Survival analysis of germination data in response to temperature for Ornithopus species and other temperate pasture legumes

Experiment 1: germination, emergence and early seedling development

The goal of these experiments (reported collectively as Experiment 1) was to examine variation in germination and emergence rate and early seedling growth in response to temperature between yellow serradella (Ornithopus compressus L.) and French serradella (Ornithopus sativus Brot.). Their response was benchmarked against a set of reference species commonly used in Australian temperate pasture systems: burr medic (Medicago polymorpha L.), lucerne (Medicago sativa L.) and subterranean clover (Trifolium subterraneum L.). The experiment was split into two components based on the test method:

The experiments were conducted in climate-controlled growth rooms at The University of Western Australia, Perth, Australia. Four temperature treatments were used (max/min, °C): (1) 10/5; (2) 15/10; (3) 20/15; and (4) 25/20 with a 12 h day/night interval. It took approximately 15 min for each cabinet temperature change to occur but temperature then remained stable for the duration of the day or night period. Light intensity was 500 μmol photons m⁻² s⁻¹ (visible light range), adequate to saturate the photosynthetic system of C3 plants (Sager and McFarlane 1997). The light intensity took up to 30 min to stabilise each morning. Each of the four temperature treatments were imposed within one only of four separate plant growth cabinets, with three replicates arranged as a randomised block within each cabinet.

Table 1 lists the 10 species groups examined, including two species of serradella (Ornithopus compressus and Ornothopus sativus) and three reference pasture cultivars (Medicago polymorpha, Medicago sativa and Trifolium subterraneum). The serradella species groups consisted of the following:

Group A and Group B were delineated to assess variation in germination, emergence and early growth between cultivars that are currently (or were until recently) commercially available and those that are no longer available. Even though some cultivars in Group A were developed some time ago, the fact that they are still in commercial production may suggest that there are some favourable traits (such as even and successful establishment) that make them preferred by growers.

Seeds were analysed to demonstrate variation in seed size and nutrition as these can have an influence of seedling emergence and early vigour (Black 1957). Seed weight was determined by averaging the weight of three subsamples of 100 seeds. Seed phosphorus (P) was determined by completing a 5:1 mL nitric:perchloric acid digestion and colorimetric measurement using the yellow method (Motomizu et al. 1983). Seed nitrogen (N) was determined by analysing seeds with an Elementar (Model Vario Macro; Hanau, Germany) using the Dumas combustion method (Dumas 1831).

All seeds were removed from their pods and sterilised for 1 min in a 10% solution of bleach (NaClO). Hard-seeded species were scarified to improve germination and seeding rate was adjusted to achieve equivalent densities of germinable seed. To quantify seed germination rate, 25 germinable seeds were placed into 90 mm Petri dishes with Whatman grade 1 filter paper and wet with 3 mL of 0.5 mM CaSO₄ solution; there were three replicate Petri dishes for each cultivar. To quantify seedling emergence rate, 25 germinable seeds were also sown into the soil medium with three replicate for each cultivar. A steam-pasteurised (maintained at 70°C for 30 min) potting mix was used to ensure nutrients were not limiting for seedlings once they had exhausted their seed reserves. Eighty-eight seedling trays (345 L × 295 W × 50 D mm) were steam-pasteurised and 3.5 kg of potting mix weighed into each tray. A row each of five cultivars was sown to a depth of 1 cm in each tray in a randomised and replicated design. After sowing, peat-based rhizobia (Becker Underwood) was applied at a rate of 0.5 g per 9 L watering can. A combination of Group S (WSM471), Group C (WSM1325), Group AL (RRI128) and Group AM (WSM1115) was applied to accommodate the rhizobia requirements of the Ornithopus, Trifolium and Medicago species.

Experiment 2: relative growth rates (RGR) and leaf gas exchange

Experiment 2 assessed RGRs and leaf gas exchange among serradella cultivars at cool temperatures compared to ‘ideal’ growing temperatures. The growth assessments occurred on two cultivars each of yellow serradella (Santorini and Avila) and French serradella (Margurita and Serratas), benchmarked against a winter-active lucerne cultivar (SARDI 10) and a subterranean clover cultivar (Woogenellup), which is also known for good winter vigour (Collins et al. 1996). Two temperature treatments were selected based on preliminary data from the Petri dish experiment and published literature. Although temperatures below 10°C maximum and 5°C minimum are common throughout southern Australia, they were deemed too cold for experimental purposes as little or no growth would occur and identifying differences between species would be difficult. Maximum temperatures of 20–25°C are considered optimal for legumes (Fukai and Silsbury 1976; McWilliam 1978). Hill and Luck (1991) showed that temperate pasture legumes produced the highest DM at 24/20°C (max/min) compared with lower temperatures. However, given that we had strong indications of better early growth at 20/15°C than 25/20°C for serradella in Experiment 1, we opted for an intermediate temperature range of 23/18°C to compare with growth at 15/10°C. There were five harvest times (weekly from 3 to 7 weeks after sowing) and three replicates of each cultivar × temperature × harvest time combination. The pots for each temperature treatment were placed randomly in a single growth cabinet and pot location re-randomised weekly.

Each pot (polyvinyl chloride; 200 mm (H) ×90 mm (D)) was filled with a 1.2 kg mixture of sandy loam and potting mix. The drained upper limit (field capacity) of the potting soil was estimated by determining the amount of water held per gram of dry soil after pots had been watered to saturation and then allowed to drain for 72 h (i.e. until free drainage had ceased). Pots were then maintained at field capacity for the duration of the experiment. Nutrient analyses indicated that the soil contained sufficient nutrients for plant growth over the duration of the experiments (data not shown). All seeds were removed from their pods and sterilised for 1 min in a 10% solution of sodium hypochlorite (NaClO); hard-seeded serradella cultivars required scarification. Ten seeds were sown in pots to a depth of 1 cm. To mimic sward conditions, pots were fitted with reflective sleeves made of insulation sarking that were raised regularly to match canopy height (Jeffery et al. 2017).

The pots were kept in darkened plant growth cabinets at 15°C until the cotyledons started emerging. At this stage, lighting was imposed and temperatures were changed to 23/18°C in both cabinets to encourage good early growth. Plants were grown for 3 weeks before an initial harvest (21 days) where shoots were cut at the soil surface and roots were washed from the soil medium over a 2 mm sieve before being oven-dried at 70°C for 3 days and weighed. Post-harvest, one growth cabinet was reduced to the low temperature (15/10°C) for the remainder of the experiment. Three replicates of each cultivar were harvested from each of the two temperature treatment cabinets every 7 days. Thus, plant measurements and destructive harvests were completed at 21, 28, 35, 42 and 49 days after sowing.

Statistical analysis

Experiment 1: germination and emergence in response to temperature

Germination

Germination was significantly slower at 10/5°C than at the other temperatures (Fig. 1a, b). Yellow serradella (Group A and Group B) and Group B French serradella cultivars generally germinated faster than burr medic and subterranean clover, but not lucerne (Fig. 1a, b). The Group B yellow serradella cultivars germinated more rapidly than the Group A cultivars at the lowest (10/5°C) and highest (25/20°C) temperatures. The cool temperature reference did not differ from the warm temperature reference, except at the highest temperature, where the cool temperature reference germinated more rapidly. Among French serradella, Group B cultivars germinated faster than Group A cultivars at the lowest temperature but were similar at the other temperatures. The French serradella groups germinated more rapidly than burr medic at the warmest temperature but did not differ from lucerne (except at 25/20°C where they were slower to germinate) or subterranean clover (except at 20/15°C where they were faster to germinate). Yellow serradella germinated more rapidly than French serradella at 15/10°C and 25/20°C (Fig. 1, Supplementary Table S3).

Fig. 1.

(a) Heatmap displaying the extent and direction of Wij statistics of Steel-Dwass-Critchlow-Fligner test for germination. The map compares the variation in germination time (days) in response to temperature (10/5, 15/10, 20/15 and 25/20°C, max/min) among species groups from yellow serradella, French serradella and reference cultivars (burr medic, subterranean clover and lucerne). Colour coding indicates the extent of the variation among groups (compare x-axis then y-axis), with red indicating slower germination time and blue indicating faster germination time. White indicates no significant difference among groups. The harmonic mean and s.d. are also presented. Only data from germinated seeds are included (see Table 2 for survival analysis, including data from non-germinated seeds). (b) Nested plots demonstrate the influence of temperature treatments on the harmonic mean of germination time for each species group. See Table 1 for the description of species groups.

Emergence

Emergence was also significantly slower at 10/5°C than at the other temperatures (Fig. 2a, b). Yellow and French serradella species groups generally emerged faster than subterranean clover at cooler temperatures (10/5°C, 15/10°C). However, they emerged more slowly than lucerne at warmer temperatures (20/15°C, 25/20°C). For serradella species groups, the fastest rate of emergence was at 20/15°C and emergence at 25/20°C was similar to that achieved at 15/10°C. Group B yellow serradella cultivars emerged faster than Group A cultivars at all temperatures and subterranean clover at the cool temperatures. Despite a lower mean emergence time for the cool temperature reference compared to the warm temperature reference at all temperature treatments, they did not statistically differ (Fig. 2, Table S3). There was no difference in emergence time among French serradella groups. Group B yellow serradella cultivars emerged slower than Group B French serradella cultivars at 15/10°C and 20/15°C.

Fig. 2.

(a) Heatmap displaying the extent and direction of Wij statistics of Steel-Dwass-Critchlow-Fligner test for emergence. The map compares the variation in emergence time (days) in response to temperature (10/5, 15/10, 20/15 and 25/20°C, max/min) among species groups from yellow serradella, French serradella, and reference cultivars (burr medic, subterranean clover, and lucerne). Colour coding indicates the extent of the variation among groups (compare x-axis then y-axis), with red indicating slower emergence time and blue indicating faster emergence time. White indicates no significant difference among groups. The harmonic mean and s.d. are also presented. Only data from emerged seeds are included (see Table 2 for survival analysis, including data from non-emerged seeds). (b) Nested plots demonstrate the influence of temperature treatments on the harmonic mean of emergence time for each species group. See Table 1 for the description of species groups.

Rate of shoot and root dry mass production

The impact of temperature treatment on the shoot and root DM production rate varied among species groups (species group × temperature treatment interaction (P < 0.001)) (Fig. 3), with this interaction largely driven by the greater DM response of subterranean clover in the cooler temperature treatments than the warmer temperature treatments. No significant variation in the shoot or root DM production rate occurred among serradella species groups at any temperature. At cool temperatures (i.e. 10/5°C and 15/10°C), subterranean clover had the greatest shoot and root DM, but the remaining species groups did not differ. At 20/15°C, subterranean clover had greater shoot and root DM than serradella species groups but similar shoot DM to lucerne. At 25/20°C, lucerne had greater shoot DM than most serradella species (except Portuguese cultivars) but did not differ significantly from the other reference species.

Fig. 3.

The effect of four temperature treatments (10/5, 15/10, 20/15 and 25/20°C, max/min) and 10 species groups on the rate of shoot (top green bars) and root (bottom beige bars) dry mass production from the time of emergence to 21 days after sowing. Values are species group means (+s.d.). There was a significant (P < 0.001) temperature treatment × species group interaction for total plant DM but only species group differences within each temperature treatment are presented. Temperature treatment means annotated with the same capital letter at the top of the graph do not differ at P < 0.05. Significant (one-way ANOVA, P < 0.05) variation among species groups for shoot or root dry mass within each temperature treatment is denoted by different lowercase letters (Games-Howell post hoc test).

A significant interaction of temperature and seed nitrogen (N) (P = 0.003) and temperature and seed phosphorus (P) (P < 0.001) occurred for shoot DM production rate, primarily driven by temperature (F ≥ 304), not seed N (F = 10) or seed P (F = 13). Moreover, when we applied linear regression to each temperature treatment, no significant relationships occurred between shoot DM production rate and seed P (Fig. 4), except for a weak positive relationship at 10/5°C (R² = 0.10). In contrast, shoot DM production rate and seed N had a weak but positive correlation for all temperature treatments, which was strongest at 20/15°C (Fig. 4).

Fig. 4.

The relationship between shoot dry mass and (a) seed nitrogen concentration and (b) seed phosphorus concentration with four temperature treatments (10/5, dark blue; 15/10, light blue; 20/15, green; and 25/20°C, orange; max/min) for yellow serradella, French serradella and reference species groups. The correlation coefficient for each temperature response is shown in the graph if the relationship was significant at P < 0.05. Each data point represents a replicate (n = 3). Significant (P < 0.05) effects of temperature treatment are denoted by different capital letters (2-way ANCOVA).

Rate of leaf appearance and total dry mass

Each temperature treatment had the number of days to leaf appearance recorded over the 21-day experimental period. Leaf appearance was deemed to occur when >50% of the plants within a row had reached a specific leaf stage. At 10/5°C, no species group had >50% of seedlings with an open first leaf during the experimental period (Fig. 5). At 15/10°C, only the reference species produced a first (unifoliate) leaf. No serradella species group reached the third leaf stage in any temperature treatment, whereas the reference species reached the third (all species) and fourth (subterranean clover only) leaf stages.

Fig. 5.

Time of leaf appearance over the 21-day growth period post-sowing relative to shoot DM for species groups from yellow serradella (a, b, c, d), French serradella (e, f, g) and reference groups (h, i, j) with four temperature treatments (10/5, dark blue; 15/10, light blue; 20/15, green; and 25/20°C, orange; max/min). Different letters denote significant temperature treatment differences (ANCOVA with Games-Howell post hoc test). Numbering within line graphs represents the mean date that ≥50% of plants reached a leaf stage (1, first leaf; 2, second leaf; 3, third leaf; and 4, fourth leaf).

For shoot DM (which includes the effect of delayed germination and/or emergence), significant (ANCOVA P < 0.001) temperature treatment differences occurred between species groups (Fig. 5). Serradella species groups generally had greater shoot DM at 20/15°C than 25/20°C, whereas the reference species did not significantly differ at these temperatures. At 10/5°C, subterranean clover had approximately double the shoot DM of serradella species groups, but no significant differences occurred among species groups at 15/10°C (Table S1). At 20/15°C and 25/20°C, all reference species had more shoot DM than serradella species groups (except Group A French serradella cultivars at 25/20°C).

Experiment 2: temperature effects beyond the seedling stage

Experiment 2 compared shoot RGR of two yellow serradella and two French serradella cultivars with subterranean clover and lucerne from 21 to 49 days after sowing. A positive RGR response occurred for the higher temperature treatment (23/18°C; P = 0.006, two-way ANOVA) but did not differ between cultivars. One-way ANCOVA and pairwise comparisons at 15/10°C demonstrated that lucerne and subterranean clover had greater RGR than all serradella cultivars except ‘Avila’ (Fig. 6). On closer analysis, this result primarily occurred because subterranean clover had more initial shoot DM at the start of the measurement period (i.e. after 21 days) and lucerne had more shoot DM at the last harvest than serradella cultivars (Table S2). At 23/18°C, lucerne had greater RGR than all other cultivars. The RGR of subterranean clover and serradella cultivars did not differ (except for cv. Serratas, which was lower). When we examined the raw DM values, it was evident that temperature affected lucerne the most, with DM at 15/10°C only 60% of that achieved at 25/20°C, compared to serradella (77–85%) and subterranean clover (96%) (Table S2).

Fig. 6.

Effect of temperature (23/18°C (red line and red circles) and 15/10°C (blue dash line and blue squares)) and harvest time on relative growth rate (RGR) of shoots (log shoot DM (g)) for six cultivars. Yellow serradella cvs (a) Avila and (b) Santorini; French serradella cvs (c) Margurita and (d) Serratas; lucerne cv. (e) SARDI 10; and subterranean clover cv. (f) Woogenellup. Each symbol represents a replicate (n = 3). Different letters indicate significant differences between cultivars at 15/10°C (lower case letters) and 23/18°C (capital letters) based on a comparison of the least square means and Tukey’s post hoc test.

Physiological measurements

A significant cultivar × temperature treatment interaction occurred for all physiological measurements (photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO₂). While no significant differences occurred between cultivars at 15/10°C, significant differences occurred between cultivars at 23/18°C (Fig. 7a–d). Subterranean clover and lucerne had higher photosynthetic rates (CO₂ assimilation) at 23/18°C than French serradella (25 vs 17–20 μmol CO₂ m⁻² s⁻¹) but did not differ from yellow serradella (21–23 μmol CO₂ m⁻² s⁻¹). Yellow serradella had a higher photosynthetic rate at 23/18°C than 15/10°C, but French serradella was not responsive to temperature (Fig. 7a).

Fig. 7.

Effect of two temperature treatments (15/10 (blue bars to left) and 23/18°C (red bars to right for each species) (max/min)) and four species: yellow serradella (YS); French serradella (FS); lucerne (L); and subterranean clover (SC) on leaf gas exchange measurements. (a) CO₂ assimilation, (b) stomatal conductance, (c) transpiration rate and (d) intercellular CO₂. Different letters within plots indicate significant (P < 0.05) temperature × species differences (Games-Howell post hoc test).

For stomatal conductance and transpiration rate, the significant cultivar × temperature treatment interaction was primarily driven by greater values for subterranean clover at 23/18°C than 15/10°C, as temperature did not affect stomatal conductance or transpiration rate for serradella cultivars or lucerne (Fig. 7b, d). Subterranean clover and lucerne had up to double the stomatal conductance at 23/18°C (0.76 and 0.55 mol m⁻² s⁻¹, respectively) than Ornithopus spp. (0.23–0.36 mol m⁻² s⁻¹), with subterranean clover higher than all other cultivars and nearly double the stomatal conductance achieved at 15/10°C (Fig. 7b).

For intercellular CO₂, no significant differences occurred among species at 15/10°C (range 290–309 μmol mol⁻¹). However, at 23/18°C, intercellular CO₂ decreased for yellow and French serradella cultivars, but not lucerne and subterranean clover (Fig. 7c).

Seedling germination and emergence

In regions of southern Australia that experience cool winter temperatures, the use of serradella species is adversely affected by anecdotal reports of poor establishment and low winter growth rates relative to other temperate pasture legumes (Rossiter et al. 1985). However, we found no evidence that cool temperatures, in isolation, limit the early productivity of serradella species relative to other commonly used temperate pasture legumes.

Cool temperature treatments delayed germination rates by 1–4 days among the species groups studied, compared to the fastest germination rate in the warm temperature treatments. Rapid and uniform germination is generally a favourable trait for seedling establishment (Covell et al. 1986; Blum 2018) and species competitiveness. Serradella species groups demonstrated (with a higher hazard ratio) that they were more likely to germinate at any temperature (and more rapidly in the case of yellow serradella) than burr medic or subterranean clover. However, warmer temperatures (i.e. 25/20°C, max/min), delayed serradella emergence, which may place some cultivars at a competitive disadvantage in relation to faster emerging weeds and/or grasses.

The most rapid emergence for serradella species groups occurred at 20/15°C (French serradella groups > yellow serradella groups). For some serradella species groups, the emergence rate at 15/10°C did not significantly differ from that at 25/20°C. At these temperatures, fast-growing species such as lucerne emerged from the soil within 2 days after sowing, whereas serradella did not emerge until 8–12 days. Such a delay is not insignificant, with differences of only one day contributing to improved leaf area in cereals (Richards et al. 2002). Genetic studies have subsequently been undertaken to improve the germination and emergence of agronomically important seed crops (Finch-Savage et al. 2010; Zhang et al. 2015). While similar work has not been specifically undertaken for pasture species such as serradella, their characteristic seed germination and emergence patterns are important as they may influence their germination and establishment success on farms.

As seeds were scarified before sowing, the delayed emergence pattern observed in some serradella cultivars cannot be due to variation in seed coat permeability but may reflect some physiological dormancy. Delayed seedling emergence can be an adaptive response to false breaks (i.e. unseasonal rainfall events in summer and/or early autumn that causes pasture seeds to germinate and seedlings to emerge but insufficient further rainfall occurs to ensure seedling survival) (Revell et al. 1998; Dear et al. 2002). In this case, by remaining dormant in the soil, the species may have a greater chance of survival than for species where seeds uniformly emerge. In summary, cool temperatures do not limit serradella germination or emergence relative to other pasture legumes, but warm temperatures (i.e. ≥25°C) delay emergence.

The pairwise comparisons in the heat maps (Figs 1 and 2) reveal the differences between species groups (Table S3). For example, Group B yellow serradella often had more rapid germination and emergence than Group A, presumably a collateral consequence of selection/breeding for improved agronomic fitness. In comparison, minor variation occurred between Group A and Group B cultivars of French serradella for germination or emergence. Late-maturing Portuguese cultivars of French serradella were slower to germinate than Group B French serradella cultivars, but this did not result in a slower emergence time.

We specifically included two yellow serradella accessions collected at locations differing by >5°C in winter temperatures to examine whether provenance was likely to have an impact on germination or emergence at low temperatures. However, no significant differences were evident between these accessions for germination or emergence at any temperature (except for the cool temperature reference germinating faster at warm temperatures than cool temperatures). These results are indicative only, and analysis of a greater suite of germplasm is required to assess whether this is a useful selection method for this trait.

Early dry matter production and leaf development

At temperatures considered sub-optimal for plant growth (i.e. <10°C daily maximum), all pasture species had slow establishment and early growth, with the early DM productivity of serradella comparable to the reference species at low temperatures. Maximum shoot DM for serradella species groups occurred at 20/15°C. Inevitably, late sowing times or late breaks in the season will result in low pasture productivity, regardless of the legume species used. However, at warmer temperatures (i.e. ≥25°C daily maximum), serradella produced up to three times less shoot DM than subterranean clover and lucerne due to delayed emergence and a slower growth rate. In contrast, DM production of subterranean clover plateaued at the warmest temperature, and that of lucerne increased progressively with increasing temperature (McWilliam et al. 1970).

A likely factor for the greater growth rates of burr medic, lucerne, and subterranean clover is their ability to initiate leaf development faster than serradella (Marañón and Grubb 1993), providing a competitive advantage because energy production through photosynthesis (beyond what is produced by cotyledons) can commence earlier. No leaves appeared (i.e. >50% of plants with fully open first leaf) within 21 days in the serradella species groups at the two coolest temperatures (10/5°C and 15/10°C), whereas all reference species had at least the first (unifoliate) leaf by 16–19 days at 15/10°C. Within the optimum temperature treatment for serradella (20/15°C), first leaf development occurred approximately 14 days after sowing, with subterranean clover producing its first trifoliate leaf (in addition to the unifoliate leaf) within this time. The superior growth rates of reference species at warm temperatures resulted in up to three trifoliate leaves (in addition to the unifoliate leaf) within 21 days, likely providing a competitive advantage over serradella in warm environments not limited by water availability.

Seed reserves

Large seeds (with greater nutrient reserves) can improve early pasture productivity (Kidd and West 1919; Black 1956). In our study, the differences in early shoot DM production (up to 21 days) positively correlated with seed N, with a stronger relationship at warmer temperatures (Fig. 4). Serradella cultivars had minimal variation in seed size and nutrient reserves (Table 1). For this reason, coupled with low variation in germination rates at cool temperatures (Young et al. 1970), it is unlikely that serradella species can be improved in this regard.

Relative growth rates

In Experiment 2, serradella cultivars generally had lower RGR than lucerne and subterranean clover beyond the first 21 days of development when grown in cool conditions (15/10°C). However, the raw shoot DM values (Table S2) indicate no significant differences between cultivars 42 days after sowing; it was only at the final sampling time (day 49) that lucerne had significantly greater DM than serradella and subterranean clover. Under warmer conditions (23/18°C), lucerne had far greater RGR and overall DM production than any other species, with no difference between serradella and subterranean clover.

Shoot RGR for all species was greatest at 23/18°C, but the rate of DM increase (indicated by variation in slopes in Fig. 6) for lucerne and subterranean clover differed among the temperature treatments. For lucerne, differences in RGR at the two temperatures increased over time, while the opposite was true for subterranean clover, which had a similar RGR for both temperature treatments by 49 days of growth. There are two possible explanations for this observation. The first might be associated with nutrient limitations or pot space restrictions, with the faster plant growth rates at 23/18°C exhausting nutrient reserves faster than at 15/10°C. The second relates to full canopy closure and a potential reduction in light interception occurring earlier at 23/18°C than 15/10°C. Light interception by the plants mimicked growth in a pasture sward due to the use of reflective sleeves raised regularly to just below canopy height. Once each micro-sward reaches canopy closure, the RGR should decrease due to the shading of lower leaves. However, due to differences in leaf architecture among the three species, full canopy closure only occurred for subterranean clover, which may explain the overlap of shoot RGR in the two temperature treatments.

The greatest negative impact on DM production (i.e. greatest disparity between RGR) at cool temperatures was for lucerne, primarily because its potential DM productivity at warm temperatures is much greater than the other species. Our results also revealed that cool temperatures limited the productivity of the tested reference species, not just serradella, a notion supported by the lack of variability in winter growth rates recorded among pasture species (Saul and Sargeant 2013). The anecdotal observations of poor growth rates by serradella under cool conditions may be due to other co-occurring factors, such as a lack of waterlogging tolerance (Kidd et al. 2020) or soil-borne disease pressure under the cool, wet conditions of autumn–winter.

Leaf physiology

Despite the lack of water stress in Experiment 2, there was no significant change in stomatal conductance or photosynthetic rate for Ornithopus spp. While such an adaptive response may limit the early season productive potential of Ornithopus spp., it may also slow soil water usage, allowing plants to survive longer into the growing season (when temperatures rise and soil moisture rapidly declines) than species that do not have such stomatal adjustments. In the semi-arid regions of Western Australia, where Ornithopus spp. are used widely, this adaptive response allows them to survive and set seed in autumn–spring growing seasons, which are becoming increasingly drier and of shorter duration (Asseng and Pannell 2013).