**1. Introduction**

Broadacre farming frequently occurs with livestock production throughout southeastern Australia, with the pasture phase of crop rotation sustaining both lamb and cattle enterprises [1]. Lamb and beef production account for the majority of livestock-related income in southeastern Australia (AU\$22 billion in 2017) and global demand is projected to dramatically increase over the next decade (Australian Bureau of Statistics, 2018). Legumes are integral to livestock pasture production systems through provision of high quality forage for grazing livestock. Establishment of pasture species that are non-toxic, persistent, and high in nutritional quality is therefore critical for continued improvement of livestock productivity.

Traditionally, subterranean clover (*Trifolium subterraneum* L.) and lucerne (*Medicago sativa* L.) are the most widely utilized pasture species in prime lamb, wool, and cattle producing regions across southeastern Australia. Subterranean clover is compatible across various soil types and is tolerant of pH extremes [2,3]. Lucerne is a deep-rooted perennial species frequently established across diverse rainfall regions and is suitable for neutral or mildly alkaline soils. Recently the establishment of both pasture species has proven challenging to sustain livestock production due to a range of biotic and abiotic factors. For example, lucerne, which contains high protein content, has been shown to undergo rapid fermentation in the rumen resulting in increased incidence of bloat and potential loss of nitrogen due to excretion [4]. In addition, ingestion of lucerne and subterranean clover is associated with metabolic disorders including acute inflammation of both the small and large intestine (red gut), sodium deficiency and pregnancy-related toxemia [5–7].

Increased risk of metabolic disorders, the significant cost of establishment, and low persistence of traditional legume pastures in low-rainfall regions [8,9] has led to the introduction of novel annual pasture legume species originating from Mediterranean regions of Europe and northern Africa to Australia [3]. These include *Biserrula pelecinus* L. (biserrula), *Ornithopus sativus* Brot. (French serradella), *Ornithopus compressus* L. (yellow serradella), *Trifolium glanduliferum* Boiss. (gland clover), *Trifolium spumosum* L. (bladder clover), and *Trifolium vesiculosum* Savi. (arrowleaf clover). These accessions are characterized by their adaptation to deep, acid and sandy soils, drought tolerance, weed suppressive potential, and prolonged availability as feed for livestock [10,11]. While the nutritive value of traditional pasture legumes has been studied in southeastern and Western Australia, a detailed investigation of the phenolicchemistryofannuallegumeshasnotbeenperformedwithrespecttolivestockproduction[2].

Legumes have evolved chemical defense mechanisms mediated by secondary metabolites, including phytoestrogens, to deter herbivores and plant pests [12,13]. In terms of phenolic chemistry and key secondary metabolites in pasture legumes, flavonoids represent a distinct class of secondary products with both positive and negative impacts on plant–microbial and plant–livestock interactions [14,15]. In general, they are classified by their chemical structure into subgroups including anthocyanidins and anthoxanthins (flavanones, flavans, and flavanonols) [16].

Significant levels of phytoestrogens are produced in pasture legumes including lucerne as well as various clover species and when present at significant levels can seriously reduce reproductive efficiency and livestock fertility [15,17,18]. Coumestans and isoflavone phytoestrogens are stable, non-steroidal secondary metabolites that mimic mammalian estrogen, an endogenous female sex hormone [15,19]. The a ffinity of these phytoestrogens in binding estrogen receptor-β can result in reproductive abnormalities during embryo development, and infertility in both sexes of grazing livestock [20]. Isoflavone phytoestrogens are typically stored either as glycosides or aglycones in pasture legumes [15,21].

Coumestans are non-flavonoid phytoestrogens, and include coumestrol and 4-methoxycoumestrol, first isolated from white clover (*Trifolium repens* L.) and lucerne (*Medicago sativa* L.) in 1957 [22]. These polycylic aromatic metabolites are closely related biosynthetically to flavonoids (Figure 1). Elevated concentrations of phytoestrogens, including isoflavones, coumestrol, and related metabolites, from ingestion of fodder or feedstock have been implicated in estrogenic clinical signs in livestock as edematous vaginal and cervical tissue, hypertrophy of mammary glands, and milky secretions from elongated teats. Phytoestrogens in forage may also cause adverse e ffects in ovarian function resulting in loss of fecundity or early embryonic death [23]. Livestock grazing various *Trifolium* species exhibit varying tolerance to coumestans, which typically range in concentration from 25 to 200 mg kg−<sup>1</sup> dry matter (DM) [15]. The presence of coumestrol at concentrations greater than ≈40 mg kg−<sup>1</sup> DM in plant tissues is associated with reproductive ine fficiency in sheep and cattle through disruption of several endocrine mechanisms [24,25].

**Figure 1.** Phytoestrogens found in abundance in lucerne (*Medicago sativa*) and subterranean clover (*Trifolium subterraneum*).

Isoflavonoids are 3-phenylchromen-4-ones (3-phenyl-1,4-benzopyrone) and are important in regulating numerous interactions in higher plants. The isoflavone biosynthetic pathway is one of the most well elaborated pathways in plant secondary metabolism due to the importance of isoflavones as chemoattractants for rhizobia and their involvement in plant defense. Isoflavones are mainly derived from the phenylpropanoid pathway but can be generated through multiple pathways in many plant species [26]. Legumes possess a unique enzyme, isoflavone synthase (IFS), which is a cytochrome P450 monooxygenase that catalyzes the 2, 3 migration of the B-ring of naringenin or liquiritigenin, resulting in the production of various biologically active isoflavonoids [27]. The genes encoding enzymes in this pathway are tissue specific and are regulated both spatially and temporally in legumes [28]. Such catalytic enzymes are induced by various stress factors influencing plant condition, including climate, temperature, soil moisture availability, nutrient deficiency, and herbivory [29].

Polyphenolic compounds, including condensed tannins (proanthocyanidins), are another key group of metabolites possessing a range of biological and nutritional properties in grazing livestock. These compounds vary with regard to chemical structure, plant source, and target animal species [30]. For example, monomeric phenolic acids in forages are associated with enhanced milk production and acid-base imbalance in the rumen following microbial degradation [31]. Traditionally, the proanthocyanidins, which are oligomeric polyphenols, have been considered as anti-nutritional factors leading to reduced palatability of forages, but recent research has shown they provide several potential advantages to grazing livestock, including reduced risk of bloat at moderate intake of 3–4% of dry matter (DM) [32] and increased weight gain, while reducing greenhouse gas emissions [33–35] and parasite burden [36,37]. However, feed composition, age and physiological status of the animal are some of the key factors need to be taken into account while studying the effects of plant secondary metabolites on livestock [38,39].

There is a marked lack of information on the phytochemical profiles of aerial tissues of annual pasture legumes, particularly those recently introduced to the southern hemisphere from the Mediterranean. Recent advances in quadrupole time-of-flight mass spectrometry (MS-QToF) instrumentation have resulted in increasing usage of time-of-flight mass spectrometry (MS-ToF) instruments as quantitation tools as well as prediction of molecular formulae because of their high resolving power and wide dynamic range [40,41]. To broaden our understanding of the secondary chemistry of novel pasture legumes in Australia, we took a metabolomics approach using LC-MS-QToF to investigate (a) the distribution of secondary metabolites that may impact livestock performance, including flavonoids and phytoestrogens (coumestrol, 4-methoxycoumestrol, formononetin, genistein and daidzein), in above-ground tissues of selected traditional and newly introduced annual pasture legumes grown under field conditions in southern Australia; (b) the total polyphenol and proanthocyanidin content to better delimit their prevalence in these pasture

species, and (c) the biosynthetic pathways associated with the production of flavonoids and certain phytoestrogens in these plants.
