxicity and larval ontogeny[END_REF]. While some cardenolides like frugoside are stored intact, others such as voruscharin are converted to other forms. We found that the preferentially sequestered calactin and calotropin were less toxic to monarchs than voruscharin, but conversion or storage imposed a significant burden for caterpillars in terms of reduced growth rate that was stronger than direct effects of voruscharin itself. Ultimately the benefits of sequestering calactin and calotropin may outweigh the costs we have described here. Recent research indicates substantial genetic variation in monarchs for sequestration [START_REF] Freedman | Host plant adaptation during contemporary range expansion in the monarch butterfly[END_REF] and our future work will address selection on this ability.

It is interesting to note that the basic conditions which mimic the pH of the monarch gut hydrolyze voruscharin or uscharin to uscharidin. Our data support this as the first step in the hypothesized pathway to derive calactin and calotropin (Fig. S5). Acid hydrolysis of the 3thiazoline ring has also been reported for the conversion of uscharin into uscharidin [START_REF] Seiber | Cardiac glycosides (cardenolides) in species of Asclepias (Asclepiadaceae)[END_REF], and the enzymatic reduction of uscharidin to calactin and calotropin was demonstrated in vivo using monarch gut homogenates [START_REF] Seiber | Pharmacodynamics of some individual milkweed cardenolides fed to larvae of the monarch butterfly (Danaus plexippus L.)[END_REF][START_REF] Marty | Metabolism of uscharidin, a milkweed cardenolide, by tissue homogenates of monarch butterfly larvae, Danaus plexippus L[END_REF]. Perhaps it seems odd that a novel and highly potent chemical defense produced by Asclepias is degraded passively in the monarch gut. One possible explanation for this observation is that the degradation is not immediate and the further enzymatic modification is costly. Depending on the hydrolysis rate and temperature in the gut, all of the voruscharin may not be converted to uscharidin, potentially imposing a cost for monarchs. Spontaneous and environment-dependent degradation of plant defense compounds is not unique to cardenolides, and has been shown to have various effects in other systems [START_REF] Wouters | Plant defense and herbivore counter-defense: benzoxazinoids and insect herbivores[END_REF][START_REF] Magalhães | Biochemical basis of the toxicity of manipueira (liquid extract of cassava roots) to nematodes and insects[END_REF][START_REF] Winde | Insect herbivore counteradaptations to the plant glucosinolate-myrosinase system[END_REF].

Given the high chemical diversity of hundreds of distinct cardenolides in Asclepias (42), it is puzzling that very few cardenolides containing a 3-thiazoline or 3-thiazolidine ring have been found (e.g., uscharin, voruscharin, and labriformin) [START_REF] Seiber | Cardiac glycosides (cardenolides) in species of Asclepias (Asclepiadaceae)[END_REF][START_REF] Abe | An androstane bioside and 3 '-thiazolidinone derivatives of doubly-linked cardenolide glycosides from the roots of Asclepias tuberosa[END_REF]. So far these have been found in some of the major host plants of monarchs, as labriformin occurs in A. syriaca [START_REF] Malcolm | Cardenolide fingerprint of monarch butterflies reared on common milkweed, Asclepias syriaca L[END_REF], but the evolutionary history of these compounds and why they are not more common is thus far unclear. Although the biosynthesis of cardenolides in Asclepias has been little-studied [START_REF] Groeneveld | Cardenolide biosynthesis from acetate in Asclepias curassavica[END_REF], and the pathway leading to voruscharin in particular is unknown, we speculated that formation of the thiazolidine ring likely involves the condensation of cysteine with the carbonyl group in position 3' of uscharidin (Fig. S3). Regardless of the specific biosynthetic origin, our analysis of correlations among cardenolides (Table S1) suggests the biosynthetic steps involved in the thiazolidine ring formation may be regulated independently from the pathways that produce the bulk of other A. curassavica cardenolides.

There has been a lack of clarity on the relative importance of flavonoid oviposition stimulants (quercetin glycosides) and cardenolides (which do not occur on the surface of milkweed leaves) for egg laying. On the one hand, monarchs tend to oviposit on intermediate cardenolide concentration plants [START_REF] Oyeyele | Cardiac glycosides and oviposition by Danaus plexippus on Asclepias fruticosa in south-east Queensland (Australia), with notes on the effect of plant nitrogen content[END_REF][START_REF] Zalucki | Oviposition by Danaus plexippus in relation to cardenolide content of three Asclepias species in the southeastern USA[END_REF], yet on the other hand, there does not appear to be a consistent relationship between cardenolides and quercetin glycosides [START_REF] Agrawal | Phylogenetic trends in phenolic metabolism of milkweeds (Asclepias): Evidence for escalation[END_REF][START_REF] Haribal | Identification and distribution of oviposition stimulants for monarch butterflies in hosts and nonhosts[END_REF]. In the current study, although we were able to show that an isolated quercetin-rhamno-hexoside was indeed stimulatory for oviposition, quantitative variation in quercetin glycosides measured in leaves did not predict egg laying in our trials. We speculate that quercetin glycosides, although stimulatory in isolation, occur in much too low concentrations to be drivers of egg laying. Nonetheless, we did find that monarchs laid the most eggs on intermediate cardenolide plants as expected based on past observations. Our plants were not flowering during the oviposition trial, so nectar was not a potential cue, although other research indicates that nectar cardenolides impact monarch oviposition [START_REF] Jones | Consequences of toxic secondary compounds in nectar for mutualist bees and antagonist butterflies[END_REF].

Finally, although we do not know how monarchs sense leaf cardenolides (or what correlated traits they detect), monarchs' preference for intermediate cardenolide plants supports the notion that specialist herbivores must balance the benefits of plant secondary compounds for sequestration with the burden that these same compounds impose. Thus, perhaps there is room for additional coevolution between these highly adapted herbivores and their toxic host plants. One challenge for monarchs is that there is relatively little evidence of local population differentiation or adaptation to particular host plants [START_REF] Zhan | The genetics of monarch butterfly migration and warning colouration[END_REF][START_REF] Freedman | Host plant adaptation during contemporary range expansion in the monarch butterfly[END_REF]. This is likely the outcome of their complex annual migratory cycle, where butterflies encounter diverse milkweeds and yet geographically distinct breeding populations intermix during migration and overwintering. Thus, although monarchs themselves are specialists on a genus of plants, that genus contains chemically diverse cardenolide toxins for which there may not be a single insect counter-ploy [START_REF] Seiber | Cardiac glycosides (cardenolides) in species of Asclepias (Asclepiadaceae)[END_REF][START_REF] Rasmann | Latitudinal patterns in plant defense: evolution of cardenolides, their toxicity and induction following herbivory[END_REF]. In fact, recent work has also shown that alternative defenses may result in some plants being ecological traps where monarch oviposition is followed by poor caterpillar performance [START_REF] Jones | Beyond preference and performance: host plant selection by monarch butterflies, Danaus plexippus[END_REF][START_REF] Ditommaso | Oviposition preference and larval performance of monarch butterflies (Danaus plexippus) on two invasive swallow-wort species[END_REF]. Thus, we conclude by speculating that although monarchs are highly adapted to Asclepias spp. overall, they may not be able to strongly adapt to any one plant species. The ratio of cardenolide sequestration by monarchs to host plant cardenolides is the highest for A. syriaca and A. curassavica [START_REF] Malcolm | Milkweeds, monarch butterflies and the ecological significance of cardenolides[END_REF], suggesting that coevolution may be primarily occurring between monarchs and these two species. And strong selection by monarchs on particularly important host plant species (e.g., A. syriaca and A. curassavica) may stimulate adaptation in these plant species. Thus, coevolution in this system is decidedly diffuse. The geographical availability of multiple milkweed species is a buffet for monarchs, but perhaps also a limitation preventing stronger specialized insect adaptation.

Materials and methods

Plant growth

In April 2016 we germinated a diverse pool of Asclepias curassavica seeds, collected from several sites in the southern USA, Mexico, and obtained from commercial seed suppliers. Our goal was to have diversity within the species, and there was substantial phenotypic variation among collections (e.g., in flower color and leaf shape), but all were confirmed to be A. curassavica based on flowers. In total, we grew approximately ten plants were from each of 22 collections. Plants were grown as described in Rasmann and Agrawal [START_REF] Rasmann | Latitudinal patterns in plant defense: evolution of cardenolides, their toxicity and induction following herbivory[END_REF], with surfacesterilized seeds that were nicked, stratified at 4 °C for four days, germinated in petri dishes at 10 28 °C, grown in potting soil (Metro-Mix, Sun Gro Horticulture) in plastic pots (500 ml) in a high light growth chamber (at least 350 microeinsteins, 16 h daylight, 26 °C day : 22 °C night). Plants were given two applications of dilute fertilizer (N:P:K 21:5:20, 150 ppm N [μg/g]) over one month of growth, after which they were clipped to just above the cotyledons. Clipped leaf tissue was freeze-dried and analyzed for cardenolides by HPLC (see below). Plants were uppotted and moved to a rooftop greenhouse at Cornell University. A total of 212 A. curassavica plants regrew in the greenhouse.

Initial insect bioassays

Two assays were conducted. In the first, adult oviposition was assessed by releasing 10 mated female monarchs (from a laboratory colony) into the 56 m 2 greenhouse. After 24 hours, over one third of the plants had at least one egg laid on them and the trial was ended. Plant height was measured, eggs were removed from plants with tweezers, and the second assay was initiated to assess caterpillar growth. Neonate monarch caterpillars were introduced to 70 of the plants and growth rate was measured as fresh mass eight days later when the caterpillars had entered the fourth instar (when monarchs typically begin to move between plants). Caterpillars were freeze-dried, their guts were removed by dissection under a microscope, and the rest of the body was used to assess cardenolide sequestration. We then estimated the total cardenolides sequestered by HPLC (see below the cardenolide quantification). This design allowed us to connect individual variation in plant chemistry, caterpillar growth, and sequestration.

Cardenolides and flavonols isolation and identification

We authenticated chemical structures using a set of authentic standards from Zhang et al. [START_REF] Zhang | Structures, chemotaxonomic significance, cytotoxic and Na+, K+-ATPase inhibitory activities of new cardenolides from Asclepias curassavica[END_REF] and Petschenka et al. [START_REF] Petschenka | Relative selectivity of plant cardenolides for Na+/K+-ATPases from the monarch butterfly and non-resistant insects[END_REF] and matched retention times, mass spectrometry exact masses (HRMS) and MS fragmentation profiles for frugoside, asclepin, calactin, calotropin in samples, and for purified compounds voruscharin, uscharin, and two flavonols quercetinrhamno-hexoside and quercetin-rhamno-di-hexoside. HRMS/MS data of voruscharin, uscharidin, calactin and calotropin are detailed in Table S7 and their respective MS 2 can be found in Fig. S7-S9. Fig. S10-S11 give the HRMS/MS data for the quercetin glycosides.

Frugoside, calactin, and calotropin were purified from the bioassay monarch caterpillars raised on A. curassavica (3.1 g dry tissue with the guts). The pooled extract was de-fatted with 50mL hexane and taken to dryness. The remaining residue was suspended in 9mL of 16% acetonitrile and water, sonicated for 30sec, vortexed, centrifuged at 20,800g for 12min and treated by adding 24uL of 40% lead acetate. This step clears the sample of pigments and other interfering compounds. The vial was then lightly vortexed and allowed to sit for a minimum of 10min to allow unwanted compounds to complex. The vial was then centrifuged at 20,800g for 12min and the supernatant transferred to a new vial. To bind up any remaining lead ions, we added an addition of 1200uL of 5% sodium sulfate. After a 10min wait, the vial was again centrifuged at 20,800g for 12min and the supernatants transferred to a new vial. The vial was then taken to dryness and the residue re-suspended in 9mL of methanol by sonication and vortexing. The vial was then centrifuged at 20,800g for 12min and the supernatant transferred to a new vial. The methanolic supernatant was transferred to a new vial and taken to dryness. We then added 16% acetonitrile to water and the residue was re-suspended by sonication and vortexing. The sample was again centrifuged at 20,800g for 12min and the supernatant saved for preparative HPLC fractionation. Uscharin and voruscharin were purified from >40mL of fresh A. curassavica latex collected from greenhouse grown plants.

All prepared samples were injected into an Agilent 1260 series preparative LC system with an Agilent 21.2mm x 150mm, C-18, 5um column. Each first-pass injection was eluted at a constant flow rate of 14.87mL/min with a gradient of acetonitrile and water as follows: 0-2min at 16% acetonitrile; 2-25min from 16% to 70%; 25-30min from 70% to 95%; 30-35min at 95%. Target peaks were detected at 218nm. In most cases, each first-pass target fraction needed to be dried down, re-suspended in 16% acetonitrile and re-injected for further cleanup. Fractions needing re-injection often required modifications to the Method gradients to increase column retention times to help isolate target peaks. The isolated fractions were pooled, dried, and resuspended in 0.5 ml of 100% MeOH, and then analyzed on a Thermo Scientific QExactiveTM hybrid quadrupole-orbitrap LC-MS system in positive ionization mode (see below the LC-HRMS instrument parameters). Compounds were identified by their exact parental mass ([M+H] + ), as well as the corresponding sodium adduct ([M+Na] + ).

Reversed-phase chromatography was performed using a Dionex 3000 LC coupled to an Orbitrap Q-Exactive mass spectrometer controlled by Xcalibur software (ThermoFisher Scientific). Methanolic extracts were separated on an Agilent Zorbax Eclipse XDB-C18 column (150 mm x 2.1 mm, particle size 1.8 µm) maintained at 40 °C with a flow rate of 0.5 mL/min. Solvent A: 0.1% formic acid (FA) in water; solvent B: 0.1% formic acid in acetonitrile. A/B gradient started at 5% B for 2 min after injection and increased linearly to 98% B at 11 min, followed by 3 min at 98% B, then back to 5% B over 0.1 min and finally at 5% B held for an additional 2.9 min to re-equilibrate the column. Mass spectrometer parameters: spray voltage (-3.0 kV, +3.5 kV), capillary temperature 380 °C, probe heater temperature 400 °C; sheath, auxiliary, and sweep gas 60, 20, and 2 AU, respectively. S-Lens RF level: 50, resolution 240,000 at m/z 200, AGC target 3E6. Each sample was analyzed in negative and positive electrospray ionization modes with m/z ranges 70-1000 for reversed-phase, 120-800 for reversed-phase post-column ion pairing, and 70-700 for normal-phase. Parameters for MS/MS (dd-MS2): MS1 resolution: 60,000, AGC Target: 1E6. MS2 resolution: 30,000, AGC Target: 2E5, maximum injection time: 50 msec, isolation window 1.0 m/z, stepped normalized collision energy (NCE) 10, 30; dynamic exclusion: 1.5 seconds, top 5 masses selected for MS/MS per scan. LC-MS data were analyzed using MZmine software (see below) and MS 2 spectra were obtained via Excalibur software (ThermoFisher Scientific).

Cardenolide and flavonol quantification

We used an Agilent 1100 HPLC with diode array detector and a Gemini C18 reversedphase, 3 um, 150 mm x 4.6 mm column. We use 50 mg of pulverized tissue for analyses by adding 1.5 ml of 100% methanol, a 20 ug digitoxin spike as internal standard, and 20 FastPrep beads. Cardenolides were then extracted by agitating twice on a FastPrep-24 homogenizer for 45 s at 6.5 m/s and then centrifuged at 20,800 g for 12 min. Supernatants were dried down in a vacuum concentrator at 35C and were resuspended in 200ul methanol, filtered using 0.45 um hydrophilic membranes, and 15ul was inject into the HPLC running a constant flow of 0.7 ml/min with a gradient of acetonitrile and water as follows: 0-2 min at 16% acetonitrile; 2-25 min from 16% to 70%; 25-30 min from 70% to 95%; 30-35 min at 95%; followed by 10 min reconditioning at 16% acetonitrile. Peaks were recorded at 218 nm and absorbance spectra are recorded between 200 nm to 300 nm. Peaks showing a characteristic single absorption maximum between 214 and 222 nm correspond to the unsaturated lactone indicative of cardenolides. Concentrations were standardized by peak area to the digitoxin internal standard with known concentration. Flavonols were similarly quantified from the same chromatograms based on our digitoxin internal standard.

Voruscharin painting experiment

We modeled our analysis of the fate of purified cardenolides based on Seiber et al. [START_REF] Seiber | Pharmacodynamics of some individual milkweed cardenolides fed to larvae of the monarch butterfly (Danaus plexippus L.)[END_REF]. We used monarchs from a laboratory colony (reared for 4 generations from wild collected individuals) and reared a cohort on A. tuberosa. A. tuberosa is milkweed that lacks detectable cardenolides and has the lowest inhibition of the porcine sodium-potassium pump of any milkweed species assayed to date [START_REF] Züst | Toxicity of milkweed leaves and latex: chromatographic quantification versus biological activity of cardenolides in 16 Asclepias species[END_REF]. When caterpillars newly molted to the 4 th instar, we kept them cool and without food until use (<5 hours). Our main treatments were A. tuberosa leaves (controls treated with methanol, n=5) and leaves dosed with realistic amounts of voruscharin (n=4). Briefly, because voruscharin was estimated to be 40% of total cardenolides in A. curassavica and totals an average of 1.7 ug/mg dry mass, we added 7g of voruscharin (dissolved in methanol) per cm 2 of A. tuberosa foliage. Individual caterpillars were provided two leaves at a time in petri dishes lined with filter paper After molting into 5 th instar, they were deprived of food for 8 hours and freeze-dried for analysis. Two caterpillars were simultaneously reared on A. curassavica from hatching to the 5 th instar. In addition to these positive controls, we analyzed samples from methanol treated A. tuberosa leaves, A. tuberosa leaves spiked with voruscharin, caterpillars fed each of these diets, and the frass from caterpillars fed the voruscharin diet.

Oviposition experiments

We modeled oviposition trials with isolated quercetin glycosides on Haribal and Renwick [START_REF] Haribal | Oviposition stimulants for the monarch butterfly: Flavonol glycosides from Asclepias curassavica[END_REF]. Briefly, cubes of washed green sponges were arranged on a four-arm stand inside of a collapsible field cage (Bioquip, 0.23 m 3 ). Each trial consisted of two control arms (sponge with water) and one sponge each of the two isolated quercetin glycosides (1 gram leaf equivalents); at least 4 female (all mated) and 1 male butterflies were included in each of 10 trials. Butterflies were allowed to lay eggs for a maximum of 5 hours (daylight hours). All eggs were counted on sponges for analysis.

Data pre-processing and metabolomics analysis

The acquired LC-MS data files were converted to mzXML files using the ProteoWizard MSconvert tool. LC-MS data was then preprocessed with the open-source MZmine software and consisted of peak detection, removal of isotopes, alignment, filtering, and peak filling. Peak detection was performed in three steps: (i) mass detection with noise value = 15,000; (ii) ADAP chromatogram builder with minimum group size in number of scan = 5, group intensity threshold = 25,000, minimum height = 30,000 and m/z tolerance = 10 ppm; (iii) wavelets ADAP deconvolution with S/N = 3, minimum feature height = 1,000, coefficient area threshold = 5, peak duration range = 0.01-3 min, retention time wavelet range = 0.01-0.04 min. Isotopes were removed using the isotopic peak grouper with m/z tolerance = 10 ppm, retention time tolerance = 0.5 min, maximum charge = 3. Chromatograms were aligned using the join aligner with m/z tolerance = 10 ppm, weight for m/z = 75, retention time tolerance = 0.5 min, weight for retention time = 25. Filtering minimum peak in a row = 2, minimum peak in an isotopic pattern = 2, and keep only peaks with MS2 scan. Gap filling was applied using the method peak finder with retention time correction with intensity tolerance = 10%, m/z tolerance = 10 ppm, retention time tolerance = 0.5 min. Quality control (QC) metabolites with a coefficient of variation (CV) greater than 30% were removed from the whole data matrix. In positive mode ionization the data matrices contain 7,252 and 6,795 features in mass range of 152-1185 m/z and 156-1155 m/z respectively.

Sodium-potassium pump assays

We quantified the biological activity of isolated cardenolides using Na + /K + -ATPase from the porcine cerebral cortex (Sigma-Aldrich, MO, USA) and monarch butterflies following methods of Petschenka et al. [START_REF] Petschenka | Relative selectivity of plant cardenolides for Na+/K+-ATPases from the monarch butterfly and non-resistant insects[END_REF]. Briefly, Na + /K + -ATPase activity was measured as the amount of inorganic phosphate (Pi) enzymatically released from ATP in the presence of K + (Na + /K + -ATPase active) minus the amount of Pi released in the absence of K + (Na + /K + -ATPase inactive). Each compound was dissolved fully in methanol (frugoside, calactin, and calotropin) or acetonitrile (voruscharin and uscharin, due to solubility), assayed by HPLC to determine concentration, and then dried and resuspended in 20% DMSO to a concentration of either 0.5mM (frugoside, calactin, and calotropin) or 0.1mM (voruscharin and uscharin, due to decreased solubility). We then prepared five serial dilutions to reach the following concentrations: 5x10 -5 M, 5x10 -6 M, 5x10 -7 M, 5x10 -8 M, 5x10 -9 M, to produce a six-point inhibition curve for each compound, incubated with each of the two enzyme preparations. These milkweed cardenolides were run alongside equivalent solutions of ouabain and digitoxin in 20% DMSO. Reactions were performed in 96-well microplates on a BioShake Iq microplate shaker (Quantifoil Instruments, Jena, Germany) at 200 rpm and 37 °C. Absorbance values of reactions were corrected by their respective backgrounds and dose-response curves were fitted using a non-linear mixed effects model with a 4-parameter logistic function in the statistical software R (function nlme with SSfpl in package nlme v3.1-137) based on ref [START_REF] Züst | Independent evolution of ancestral and novel defenses in a genus of toxic plants (Erysimum, Brassicaceae). eLife 9:e51712. 73 glycosides from Asclepias curassavica[END_REF]. We focus analyses on the dilution value IC50 at which the enzyme in inhibited by 50%.

Statistical approaches

Linear statistical models (regression, ANOVA) were usable for most analyses and residuals were checked for normality and heteroscedasticity. Except as noted below, all analyses were conducted in JMP Pro V14. As noted in the text, we quantified nine abundant cardenolides in the leaf tissue of A. curassavica and nine sequestered cardenolides in monarchs (which overlapped but were not identical to those in plants). As shown in the results, the concentrations of many, but not all, of the distinct cardenolides were highly correlated across individual plants. We thus first used principal components analysis to reduce the number of variables in each set and used the two PCs (eigenvalues >1) in statistical analyses (mostly regressions to explain response variables like egg number, larval mass, and sequestration). Next, to confirm a role (or lack thereof) for particular cardenolides that were strongly (or weakly) loaded on particular PC axes, we followed up with analyses using the individual compounds.

Metabolomic analyses were performed using the web-based metabolomics data processing tool MetaboAnalyst 3.0 (http://www.metaboanalyst.ca/). Briefly data were filtered using a non-parametric relative standard deviation (MAD/medium), normalized by sum, log transformed and auto scaled. The metabolomes of each group of samples were analyzed with a Bray-Curtis distance matrix on normalized chemical data and visualized using principal coordinates analyses (PCoA). Statistical tests were conducted using PERMANOVA.

We used a Poisson regression to predict egg count data which ranged from 0-6 eggs per plant; a squared term was include in the model both because past work [START_REF] Oyeyele | Cardiac glycosides and oviposition by Danaus plexippus on Asclepias fruticosa in south-east Queensland (Australia), with notes on the effect of plant nitrogen content[END_REF][START_REF] Zalucki | Oviposition by Danaus plexippus in relation to cardenolide content of three Asclepias species in the southeastern USA[END_REF] and the current results fit a quadratic model. isolation, and identification of the quercetin glycosides. A.P.H. supported most of the experiments and conducted all sodium-potassium pump assays. R.A.W. conducted caterpillar sequestration analyses on HPLC-UV and isolated the cardenolides. R-W.J. provided cardenolide standards. C.D. conducted the LC-HRMS analysis of cardenolides, analyzed the metabolomics data, designed and performed the voruscharin degradation experiment, and proposed the biosynthetic pathways. A.A.A. designed the research, conducted the monarch bioassays on purified compounds, and wrote the manuscript (all authors contributed to the writing and editing). Competing interests: The authors declare no competing financial and non-financial competing interests. Data and materials availability: All data is available in the main text or in the supplementary materials.

Supplementary Materials: Figures S1-S11

Tables S1-S7 visualized with a Bray-Curtis distance matrix. Elipses represent the region of 95% confidence. B) Voruscharin was converted to calactin and calotropin when fed to monarch caterpillars. Shown are means ± SE concentrations as determined by UV-HPLC (n=3-9). Data bars very close to zero had no detectable cardenolides. Note that the caterpillars fed A. curassavica were reared on this diet from hatching and had an order of magnitude higher cardenolides than other treatments which were dosed only during the fourth instar. C) The basic skeleton of cardenolides and the chemical structures of calactin, calotropin, and voruscharin. Fig. 3. The difference in inhibitory impacts of isolated cardenolides on the highly sensitive porcine Na + /K + -ATPase versus the milkweed-adapted monarch butterfly Na + /K + -ATPase. Data are presented as the molar concentration of plant toxin necessary to cause 50% inhibition of the animal enzyme, or IC 50 , on the Y axis. Thus, higher values on the log scale Y axis indicate that the enzyme is more resistant to the cardenolide. The monarch Na + /K + -ATPase is substantially more resistant (it typically takes a higher concentration of the plant compounds to inhibit the enzyme). Sequestered+ indicates that these compounds are sequestered intact from consumed leaves as well as converted products from consumed voruscharin and uscharin. Each bar is a mean of 3-9 replicates (each based on a 6-concentration inhibition curve) ± SE (where not visible, SEs are too small). The arrow representing the polarity of cardenolides is based on elution through a C18 HPLC column (apolar stationary phase). . Table S3. PERMANOVA results analyzing metabolic data based on Bray-Curtis dissimilarities. [START_REF] Malcolm | Milkweeds, monarch butterflies and the ecological significance of cardenolides[END_REF]

Fig. 1 .

 1 Fig. 1. Chemical conversion of milkweed cardenolides by monarch caterpillars. A) A visualization of metabolomic data showing the differences in the chemical composition across sample groups (n=4 per group, significance tested by PERMANOVA). After data curation over 7000 chemical features (m/z) were generated with MS data collected in positive ionization mode and

Fig. 2 .

 2 Fig.2. Connections between plant cardenolides, monarch sequestration, and monarch growth. A) Sequestration was predicted by concentration of the dominant plant cardenolide, voruscharin, although this compound was itself not sequestered (it was converted to calactin and calotropin). B) In multiple regression, sequestered cardenolides were predictive of monarch growth (whereas plant cardenolides were not), indicating a cost of converting or storing cardenolides. The weak relationships of growth predicted by total leaf cardenolides and leaf voruscharin are shown in Fig.S6.

Fig. S2 .

 S2 Fig. 4. Monarch oviposition was a quadratic function of total leaf cardenolides, with the highest number of eggs laid on intermediate cardenolide plants. Ten butterflies were released to freely oviposit in a large greenhouse common garden (n=212 plants). Although raw data are shown, plant height was included and was significant in the statistical model. Zero egg values have been jittered.

Fig. S3 .

 S3 Fig. S3. Voruscharin putative biosynthesis in Asclepias curassavica and its degradation pathway in monarch gut.The functional group in position C3' is highlighted in yellow to show the successive steps leading from calactin/calotropin to voruscharin.

Fig. S5 .Fig. S6 .

 S5S6 Fig. S5. Voruscharin stability assay at different pH values. Chromatograms of voruscharin (A) and uscharidin (B), and corresponding epimers, after being stored at different temperatures and pH values. The control was stored at -20°C for 24h. The epimer's retention times was anticipated based on previous results of 2'-epi-uscharin (1).

Fig. S7 .

 S7 Fig. S7. MS/MS product ion mass spectrum from [M+FA-H] -adduct of voruscharin.The MS 2 spectrum of voruscharin was deposited to the GNPS database. https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB00005724352#%7B%7D

Figure S10 .

 S10 Figure S10. MS/MS product ion mass spectrum from [M+H] + adduct of quercetin-rhamno-hexoside. Based on previous phytochemical studies of characterized flavonols in A. curassavica (2) the structure is either quercetin 3-O--(2"-O--L-rhamnopyranosyl)--D-galactopyranoside or quercetin 3-O--(2"-O--L-rhamnopyranosyl)--D-glucopyranosyl (rutin).

Figure S11 .

 S11 Figure S11. MS/MS product ion mass spectrum from [M+H] + adduct of quercetin-rhamno-di-hexoside. Based on previous phytochemical studies of characterized flavonols in A. curassavica (2) the structure is either quercetin 3-O-(2",6"di--L-rhamnopyranosyl)--D-galactopyranoside or quercetin 3-O-(2",6"-O-di--L-rhamnopyranosyl)--D-glucopyranoside.

Table S4 . The relative inhibitory potential of seven isolated cardenolides on the sensitive 32 porcine and typically-resistant monarch butterfly Na-K-ATPase (See also Figure 3). 33

 S4 25IC 50 is the concentration (M) at which the enzyme is inhibited by 50%. Sequestered+ indicates 34 that these compounds are sequestered intact from consumed leaves as well as converted 35 products from consumed voruscharin and uscharin.

	36	Source of variation	DF	SumSq		MeanSqs	F. model	R	2	P
	37	All chemical extracts Residuals	4 15	0.067220 0.036563		0.0168050 0.0024376	60.8942	0.6477 0.3523	0.001
	38	Total	19	0.103783					1.0000
		Pairwise		Porcine		Monarch	IC 50	Total
		LAtV × MAt	1	0.039476 ATPase (IC 50 ) 0.039476	3.429 ATPase (IC 50 )	0.36367 ratio	0.037 n
		LAtV × MAc LAtV × MAtV Ouabain (standard)	1 1	0.045634 0.026582	0.81	0.045634 0.026582		2.1744 1.7521 122.09	0.266 0.22601 151	0.123 0.143 11
		LAtV × FMAtV Digitoxin (standard)	1	0.020933	0.49	0.020932		1.3962 16.25	0.18877 33	0.181 12
		MAt × MAc MAt × MAtV Frugoside (sequestered) 1 1	0.024287 0.026924	0.37	0.024287 0.0269240	2.2136 6.3835 27.80	0.26951 0.51548 76	0.121 0.002 8
		MAt × FMAtV Calotropin (sequestered+) 1	0.033727	0.27	0.033727		4.3275 10.90	0.41902 40	0.036 6
		MAc × MAtV MAc × FMAtV Calactin (sequestered+) 1 1	0.028296 0.046877	0.24	0.0282957 0.046877	3.4997 3.2347 3.21	0.3684 0.35028 13	0.024 0.033 6
		MAtV × FMAtV Uscharin (unsequestered) 1	0.021856	0.78	0.0218561	2.2519 1.00	0.27289 1	0.038 12
		Voruscharin (unsequestered)		0.46			2.02	4	26
										48

Table S5 . Poisson GLM model fits for predicting monarch oviposition on 212 Asclepias

 S5

https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID=CCMSLIB00005724354#%7B%7D

LAtV: Leaf of A. tuberosa painted with voruscharin; MAt: Monarchs on A. tuberosa;

MAc: Monarchs on A. curassavica; MAtV: Monarchs on A. tuberosa painted with voruscharin;

FMAtV: Frass from monarchs fed A. tuberosa painted with voruscharin.

DF -degrees of freedom; SumSq -sum of squares; MeanSqr -mean squares.

P-values based on 999 permutations (lowest P-value possible 0.001).

curassavica plants in a common greenhouse environment.

Molecular mass 756 g/mol

Molecular mass 610 g/mol

Acknowledgements

We thank Bennett Fox and Frank Schroeder for unfailing assistance with the mass spectrometry of cardenolides at the Boyce Thompson Institute and Nicolas Baert for mass spectrometry analyses of quercetin glycosides at the Cornell Chemical Ecology Core Facility. Chelsea Lee and Steven Broyles helped with latex collection. The manuscript was improved by comments from two anonymous reviewers, Nathaniel Carlson, Tyler Coverdale, Arielle Johnson, Xosé López-Goldar, Jennifer Thaler and Shirley Zhang. Frances Fawcett drew the monarch in the figures. Funding: This research was supported by a grant from the National Science Foundation to A.A.A. (IOS-1907491). Author contributions: K.B. conducted the plant grow up, monarch bioassays on whole plants, and plant HPLC-UV analysis. M.H. helped with the quantification,

Supplementary Information for

Cardenolides, toxicity and the costs of sequestration in the coevolutionary interaction between monarchs and milkweeds

Anurag A. Agrawal 1,2* , Katalin Böröczky 1 , Meena Haribal 3 , Amy P. Hastings 1 , Ronald A. White 1 Ren-Wang Jiang 4 , Christophe Duplais 5 Email: agrawal@cornell.edu This PDF file includes: S4. The relative inhibitory potential of seven isolated cardenolides on the sensitive porcine and typically-resistant monarch butterfly Na-K-ATPase (See also Figure 3

2

Where compounds were not identified their retention time is given. Four non-significant 3 correlations are highlighted in grey.