Species‐specific thermal thresholds for postdispersal embryo growth in Apiaceae

Abstract

Premise

Temperature is a primary regulator of seed development. In seeds with morphological (MD) or morphophysiological (MPD) dormancy, embryo elongation represents a distinct postdispersal developmental phase that precedes germination. However, the thermal thresholds governing this embryo growth phase remain poorly quantified. We quantified species‐specific embryo‐growth thermal niches to provide a mechanistic framework for understanding regeneration timing and its evolutionary constraints.

Methods

We estimated cardinal temperatures—base (T _b), optimum (T _o), and maximum (T _m)—for embryo growth in 10 Apiaceae species, a family in which MD and MPD are very common due to the presence of underdeveloped embryos. Seeds were incubated at five temperatures (5–25°C) with and without gibberellic acid (GA₃). Embryo growth rates were modeled using nonlinear thermal performance curves within a multimodel inference framework, followed by phylogenetic signal analyses.

Results

Substantial interspecific variation was found, with T _b ranging from 0 to 6.5°C, T _o from 5 to 25°C, and T _m from 21.5 to 31°C. GA₃ generally increased growth rates and widened thermal ranges by reducing T _b and raising T _m, though responses were strongly species‐specific. Phylogenetic analyses revealed significant signal for all thermal thresholds (T _b, T _o, T _m), indicating that evolutionary history constrains these thermal niches for postdispersal embryo growth.

Conclusions

Postdispersal embryo growth constitutes a distinct, quantifiable thermal niche rather than a mere proxy for germination. By explicitly treating postdispersal embryo growth as a distinct thermal niche rather than a proxy for germination, this study extends thermal threshold theory to an overlooked developmental phase and provides a mechanistic framework for understanding dormancy release and regeneration timing under variable climatic conditions.

Functional variation in plant traits plays a central role in shaping species distributions across environmental gradients and mediating responses to climate change (Violle et al., 2014). Among these traits, temperature‐dependent developmental processes are particularly influential because they regulate the timing and success of key life‐history transitions, including seed germination and early seedling establishment (Bykova et al., 2012; Fernández‐Pascual et al., 2019; Walker et al., 2025). Importantly, temperature sensitivity extends beyond germination sensu stricto because in some species it also affects earlier postdispersal seed developmental stages that determine whether and when germination can occur. Despite their ecological significance, the physiological thresholds governing postdispersal temperature‐driven seed developmental processes remain poorly quantified for many taxa, limiting our ability to predict regeneration dynamics under shifting climatic conditions (Bykova et al., 2012; Blandino et al., 2025).

Seed germination sensitivity to temperature is commonly described using thermal thresholds, the base temperature (T _b), below which development ceases; the optimum temperature (T _o), at which developmental rates are maximized; and the maximum temperature (T _m), above which development is inhibited (Garcia‐Huidobro et al., 1982; Watt and Bloomberg, 2012; Maleki et al., 2024). These variables define a species’ thermal niche and provide a mechanistic framework for understanding how temperature regulates developmental timing (Watt and Bloomberg, 2012; Rosbakh and Poschlod, 2015; Maleki et al., 2024). Thermal thresholds are therefore key functional traits linking reproductive ecology, species persistence, and biogeographical responses to climate change (Walck et al., 2011; Baskin and Baskin, 2014; Cochrane et al., 2015; Parmesan and Hanley, 2015) and may be constrained by shared evolutionary history. Thermal performance of temperature‐dependent developmental processes is commonly described using mathematical functions that relate developmental rate to temperature (Shafii and Price, 2001; Soltani et al., 2006). These models provide estimates of biologically meaningful thermal thresholds, including the base (T _b), optimum (T _o), and maximum (T _m) temperatures that characterize species‐specific thermal niches. While such approaches have been widely applied to germination, recent studies indicate that similar frameworks can be extended to postdispersal embryo growth by modeling embryo development rates as a function of temperature (Wawrzyniak et al., 2023). While thermal thresholds have been widely applied to describe germination responses, their application to postdispersal embryo growth, a distinct developmental phase in seeds with underdeveloped embryos, remains limited (Porceddu et al., 2017; Wawrzyniak et al., 2023; Walker et al., 2025).

Seed dormancy represents a key ecological strategy that delays germination until conditions favor seedling establishment and survival (Gremer et al., 2020; Pausas et al., 2022; Lamont and Pausas, 2023). In species with morphological dormancy (MD) or morphophysiological dormancy (MPD), dormancy is associated with an underdeveloped embryo that elongates after dispersal before germination can proceed. This postdispersal embryo growth phase is strongly temperature‐dependent and governs both the rate and timing of dormancy release, functioning as a developmental filter on regeneration timing (Vandelook et al., 2007a; Phartyal et al., 2009; Porceddu et al., 2017; Wawrzyniak et al., 2023; Blandino et al., 2025). The distinction between embryo growth and germination in seeds with underdeveloped embryos has long been recognized, with classical work by Nikolaeva (1969, 1977) demonstrating that embryo elongation must precede radicle emergence and that these processes may be regulated by different environmental conditions. Although intraspecific variation in germination timing is known to be related to climatic seasonality and habitat conditions (Allen and Meyer, 1998; Donohue, 2005; Wagmann et al., 2012), comparable variation in postdispersal embryo growth traits has rarely been quantified or integrated within an evolutionary framework (Mondoni et al., 2008; Vandelook et al., 2024). In addition to temperature, the progression of dormancy release during stratification (i.e., the duration of moist incubation following imbibition under controlled temperatures) is expected to play a central role in regulating embryo development and its sensitivity to thermal conditions.

Seed thermal niches are most effectively characterized in species that display strong temperature sensitivity during germination. Species with MD or MPD are particularly suitable for such studies because their embryo development and dormancy release are tightly regulated by temperature. Members of the Apiaceae family commonly have underdeveloped embryos and substantial interspecific variation in dormancy depth and embryo size (Martin, 1946; Vandelook et al., 2012; Baskin and Baskin, 2014). Most species have MD or MPD, requiring postdispersal embryo growth before germination. Despite postdispersal embryo growth before germination, seed longevity in Apiaceae is often relatively short, with many species forming transient or short‐term persistent soil seed banks (Baskin et al., 2000; Vandelook et al., 2007a, 2007b). This apparent contrast reflects differences in life‐history strategies: Although seeds may not persist for extended periods in the soil, dormancy‐breaking processes such as embryo growth can still require prolonged exposure to specific environmental conditions (e.g., seasonal temperature regimes). In some annual and biennial species, dormancy duration may extend across multiple seasons, allowing seeds to delay germination until favorable conditions arise, even if individual seeds do not remain viable for long periods (Baskin and Baskin., 2014). In addition to temperature, hormonal regulation, particularly by gibberellins, plays a central role in coordinating embryo growth and dormancy release by modulating cell elongation, reserve mobilization, and embryo–endosperm interactions (Finch‐Savage and Leubner‐Metzger, 2006; Walker et al., 2025). Exogenous application of gibberellic acid (GA₃) can promote embryo growth and germination in a dormancy‐dependent manner, being most effective in seeds with nondeep physiological dormancy, whereas in seeds with MD or MPD it contributes to the regulation of embryo growth but may not fully substitute for environmental cues required for germination (Baskin and Baskin, 2014; Walker et al., 2025). However, whether GA₃ primarily accelerates embryo growth within an existing thermal niche or alters apparent thermal thresholds (T _b, T _o, T _m) of embryo development remains poorly resolved.

Intraspecific variation in seed germination timing, typically regulated by seed dormancy, is largely driven by climatic conditions and, in particular, by the degree of climate seasonality. Optimal primary dormancy is expected to correspond to the duration of the period following seed release that is unfavorable for seedling establishment, resulting in population‐level differentiation along climatic gradients (Allen and Meyer, 1998; Wagmann et al., 2012). Evidence further indicates that intraspecific variation in germination characteristics often coincides with differences in associated seed morphological traits, reflecting coordinated evolutionary responses to environmental heterogeneity (Vandelook et al., 2024). While such within‐species variation can be substantial, the extent to which comparable variation in postdispersal embryo growth traits translates into consistent differences among species remains unclear. Here, we focus explicitly on interspecific variation in thermal niche traits for embryo growth, rather than population‐level differentiation, to evaluate whether species exhibit distinct and evolutionarily structured thermal requirements for postdispersal embryo growth. Variation in germination and embryo growth among populations or ecotypes has also been linked to contrasts in postdispersal environments, primarily shaped by habitat conditions (Donohue, 2005; Gimenez‐Benavides et al., 2005; Mondoni et al., 2008; Porceddu et al., 2017). Moreover, phenotypic trait variation within species can be as pronounced as variation among species (Albert et al., 2010), suggesting that intraspecific diversity may play a comparably influential role in shaping community composition and ecosystem functioning (Palkovacs et al., 2009; Govaert et al., 2016; Des Roches et al., 2018).

In this study, we quantified species‐specific thermal niches for postdispersal embryo growth in 10 Apiaceae species by estimating cardinal temperatures under controlled temperatures in the presence and absence of GA₃. Specifically, we aimed to (1) define the thermal thresholds governing postdispersal embryo growth for each species, (2) assess interspecific variation in these thresholds and their sensitivity to hormonal regulation, and (3) evaluate the extent to which embryo growth thermal traits are constrained by shared evolutionary history. We tested three hypotheses: (H1) Postdispersal embryo growth is jointly regulated by temperature and dormancy‐release progression; therefore, embryo growth rate will vary with temperature and stratification duration, and these effects will interact. (H2) GA₃ enhances embryo developmental potential; therefore, GA₃ will generally increase embryo growth rates and is expected to shift apparent thermal thresholds by lowering T _b and/or extending the upper thermal range (higher T _m), although the magnitude and direction of change may be species‐specific. (H3) Embryo growth thermal niche traits are evolutionarily constrained; therefore, T _b, T _o, T _m, and derived niche metrics will exhibit significant phylogenetic signal across species. By explicitly treating postdispersal embryo growth as a distinct, quantifiable developmental thermal niche, rather than as a surrogate for germination, we provide a mechanistic and eco‐evolutionary framework for understanding regeneration timing in species with underdeveloped embryos.

MATERIALS AND METHODS

Study system

Apiaceae (syn. Umbelliferae) are a large and cosmopolitan family of angiosperms comprising 466 accepted genera and approximately 3820 species (Plunkett et al., 2018; Clarkson et al., 2021). Iran represents one of the primary centers of Apiaceae diversity in Asia (Pimenov et al., 2004), encompassing 114 genera and 363 species, of which 12 genera and 114 species are endemic to Iran (Ajani et al., 2008; Mozaffarian, 2013; Moazzami Farida et al., 2018). Globally, Apiaceae species are predominantly distributed across temperate regions of the northern hemisphere, extending into tropical high‐altitude ecosystems (Spinozzi et al., 2021; Mohammadianfar et al., 2024). Many species of this family hold substantial economic and cultural importance, being widely cultivated and utilized as foods, spices, and medicinal herbs (Thiviya et al., 2021; Jayakodi et al., 2024). Fruits of the Apiaceae are typically schizocarps that separate into two mericarps, each containing a seed with abundant nuclear endosperm and a small, linear embryo (Vandelook et al., 2012). Seeds of many Apiaceae species are relatively short‐lived within soil seed banks (Baskin et al., 2000; Vandelook et al., 2007a, 2007b; Vandelook and Van Assche, 2008), whereas those of annual and biennial species can retain dormancy for up to a decade (Baskin et al., 2004; Baker et al., 2005; Ronse et al., 2024). Because seeds of this family possess underdeveloped embryos of varying morphology (e.g., linear or spatulate; Martin, 1946) and typically have either MD or MPD (Baskin and Baskin, 2014), Apiaceae provide an ideal system for studying postdispersal embryo growth. In this study, we therefore focused on members of this family, using the relative embryo size, expressed as the ratio of embryo length to seed length (E:S ratio), to quantify embryo growth. In addition, cardinal temperatures for embryo growth were estimated by measuring embryo growth rates at constant temperatures and modelling growth rate as a function of temperature.

Seed collection

Seeds of 10 Apiaceae species were collected during their natural dispersal period in summer 2023 from wild populations in their natural habitats across different regions of Iran (Table 1). Species were randomly selected from locally available Apiaceae populations during the sampling period, subject to the presence of sufficient mature seeds. For each species, seeds were collected directly from approximately 25–50 mature, healthy, naturally growing plants in each population. The base populations were selected to represent typical, naturally established stands of each species in the field. To capture population‐level variation and ensure adequate seed quantity, we randomly sampled individual plants across each site, maintaining a minimum distance of 5 m between plants to avoid sampling from potentially clonal individuals. Seeds from all sampled plants of each species were pooled (bulk‐collected) to form a single representative seed lot per species. After collection, seeds were air‐dried at ambient temperature and subsequently stored dry in a refrigerator at 3°C until the beginning of the experiments. Seed mass for each species was measured before the experiments (Table 1). Seeds were stored in dry, controlled conditions before the start of the experiments. While dry storage may influence dormancy status in some species, seeds with MD and MPD primarily require moist conditions for embryo growth and dormancy release; therefore, experimental treatments were expected to be the dominant drivers of developmental responses observed in this study. All experiments were done during fall and winter 2023 in the Seed Laboratory of the Faculty of Agricultural Technology, Aburaihan Campus, University of Tehran, Iran.

Table 1.

Collection information for the 10 collected species of Apiaceae used to estimate embryo growth.

Taxon	Seed mass (mg)	Collection date	No. of plants sampled	Country	Latitude	Longitude	Dormancy class
Anethum graveolens L.	1.3	6/30/2023	25–50	Iran	32.6539 N	51.666E	MD
Astrodaucus orientalis (L.) Drude	6.7	7/13/2023	25–50	Iran	37.9669 N	48.1983E	MD
Coriandrum sativum L.	9.4	8/15/2023	25–50	Iran	32.6539 N	51.666E	MD
Cuminum cyminum L.	2.6	6/14/2023	25–50	Iran	35.4669 N	51.6861E	MPD
Dorema aucheri Boiss.	8.6	8/12/2023	25–50	Iran	30.8356 N	51.5411E	MPD
Foeniculum vulgare Mill.	8.7	8/9/2023	25–50	Iran	35.4669 N	51.6861E	MPD
Kelussia odoratissima Mozaff.	28.8	8/20/2023	25–50	Iran	32.6586 N	49.863E	MPD
Petroselinum crispum (Mill.) Fuss	1.7	7/29/2023	25–50	Iran	32.6539 N	51.666E	MPD
Pimpinella anisum L.	1.6	7/20/2023	25–50	Iran	33.642 N	50.079E	MD
Trachyspermum ammi (L.) Sprague	0.7	7/17/2023	25–50	Iran	35.4669 N	51.6861E	MD

Postdispersal embryo growth measurements

The experiment was set up as a completely randomized design with 10 replicate Petri dishes per species and treatment. For each species, 350 seeds were evenly distributed among the 10 replicate dishes (35 seeds per dish) and placed on two layers of filter paper moistened with either distilled water containing carboxin–thiram fungicide (1000 ppm; control) or 100 ppm GA₃ (dissolved in carboxin–thiram at 1000 ppm; Soltani et al., 2022) solutions and incubated in darkness for 2 months at constant 5°C, 10°C, 15°C, 20°C, or 25°C as stratification treatments. The plates were checked daily, and each dish was moistened with the same solution as needed to keep the filter paper moist. To maintain consistent treatment conditions, the solution in each Petri dish was periodically removed and replaced with fresh solution of the same composition and concentration (including GA₃ where applicable), ensuring that hormone concentrations remained constant throughout the experiment. Each week, 10 seeds per species (N = 10 species), with intact, white, and firm embryos, were randomly selected from control and GA₃ treatments at each temperature. All 10 seeds were cut longitudinally using a scalpel and photographed with a digital camera mounted on a binocular dissecting microscope (Nikon, 100× magnification) equipped with a calibrated micrometer. Embryo length, seed length, and mericarp length were measured using the image analysis software Digimizer (version 4.6). Embryo growth was monitored weekly for 8 weeks until no further elongation was observed, and measurements were expressed as the ratio of embryo length ‐to seed length (E:S) for subsequent analyses. The ratio of embryo length to mericarp length (E:M) was also calculated (Nakabayashi et al., 2025).

During the 8‐week stratification, the seeds of some species gradually germinated over time. Although germination during stratification was not the primary focus of our study, the number of germinated seeds was recorded, and the germination percentage was calculated (Appendix S1).

Thermal threshold parameter estimation

In this study, “thermal niche” refers to the range of temperature conditions that permit and regulate postdispersal embryo growth, rather than to final germination outcomes. In species with MD or MPD, embryo growth represents a prerequisite developmental phase for germination; therefore, environmental conditions that support or constrain embryo development constitute a fundamental component of the germination niche, even when germination is not completed within the experimental timeframe. Accordingly, thermal thresholds were estimated based on embryo growth rates, independent of final germination percentages.

To estimate thermal threshold parameters for postdispersal embryo growth, embryo growth rate (EGR) was quantified at constant temperatures and modeled as a function of temperature using nonlinear thermal performance curves. The embryo growth rate (EGR; E:S ratio day⁻¹) inside the seed was estimated as the slope of a linear regression between E:S ratio and number of stratification days for each species, stratification temperature, and GA₃ treatments, separately (Appendices [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link]). To estimate the thermal threshold parameters for postdispersal EGR, we regressed EGR against temperature using the following model:

$$\text{EGR }=\mathrm{f}\left(\mathrm{T}\right)\text{EGRmax},$$

where f(T) is a temperature function that ranges between 0 at the base and maximum temperature and 1 at optimal temperature(s) and EGR_max is the inherent maximum rate of embryo growth at optimal temperature. The reciprocal, 1/EGR_max, represents the minimum time (days) required for postdispersal embryo growth at the optimum temperature.

To quantify the relationship between temperature and embryo growth rate, we fitted a comprehensive suite of nonlinear thermal performance curve (TPC) models using the R package rTPC (Padfield et al., 2021). This framework provides a standardized implementation of commonly used mechanistic and phenomenological models describing unimodal temperature–performance relationships across biological systems (Angilletta, 2009; Dell et al., 2011). We evaluated 23 candidate TPC models, including Gaussian‐type, Brière‐type, Sharpe–Schoolfield derivatives, and other asymmetric and mechanistic formulations. These models differ in their assumptions regarding thermal asymmetry, performance decline at high temperatures, and the explicit representation of lower and upper thermal limits. The inclusion of multiple models reduces dependence on any single functional form and allows uncertainty in model structure to be propagated into parameter estimates (Burnham and Anderson, 2002; Padfield et al., 2021).

All models were fitted using nonlinear least squares with multistart optimization implemented via the R package nls.multstart (Padfield et al., 2021). For each model, parameters were estimated using repeated optimization from randomly sampled starting values drawn within biologically informed bounds. Starting values and parameter limits were obtained using model‐specific helper functions (get_start_vals, get_lower_lims, get_upper_lims) provided by the rTPC package, ensuring numerical stability and biological plausibility of fitted parameters. Each model was fitted with 3–6 iterations per parameter, depending on model complexity. Convergence diagnostics were evaluated based on successful minimization and finite parameter estimates (Padfield et al., 2021). Models failing to converge or producing nonfinite estimates were retained only for visualization purposes and excluded from model averaging where appropriate.

Model performance was assessed using Akaike's information criterion corrected for small sample size (AICc), with AIC and Bayesian information criterion for reference (Burnham and Anderson, 2002). The AICc was selected as the primary metric due to the low number of observations relative to model complexity. Models were ranked based on AICc, and Akaike weights were calculated to quantify relative model support. Rather than relying on a single “best” model, we adopted a multimodel inference framework, recognizing that multiple models may provide comparable support given limited data and shared explanatory power (Burnham and Anderson, 2002).

For each fitted model, biologically meaningful thermal parameters were extracted using the function calc_params in the package rTPC. These included: base temperature (T _b): the estimated lower temperature limit for positive growth, thermal optimum (T _o): the temperature at which growth rate is maximized, maximum thermal maximum (T _m): the upper temperature limit for positive growth, and maximum growth rate (R _max). Derived metrics were also calculated to characterize thermal niche properties for embryo growth: thermal performance breadth (T _m − T _b) representing the temperature range permitting growth, and thermal safety margin (T _m − T _o), indicating proximity of optimal performance to upper thermal limits (Sunday et al., 2015; Sentinella et al., 2020).

To account for uncertainty in model structure, we averaged thermal performance parameters across models using AICc‐based weights (Burnham and Anderson, 2002). Before this averaging, models with non‐finite estimates of T _b, T _o, T _m, or R _max were removed. Weighted means were then calculated for all thermal traits, and T _b estimates below 0°C were constrained to zero to maintain biological realism. This model‐averaging approach provides robust parameter estimates that integrate uncertainty across competing hypotheses regarding the shape of the temperature–growth relationship, an approach increasingly recommended in thermal ecology and comparative physiology (Angilletta, 2009; Dell et al., 2011; Padfield et al., 2021).

Phylogenetic signal and tree construction

A phylogenetic tree for the study species was generated using the most extensive dated vascular plant megatree available from the Open Tree of Life (OpenTreeofLife et al., 2024) as the baseline (Figure 1). The ultrametric tree was assembled in R with the R package V.PhyloMaker (Jin and Qian, 2019), ensuring that all taxa from our data set were represented. This package relies on the GBOTB.extended.tre backbone, a revised and expanded form of the GBOTB tree for seed plants (Jin and Qian, 2019). Species names were standardized according to the World Flora Online taxonomic backbone (World Flora Online Consortium, 2025) and further verified with the R package expowo, which compiles and harmonizes plant diversity information (Zuanny et al., 2024). To measure phylogenetic signal for each thermal threshold parameter, the R package phytools 2.0 was used to calculate phylogenetic signal (Revell, 2024). Pagel's lambda was used as phylogenetic signal.

Figure 1.

Phylogenetic distribution of thermal threshold parameters for postdispersal embryo growth in 10 Apiaceae species. Colored heatmap cells represent species‐specific trait values, with each parameter (T _b, T _o, T _m, thermal safety margin, and thermal performance breadth) depicted using a distinct color gradient. The phylogenetic tree illustrates evolutionary relationships among species. Photographs below show representative individuals of each species included in the analysis, arranged to correspond with their phylogenetic positions.

RESULTS

Seed germination during stratification

Final germination percentages during stratification varied between the examined Apiaceae species and with temperatures and GA₃ treatments (Appendix S1). In general, germination was significantly higher at moderate temperatures (particularly 15°C and 20°C), with reduced germination at the lower (5°C) and higher temperatures (25°C). GA₃ clearly improved germination in several species (Appendix S1), particularly at mid‐range temperatures (15–20°C). For example, seeds of Trachyspermum ammi germinated up to 75% and Cuminum cyminum up to 50% at 15°C under GA₃ treatment, while Anethum graveolens and Cuminum cyminum had optimal germination (44%) at 20°C with GA₃. However, the extent of response varied among species, although species such as Pimpinella anisum and Dorema aucheri had very low germination across all treatments and temperatures, and no seeds of Kelussia odoratissima germinated, suggesting incomplete dormancy release or insensitivity to GA₃ under the tested conditions.

Estimation of embryo growth rate

Embryo growth rates at all examined temperatures were obtained using linear regressions models (Figure 2; Appendices [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link]). The progression of E:S ratio generally followed a temperature‐ and species‐specific trajectory. Embryo growth rate was the fastest at 20°C for the control and GA₃ treatment for the 10 species in 8 weeks. The lowest E:S ratio increment was ca. 0.29 at 25°C in the control in weeks 1 and 2 for K. odoratissima and the highest E:S ratio increment was at 5°C (0.89), in GA₃ treatment in week 7 for C. cyminum. No significant differences in the E:S ratio increment were observed between 5°, 10°, and 25°C. The lowest trend of increasing embryo growth occurred in K. odoratissima (0.32), followed by a similar trend of increasing embryo growth in F. vulgare and D. aucheri (0.48–0.49). A similar trend of increasing embryo growth was observed in A. orientalis and C. sativum (0.51) and in P. crispum, P. anisum, A. graveolens, and C. cyminum (0.60–0.61). The highest rate of increasing embryo growth occurred in T. ammi (0.65).

Figure 2.

Representative progression of postdispersal embryo growth across 10 Apiaceae species during incubation.

Effect of GA₃ on embryo growth

GA₃ treatment generally increased the rate of embryo growth rate and ratio of embryo length to seed length ratio (E:S) compared to the control across species and temperatures (Appendices [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link]). The lowest embryo growth rate was observed in K. odoratissima (0.01) at 20°C and 25°C for both control and GA₃ treatments, whereas the highest growth rate occurred in Foeniculum vulgare (0.27) at 15°C under GA₃ treatment. Overall, GA₃ enhanced embryo growth rates in most species; however, the magnitude and direction of the response were temperature‐ and species‐dependent. Positive GA₃ effects were particularly evident in F. vulgare at 25°C, Anethum graveolens at 5°C and 10°C, and Cuminum cyminum at 20°C. In contrast, GA₃ had little to no effect on embryo growth in Trachyspermum ammi, and growth rates were reduced in C. cyminum at 5°C, Petroselinum crispum at 15°C and 20°C, and K. odoratissima across all temperatures, indicating species‐specific constraints on GA₃‐mediated embryo development.

Thermal thresholds for postdispersal embryo growth

The thermal threshold for postdispersal embryo growth (T _b, T _o, and T _m) under different stratification conditions and GA₃ treatments varied considerably among the 10 Apiaceae species studied (Table 2, Figure 3). Among species, T _b for postdispersal embryo growth ranged from 0 to 6.5°C, T _o from 5 to 25°C, and T _m from 21.5 to 31°C. In seeds treated with GA₃, these ranges shifted: T _b varied from 0 to 4°C, and T _o and T _m ranged from 15 to 32°C (Table 2). GA₃ treatment generally altered thermal thresholds in a species‐specific manner. In most species, GA₃ application reduced T _b, except in F. vulgare and C. cyminum, indicating enhanced embryo responsiveness at cooler temperatures and an earlier onset of growth. The T _o increased in nearly all species, except A. graveolens and C. cyminum. The T _m also increased in most species, with the exceptions of A. orientalis and A. graveolens, indicating that GA₃ extended the upper thermal limit for postdispersal embryo growth.

Table 2.

The effects of GA₃ treatment on thermal performance traits, including base cardinal temperature (T _b), optimum cardinal temperature (T _o), maximum cardinal temperature (T _m), thermal safety margin (TSM), thermal performance breadth (TPB), and maximum embryo growth rate (R _max) across 10 species in the Apiaceae family.

Species	GA3	T b	T o	T m	TSM	TPB	R max
Anethum graveolens	No	0.0	12.5	43.4	31	43.4	0.217
Anethum graveolens	Yes	0.4	11.7	21.3	9.6	20.8	0.111
Astrodaucus orientalis	No	12.0	14.9	45.2	33.3	33.2	0.0216
Astrodaucus orientalis	Yes	1.9	15.6	22.4	6.7	20.45	0.172
Coriandrum sativum	No	4.34	17.2	35.5	18.3	31.1	0.028
Coriandrum sativum	Yes	0.0	18.2	35.2	17.0	35.2	0.160
Cuminum cyminum	No	0.0	25.0	30.4	5.4	30.4	0.184
Cuminum cyminum	Yes	4.2	15.5	36.4	11.4	32.1	0.205
Dorema aucheri	No	7.55	13.7	20.4	6.6	12.8	0.034
Dorema aucheri	Yes	5.0	25.0	45.1	20.1	40.1	0.170
Foeniculum vulgare	No	7.8	14.8	21.7	6.9	13.81	0.124
Foeniculum vulgare	Yes	8.6	16.9	23.1	6.1	14.44	0.296
Kelussia odoratissima	No	0.0	5.19	11.9	6.7	11.9	0.052
Kelussia odoratissima	Yes	0.0	5.0	28.0	23.0	28.0	0.130
Petroselinum crispum	No	9.22	18.9	25.7	6.7	16.4	0.203
Petroselinum crispum	Yes	0.0	25.0	27.8	2.8	27.8	0.120
Pimpinella anisum	No	2.9	14.3	22.3	8.0	19.4	0.159
Pimpinella anisum	Yes	1.4	15.0	22.6	7.6	21.1	0.201
Trachyspermum ammi	No	3.4	10.0	23.6	13.6	20.1	0.509
Trachyspermum ammi	Yes	2.7	20.4	31.9	11.5	29.1	0.121

Figure 3.

Thermal response curves of postdispersal embryo growth rate for 10 Apiaceae species in the control conditions (without GA₃; orange circles and lines) and with GA₃ treatment (blue triangles and lines). Points represent observed embryo growth rates at each incubation temperature; curves show fitted temperature response functions. Each panel corresponds to a single species, with species names indicated above the plots.

The largest GA₃ effect was observed in D. aucheri, which had substantial increases in both T _o and T _b. GA₃ also markedly increased T _o in T. ammi and T _b in K. odoratissima. In contrast, GA₃ had no discernible effect on T_b and T_o in K. odoratissima (0.0°C and 5.19–5.0°C, respectively) or on T _b in Anethum graveolens (0.0–0.4°C). T _m remained unchanged in Pimpinella anisum (22.3–22.6°C) and Coriandrum sativum (35.5–35.2°C), indicating species‐specific insensitivity to GA₃ with respect to thermal thresholds for postdispersal embryo growth.

Phylogenetic signal in thermal threshold traits

All thermal threshold traits exhibited significant phylogenetic signal (Pagel's λ, P < 0.001; Table 3, Figure 1), indicating that closely related species tend to resemble each other in their thermal threshold traits for postdispersal embryo growth. The magnitude of λ varied among the variables, ranging from 0.22 for T _b to 0.55 for thermal performance breadth (TPB). Values were intermediate for T _o (λ = 0.30), T _m (λ = 0.39), and thermal safety margin (TSM; λ = 0.40).

Table 3.

Phylogenetic signal (Pagel's λ) for thermal threshold traits in postdispersal embryo growth of Apiaceae species. T _b = base temperature, T _o = optimum temperature, T _m = maximum temperature, TSM = thermal safety margin, TPB = thermal performance breadth. All traits show significant phylogenetic signal (P < 0.001).

Trait	Pagel's λ	Log likelihood	P
T b	0.22	–28.15	<0.001
T o	0.30	–30.25	<0.001
T m	0.39	–34.13	<0.001
TSM	0.40	–37.20	<0.001
TPB	0.55	–37.61	<0.001

DISCUSSION

Temperature‐dependent embryo growth defines species‐specific regeneration niches in Apiaceae

Our results provide clear empirical support for treating postdispersal embryo growth as a distinct, temperature‐regulated developmental phase that determines species‐specific regeneration niches in Apiaceae. Across all 10 species, embryo elongation occurred before radicle emergence and responded strongly to temperature, confirming that this phase constitutes a functional bottleneck for germination rather than a passive precursor. Because Apiaceae seeds are dispersed with underdeveloped embryos, successful regeneration necessarily depends on the completion of embryo growth post‐dispersal, during which embryos utilize endosperm reserves to reach a critical size threshold (Stokes, 1953a, 1953b). From a mechanistic perspective, this developmental bottleneck reflects the requirement for sustained cell elongation and division within the embryo axis before germination competence can be achieved. Temperature therefore regulates not only metabolic rate but also the progression of embryo developmental programs, including reserve mobilization and embryo–endosperm coordination, that must be completed before radicle protrusion (Linkies et al., 2010; Steinbrecher and Leubner‐Metzger, 2017).

Consistent with our expectations, embryo growth responded strongly to temperature and GA₃, with clear evidence that treatment effects were temperature‐ and species‐dependent. The interplay between stratification, temperature, and GA₃ indicates that hormonal stimulation modifies embryo developmental progression but does not override thermal regulation. Across all 10 species, we were able to estimate cardinal temperatures (T _b, T _o, T _m) for embryo growth, revealing substantial interspecific variation in embryo‐growth thermal niches and demonstrating that GA₃ often enhanced embryo elongation and altered apparent thermal limits. The postdispersal embryo growth phase in Apiaceae is commonly associated with dormancy release induced by specific combinations of temperature and moisture, and most Apiaceae exhibit primary MPD that is broken by either low (0–10°C) or higher (>10°C) temperatures (Baskin and Baskin, 2014; Ronse et al., 2024).

Thermal thresholds reveal interspecific variation in postdispersal embryo growth

For most plant species, the optimum temperature for seed germination typically ranges from 15°C to 30°C, with maximum temperatures extending to 30–40°C (Sarmadnia, 1997; Maleki et al., 2024). However, the cardinal temperatures governing postdispersal embryo growth, particularly in species with underdeveloped embryos, have rarely been quantified. The first such study was conducted by Porceddu et al. (2017), who quantified requirements for embryo growth in Aquilegia barbaricina (Ranunculaceae), a species with morphophysiological dormancy. Under warm + cold stratification (W + C), T _b for embryo growth was estimated at 5.3°C, and under GA₃ treatment, at 5.2°C. For seeds pretreated with W + C stratification, all cardinal temperatures were calculated, with a T _o of approximately 15°C and a T _m of 29.5°C. However, T _o and T _m could not be determined for seeds treated with GA₃ alone. Similarly, Wawrzyniak et al. (2023) estimated the cardinal temperatures for embryo growth in Fraxinus excelsior L. (Oleaceae), a species with deep simple MPD. Under warm + cold stratification, T _b, T _o, and T _m were estimated at 1.4°C, 15.7°C, and 29.9°C, respectively, though the influence of GA₃ was not examined. More recently, Walker et al. (2025) provided the most comprehensive characterization to date of temperature‐dependent embryo growth in morphologically dormant seeds of Apium graveolens (Apiaceae). Using population‐based thermal time modelling across a broad temperature gradient (6–29°C), they demonstrated that embryo growth follows a thermal time‐compliant process and estimated T _b, T _o, and T _m for embryo growth at approximately 2.9°C, 21.5°C, and 28.8°C, respectively. Importantly, these values closely matched the cardinal temperatures for germination, while embryo growth had a lower thermal time requirement than radicle emergence, indicating that embryo elongation constitutes a distinct, temperature‐regulated developmental phase that precedes germination.

Considerable interspecific variability was detected in the thermal thresholds for embryo growth in the 10 Apiaceae studied here. In the control conditions, the T _b ranged from 0 to 6.5°C, T _o from 5 to 25°C, and T _m from 21.5 to 31°C. In seeds treated with GA₃, these thresholds shifted slightly, with T _b ranging from 0 to 4°C, T _o from 15 to 24.5°C, and T _m from 27 to 32°C. This wide range of cardinal temperatures indicates that embryo‐growth thermal niches are finely tuned at the species level, likely reflecting differences in embryo morphology, metabolic sensitivity, and the efficiency of reserve mobilization. Variation in T _o suggests that species differ in the temperature range that maximizes cell expansion and enzymatic activity during embryo elongation, whereas differences in T _m may reflect limits imposed by membrane stability, hormonal balance, or oxidative stress at elevated temperatures.

GA₃ modulates embryo thermal thresholds without overriding species‐specific constraints

Application of GA₃ generally increased embryo growth rates and altered thermal thresholds, but responses were highly species dependent. This pattern is consistent with the known role of gibberellins in promoting cell elongation, reserve mobilization, and embryo–endosperm interactions during dormancy release (Finch‐Savage and Leubner‐Metzger, 2006; Baskin and Baskin, 2014; Soltani et al., 2022). Mechanistically, GA₃ likely accelerates embryo growth by enhancing cell wall extensibility, stimulating hydrolytic enzyme activity in the endosperm, and promoting carbon flux to the growing embryo axis (Steinbrecher and Leubner‐Metzger, 2017; Chandrasekaran et al., 2022). However, the magnitude of these effects depends on the depth of dormancy and the developmental state of the embryo at dispersal (Baskin and Baskin, 2014).

In several studies, only the temperature ranges that promote maximum embryo growth were determined, revealing considerable interspecific variation in thermal requirements. Some species have complex temperature dependencies for embryo elongation. For instance, embryo growth in Aconitum lycoctonum occurred at temperatures below 10°C (Vandelook et al., 2009), while Campanile giganeum embryos grew best under alternating temperature regimes of approximately 20°C, followed by cooler conditions around 10°C (Phartyal et al., 2012). In Taxus baccata L., embryo growth was restricted to alternating temperatures (24/24 h cycles) within a narrow range of 15–20°C (Suszka, 1985). Embryos of Thalictrum rochebrunianum H. Lev., endemic to the Korean Peninsula, required low temperatures of approximately 5°C for elongation (Lee et al., 2015). Similarly, an optimum temperature of 2°C was reported for embryo growth in Heracleum sphondylium (Stokes, 1953a, b). For Lomatium dissectum, embryo growth was optimal between 3 and 6°C, with negligible growth at 0.5°C and 9.1°C (Scholten et al., 2009). In Conopodium majus, thermal thresholds for embryo growth varied along a latitudinal gradient, with T _o ranging from 2.5 to 5.2°C, T _m between 12 and 20.5°C, and T _b between −6.6 and −2.7°C (Blandino et al., 2025). Similarly, in Apiaceae crops Apium graveolens and Daucus carota, embryo growth and germination were optimal at 20–25°C (Finch‐Savage et al., 1998; Rowse and Finch‐Savage, 2003; Walker et al., 2021), which corresponds closely to the temperature range observed in our study.

Limited responsiveness to GA₃ indicates variation in dormancy depth among Apiaceae species

The application of GA₃ generally reduced T _b and, in most species, increased T _o and T _m, thereby broadening the thermal range suitable for effective embryo development. Such limited responsiveness suggests that, in some species, embryo growth remains constrained by structural or physiological factors that are not readily alleviated by hormonal stimulation alone, including low intrinsic growth potential, limited reserve accessibility, or the requirement for prolonged or sequential temperature cues.

The greatest GA₃‐induced effects were observed in Dorema aucheri, which showed pronounced shifts in both T _o and T _b and in Trachyspermum ammi (increased T _o) and Kelussia odoratissima (increased T _b), indicating enhanced a developmental capacity in warm conditions. In contrast, GA₃ treatment had little to no effect on T _b and T _o for Kelussia odoratissima (0.0°C and 5.0–5.19°C, respectively) or on the T _b of Anethum graveolens (0.0–0.4°C). Similarly, the T _m remained unchanged in Pimpinella anisum (22.3–22.6°C) and Coriandrum sativum (35.5–35.2°C). These species had narrow thermal performance ranges and limited responsiveness to GA₃, suggesting a more rigid dormancy mechanism or additional ecological requirements such as prolonged stratification or alternating temperature cycles (Maleki et al., 2024). These findings align with previous studies of MPD in Apiaceae, where the depth of dormancy and sensitivity to gibberellins have been shown to vary substantially among species, reflecting differences in ecological origin, seed physiology, and evolutionary history (Rolston, 1978; Finch‐Savage and Leubner‐Metzger, 2006; Baskin and Baskin, 2014).

Phylogenetic signal in embryo thermal traits suggests evolutionary constraint

All thermal threshold traits exhibited significant phylogenetic signal (Pagel's λ, P < 0.001), indicating that more closely related species tend to resemble one another more in their thermal requirements for postdispersal embryo growth. This pattern provides evidence that thermal niches for embryo growth are not evolutionarily labile but are, at least in part, constrained by shared ancestry, consistent with broader evidence for phylogenetic conservatism in plant functional traits (Linkies et al., 2010; Violle et al., 2014; Rosbakh and Poschlod, 2015; Vandelook and Carta, 2025). Such phylogenetic structure suggests that the temperature sensitivity of embryo growth reflects inherited developmental and physiological architectures, rather than solely short‐term ecological adjustment (Finch‐Savage and Leubner‐Metzger, 2006).

The strength of phylogenetic signal varied among traits, with lower values for T _b and higher values for thermal performance breadth (TPB) and thermal safety margin (TSM). This trait‐specific variation implies that different components of the embryo‐growth thermal niche are subject to distinct evolutionary pressures, a pattern commonly observed in comparative analyses of thermal and life‐history traits (Dell et al., 2011; Sentinella et al., 2020). Lower λ values for T _b may reflect adaptive flexibility in the minimum temperature required to initiate embryo growth, which could facilitate local persistence in cooler or more variable environments. In contrast, stronger phylogenetic signal in TPB and TSM suggests that the overall width of the thermal niche and the proximity of optimal growth to upper thermal limits are more evolutionarily conserved (Araújo et al., 2013), potentially reflecting constraints imposed by seed structure, embryo–endosperm interactions, and hormonal regulation (Finch‐Savage and Leubner‐Metzger, 2006; Carta et al., 2024; Walker et al., 2025).

Our findings extend previous work on phylogenetic constraints in seed functional traits by demonstrating that thermal thresholds governing postdispersal embryo growth, rather than germination sensu stricto, also have a detectable phylogenetic signal. Prior studies have documented phylogenetic structure in germination temperature thresholds, dormancy class, and relative embryo size (Vandelook et al., 2012; Rosbakh and Poschlod, 2015; Maleki et al., 2024; Vandelook and Carta, 2025), but empirical evidence for evolutionary constraint acting on embryo‐growth thermal niches has been largely absent (Porceddu et al., 2017; Wawrzyniak et al., 2023; Walker et al., 2025). By quantifying T _b, T _o, and T _m for embryo growth across multiple species and integrating these estimates within a phylogenetic framework, we provide the first direct evidence that this overlooked developmental phase might have been shaped by evolutionary history.

The presence of phylogenetic signal further suggests that embryo‐growth thermal niches may evolve more slowly than climatic conditions, potentially generating mismatches between inherited developmental thresholds and rapidly changing thermal environments (Araújo et al., 2013; Martin et al., 2023). The presence of phylogenetic signal is particularly relevant for species with morphophysiological dormancy, in which embryo growth constitutes a necessary prerequisite for germination and thus acts as a gatekeeper for regeneration timing (Baskin and Baskin, 2014; Lamont and Pausas, 2023; Maleki et al., 2024). If embryo thermal thresholds are evolutionarily constrained, shifts in regeneration phenology under climate warming may be limited by developmental inertia rather than by the capacity for rapid physiological adjustment (Crisp and Cook, 2012). Although the limited number of species included here restricts inference about macroevolutionary rates or the directionality of trait evolution, the consistent detection of phylogenetic signal across all thermal traits supports the interpretation that thermal niches for embryo growth are shaped by both evolutionary constraint and ecological filtering. As such, our results should be viewed as preliminary but robust evidence that postdispersal embryo growth represents an evolutionarily structured component of the seed regeneration niche (Vandelook and Carta, 2025). Expanding this framework to larger taxonomic scales will be essential for testing whether evolutionary conservatism in embryo thermal traits contributes to broad‐scale patterns of species distribution and climate vulnerability (Violle et al., 2014; Sentinella et al., 2020).

CONCLUSIONS

This study demonstrates that postdispersal embryo growth represents a distinct and quantifiable thermal niche in Apiaceae species with underdeveloped embryos. By estimating cardinal temperatures for embryo growth across 10 species and explicitly separating this developmental phase from germination sensu stricto, we showed that temperature regulates regeneration timing through mechanisms operating prior to radicle emergence. Substantial interspecific variation in T _b, T _o, and T _m indicates that embryo‐growth thermal niches differ markedly among species, reflecting species‐specific regeneration strategies rather than a uniform dormancy response. Gibberellic acid generally enhanced embryo growth rates and broadened the thermal range permitting growth, but its effects were strongly species dependent. In some species, GA₃ reduced the T _b and extended the upper thermal limit for embryo growth, whereas in others it had little or no effect, indicating variation in dormancy depth and hormonal sensitivity. These results demonstrate that hormonal regulation modulates, but does not override, intrinsic thermal constraints on embryo development, highlighting the interactive roles of temperature and hormonal cues within species‐specific developmental frameworks. The detection of significant phylogenetic signal across all embryo thermal traits further indicates that embryo‐growth thermal niches are shaped by shared evolutionary history. Trait‐specific differences in phylogenetic signal suggest that some components of the embryo‐growth thermal niche, particularly thermal breadth and safety margins, are more evolutionarily conserved than others. By extending thermal threshold theory to postdispersal embryo growth, this study fills a critical gap in seed developmental ecology and provides a mechanistic basis for linking dormancy release, regeneration timing, and evolutionary constraint under ongoing climate change.

AUTHOR CONTRIBUTIONS

E.S. and F.V. conceived the main idea of the study. S.N. performed the experiments. S.N., E.S., and K.M. wrote the manuscript. S.N. and K.M. created the figures and analyzed the data. All authors contributed to the review and editing of the manuscript and approved the final version.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

Appendix S1: Final germination percentage of 10 Apiaceae species following an 8‐week stratification at a constant temperature (5, 10, 15, 20, 25°C), with or without gibberellic acid treatment in the dark.

Appendix S2: Regression of stratification duration (8 weeks) at a constant temperature of 5, 10, 15, 20, or 25°C and GA₃ treatment on ratio of embryo length to seed length (E:S) in Pimpinella anisum.

Appendix S3: Regression of stratification duration (8 weeks) at a constant temperature of 5, 10, 15, 20, or 25°C and GA₃ treatment on ratio of embryo length to seed length (E:S) in Dorema aucheri.

Appendix S4: Regression of stratification duration (8 weeks) at a constant temperature of 5, 10, 15, 20, or 25°C and GA₃ treatment on ratio of embryo length to seed length (E:S) in Coriandrum sativum.

Appendix S5: Regression of stratification duration (8 weeks) at a constant temperature of 5, 10, 15, 20, or 25°C and GA₃ treatment on ratio of embryo length to seed length (E:S) in Astrodaucus orientalis.

Appendix S6: Regression of stratification duration (8 weeks) at a constant temperature of 5, 10, 15, 20, or 25°C and GA₃ treatment on embryo‐to‐seed length (E:S) ratio in Petroselinum crispum.

Appendix S7: Regression of stratification duration (8 weeks) at a constant temperature of 5, 10, 15, 20, or 25°C and GA₃ treatment on ratio of embryo length to seed length (E:S) in Foeniculum vulgare.

Appendix S8: Regression of stratification duration (8 weeks) at a constant temperature of 5, 10, 15, 20, or 25°C and GA₃ treatment on ratio of embryo length to seed length (E:S) in Anethum graveolens.

Appendix S9: Regression of stratification duration (8 weeks) at a constant temperature of 5, 10, 15, 20, or 25°C and GA₃ treatment on ratio of embryo length to seed length (E:S) in Trachyspermum ammi.

Appendix S10: Regression of stratification duration (8 weeks) at a constant temperature of 5, 10, 15, 20, or 25°C and GA₃ treatment on ratio of embryo length to seed length (E:S) in Cuminum cyminum.

Appendix S11: Regression of stratification duration (8 weeks) at a constant temperature of 5, 10, 15, 20, or 25°C and GA₃ treatment on ratio of embryo length to seed length (E:S) in Kelussia odoratissima.

Nadi, S. , Maleki K., Soltani E., Javid M. G., and Vandelook F.. 2026. Species‐specific thermal thresholds for postdispersal embryo growth in Apiaceae. American Journal of Botany 113(7): e70231. 10.1002/ajb2.70231

DATA AVAILABILITY STATEMENT

All data used in the current study can be found in the main text of the paper and in the online appendix files.