Seed Germination of Native Mediterranean Species for Establishing Self-Sustaining Urban Meadows Supporting Urban Biodiversity

Abstract

Urbanization reduces biodiversity and affects plant–insect interactions, creating a need for more functional green spaces. Urban meadows with native species are a promising option, but their use is still limited due to a variety of reasons concerning the utilization framework of suitable plant species. The present study aimed to develop seed germination protocols for 26 native Mediterranean herbaceous species originating from northeastern Greece selected to support the establishment of species-rich and self-sustaining urban meadows. To the above end, seed germination experiments were conducted ex situ under controlled environment conditions using seeds collected from the wild for each species. Seed viability was assessed using the tetrazolium (TTZ) test to determine the maximum germination potential in each case. Freshly collected seeds were stored under ambient conditions for approximately 3 months (after-ripening) prior to germination testing, which was followed by cold stratification as a pretreatment for dormancy release. The results showed high embryo viability in all species and indicated that most taxa exhibited either no dormancy or relatively shallow physiological dormancy. Germination tests revealed that 14 of the 26 species presented high germination percentages in the control treatment, which suggests that after-ripening contributed to dormancy release in a significant portion of the seed lot. However, it remains unclear whether freshly collected seeds require an initial after-ripening period before responding to cold stratification. Furthermore, cold stratification significantly enhanced germination in 12 species confirming its effectiveness as a simple and practical method for dormancy release. In addition to the seed germination results, the selected species present a wide range of functional and esthetic characteristics, including variation in plant height, flowering phenology and flower and leaf color. These traits are important for both ecological performance and visual quality in urban environments. The combination of extended flowering periods and color diversity suggests the potential for continuous floral resource availability, which can support diverse pollinator communities and, indirectly, urban fauna such as insectivorous birds. The results indicate that the studied species are suitable for biodiversity-oriented urban plantings. Their relatively shallow dormancy and ease of propagation, coupled with their functional and aesthetic traits, support their use in the development of resilient and self-sustaining urban meadows.

1. Introduction

Cities today are experiencing a dramatic shift toward urban living. Whereas only about 10% of the global population lived in cities in 1900, this proportion now exceeds 50% [1] and is expected to reach nearly 68% by 2050 [2,3]. The European Union is one of the most urbanized areas in the world as out of 447 million EU inhabitants more than 75% live in urban areas [4]. Therefore, as urban areas expand, they significantly affect land uses [5,6]. These changes alter cities’ environment, their microclimatic conditions, and soil characteristics or resource availability which have a significant impact on urban ecosystem processes, plant communities and other organisms that depend on plants [7].

Urban green spaces are usually fragmented and dominated by simplified habitats resulting from the replacement of the heterogeneous natural vegetation with intensively managed green spaces [8]. Moreover, the availability and temporal continuity of floral resources and bird nesting habitats are generally low. Urbanization typically reduces species richness, especially for predators, specialists and rare species, which can translate into reduced biodiversity, and simplify trophic relationships while favoring generalists [6,9]. It is well documented that, as urban density increases, functional type, species diversity and insect abundance are decreasing [10]. As a result, urban landscapes frequently support pollinator assemblages dominated by a limited number of generalist species, while specialist and habitat-sensitive taxa tend to decline [11]. Such changes can affect plant–pollinator interactions with potential consequences for pollination and plant community structure.

In addition to their role in pollination, insects are an essential component of urban food webs as they are an important component of insectivorous consumers’ diets which are part of the urban fauna [12], particularly birds, bats and small mammals [13]. Consequently, changes in insect communities due to urbanization may affect urban fauna both quantitatively and qualitatively. The population dynamics of urban fauna may be influenced by declines in insect biomass that reduce food availability and affect reproduction and distribution [12]. At the same time, shifts in insect diversity and the increasing dominance of a few generalist species may also alter predator diversity [14].

In this context, urban meadows can increase habitat heterogeneity and provide continuous floral and food resources throughout the growing season [15]. The use of native plant species in the establishment of urban meadows can be considered essential as they often support a greater diversity and abundance of insects because many insect species have evolved specific associations with local flora [12]. For example, flowering genera such as Achillea, Thymus, Campanula and Hypericum are widely visited by bees, hoverflies and butterflies in Mediterranean habitats [16,17]. Consequently, urban meadows as they potentially support richer insect communities indirectly also support the urban fauna that depends on them. In addition, species-rich herbaceous habitats can also benefit from urban bird assemblages. Many insectivorous species that commonly occur in Mediterranean landscapes, including swifts (Apus apus), swallows (Hirundo rustica), wagtails (Motacilla alba), larks (Alauda arvensis, Galerida cristata) and flycatchers (Muscicapa striata), forage on insects associated with open herbaceous vegetation. Moreover, many passerines, such as partridges, sparrows, finches, and buntings, mainly eat plant material-like seeds, shoots, and buds. However, they feed insects to their chicks during spring and summer. Therefore, meadow plants are essential throughout the year for many bird species as food sources and insect attractants [18].

Moreover, native plants are generally better adapted to local environmental conditions and are more closely integrated into regional ecological networks [14]. Within Europe, Mediterranean regions represent a distinct climatic and ecological area, where hot, dry summers and strong seasonality shape the plant communities. These conditions are particularly relevant when considering the design and management of urban green spaces. Consequently, native Mediterranean species can contribute to the development of resilient urban plant communities that require lower maintenance while providing important ecological resources for pollinators and other components of urban fauna.

Despite the growing recognition of the ecological value of urban meadows, an important limitation remains the limited availability of suitable native plant material for large-scale applications. Many wild species that could potentially be used in urban meadow seed mixtures remain underutilized, partly because their propagation and wider utilization requirements are usually poorly understood and documented. In restoration ecology, the development of reliable seed propagation protocols is widely recognized as a critical step for enabling the use of native plant species in ecological restoration and biodiversity-oriented landscaping [19]. This is particularly relevant for Mediterranean plants, many of which present seed dormancy that has evolved following ecological adaptation to the unpredictable Mediterranean climate [20,21]. The selection of plant species suitable for urban meadow establishment therefore requires consideration of both ecological performance and practical propagation potential. In addition, the selection of species for urban meadows involves esthetic considerations, such as variation in flower color, plant height and flowering period. These traits contribute to the visual diversity of urban plantings and may influence both public acceptance and ecological functionality, including pollinator attraction. Considering all the above, the current study aims at the development of ex situ seed propagation protocols for a variety of native (Mediterranean) and underutilized plant species via investigation of their seed germination requirements. These protocols can constitute a versatile tool that can be utilized for either restoration and conservation purposes, or for the sustainable utilization of the selected species in applications like the establishment of urban meadows. Consequently, 26 native Mediterranean species abundant in natural habitats were selected. Such species are more likely to be ecologically adapted, widely available and suitable for use in species-rich urban meadow plantings.

2. Materials and Methods

2.1. Species Selection and Seed Collection

The study focused on native and potentially underutilized plant species originating from northeastern Greece (

Table 1. Plant species examined in this study and their morphological and phenological traits, including habitat type relevant for species-rich urban meadow plantings.

A/N	Species	Habitat Type	Average Height of Mature Individuals (cm)	Flower Color	Leaf Color	Flowering Period
1	Achillea crithmifolia Waldst. & Kit.	Dry/open habitats	10–30 (40)	Pale greenish, yellow/white	Gray-green	June–August
2	Achillea millefolium L.	Dry/open habitats	10–50	White or pinkish	Green	July–September
3	Anthemis tinctoria L. (=syn. Cota tinctoria (L.) J.Gay)	Dry/open habitats	20–60	Bright yellow	Green (glaucous above)	June–August
4	Asphodeline lutea (L.) Rchb.	Dry/open habitats	60–90	Yellow	Glaucous-green	April–June
5	Armeria canescens (Host) Boiss.	Mesic habitats	15–35	Pinkish or withe	Green	May–June (July)
6	Briza media L.	Mesic habitats	15–60	Purple-tinged	Green	June–August
7	Campanula lingulate Waldst. & Kit.	Dry/open habitats	15–35	Violet-blue	Green	July–September
8	Campanula persicifolia L.	Mesic habitats	30–80	Blue	Bright green	June–August
9	Campanula trachelium L.	Moist/shaded habitats	40–100	Violet-blue	Green, hispid	July–September
10	Dianthus cruentus Griseb.	Dry/open habitats	30–60	Reddish-purple	Glaucous	End of May and mid-July
11	Dianthus gracilis Sm.	Dry/open habitats	15–40	Pink-reddish	Green to glaucous	June–August
12	Dianthus pinifolius Sm.	Dry/open habitats	15–45	Red-purple	Green	June–August
13	Digitalis lanata Ehrh.	Mesic habitats	30–60 (90)	White or cream	Green glaucous	June–August
14	Digitalis viridiflora Lindl.	Mesic habitats	40–80	Greenish yellow	Green, glaucous or sparsely pubescent	June–August
15	Epilobium angustifolium L.	Moist/shaded habitats	(30) 60–150	Pink to purple	Green (lighter below)	June–August
16	Heracleum sphondylium L.	Moist/shaded habitats	50–150	White or pinkish	Dark green	June–August
17	Hypericum maculatum Crantz	Mesic habitats	20–60	Yellow	Green	June–July (August)
18	Hypericum olympicum L.	Mesic habitats	10–35	Yellow	Glaucus	June–July
19	Hypericum perforatum L.	Mesic habitats	30–90	Yellow	Green	June–August
20	Pedicularis friderici-augusti Tomm.	Moist/shaded habitats	10–20	Pale yellow	Green	May–July
21	Potentilla recta L.	Dry/open habitats	30–70	Yellow-cream	Green to gray-green	Mid-May–early August
22	Prunella vulgaris L.	Mesic habitats	5–30	Violet-blue	Green	June–September
23	Pulsatilla helleri (All.) Willd.	Dry/open habitats	10–30	Violet-purple	Green	April–May
24	Silene coronaria (L.) Clairv.	Dry/open habitats	40–80	Deep pink-magenta	White tomentose	June–July
25	Thymus thracicus Velen.	Dry/open habitats	5–15	Pink or purplish	Dark green, sometimes reddish-purple	June–August
26	Thymus praecox Opiz	Dry/open habitats	2–10 (15)	Purple or pink	Green	June–August

). Species selection was based on criteria relevant to their potential use in urban meadow plantings. Emphasis was placed on perennial herbaceous species, as perennial plants are generally more suitable for establishing persistent meadow communities. Species were also selected based on their ecological suitability for Mediterranean urban environments, including tolerance to high summer temperatures, seasonal drought and relatively poor soil conditions that often characterize urban substrates. Habitat type classification was based on general ecological preferences reported in the literature and field observations, grouping species into broad habitat functional categories (dry/open, mesic, and moist/shaded habitats) [22,23,24].

In addition, ornamental characteristics were taken into consideration. Ornamental value in herbaceous species is commonly associated with traits such as flower color, flowering period, plant height and growth form. Therefore, key morphological and phonological traits relevant to the visual structure of urban meadow vegetation that contribute to the visual diversity of urban plantings, influencing pollinator attraction and ecological functionality, were recorded for each species (Table 1).

Mature inflorescences were collected in the broader area of East Macedonia (NE Greece), from wild populations from early July to early autumn 2024. Following collection, the material was dried and cleaned and seeds were extracted and counted at room temperature. Seed lots were stored under ambient conditions till late autumn before testing approximately 3 months later. Therefore, all germination experiments were conducted on seeds that had undergone approximately 3 months of after-ripening (dry storage) under ambient conditions prior to the application of treatments.

2.2. Seed Viability Testing

Seed viability was assessed via the tetrazolium chloride (TTZ) test, which is a rapid and reliable method to identify metabolically active tissue assessing the viability of the embryo and the potential of the seed to germinate [25]. It is a fast test that is used before the germination test which is usually a lengthy process.

Two random replicate samples of 25 seeds each were used for the estimation of seed viability for each species. Excision of the embryos was conducted with the use of a dissecting needle following abrasion of the testa in seeds that were soaked in water for 12 h. During seed dissection, seeds were grouped into two categories: seeds with embryo (filled seeds), and empty seeds (without any gametophytic tissue). Empty seeds or seeds with atrophic embryos were considered non-viable. The viability of the extracted intact embryos was determined by staining them with a 1% w/v tetrazolium chloride solution (abbr. TTZ) [26]. Metabolically active tissue that was stained red in the tetrazolium chloride solution was considered viable. Consequently, the percentage of non-viable seeds is the sum of the percentages of empty seeds and those with a non-viable embryo.

2.3. Seed Dormancy and Pretreatments Applied

Seed dormancy is a widespread adaptive trait in wild plants, evolved to delay germination until environmental conditions are suitable for seedling establishment [20]. While ecologically significant, dormancy poses major constraints for cultivation, as it can prevent uniform germination and reliable seedling production. Therefore, understanding and overcoming dormancy is a necessary step for successful propagation. The studied species generally exhibit physiological seed dormancy, as indicated by published literature, mainly at the genus level (

Table 2. Summary of the available genus-specific literature information on the documented dormancy type concerning population samples of the focal species from different geographical areas.

	Species/Genus	Reference on the Dormancy Type
1	Achillea sp.	[27]
2	Anthemis sp.	[28]
3	Asphodeline sp.	[29]
4	Armeria sp.	[30]
5	Briza media	[31]
6	Campanula sp.	[32]
7	Dianthus sp.	[33]
8	Digitalis sp.	[34]
9	Epilobium angustifolium	[35]
10	Heracleum sphondylium	[36]
11	Hypericum sp.	[37]
12	Pedicularis friderici-augusti	[38]
13	Potentilla recta	[39]
14	Prunella vulgaris	[40]
15	Pulsatilla halleri	[41]
16	Silene coronaria	[42]
17	Thymus sp.	[20]

).

Based on the available literature summarized in Table 2, most of the studied taxa are reported to present physiological dormancy or no dormancy at the genus level. Therefore, cold stratification was selected as a suitable treatment to promote seed dormancy release and germination in the selected species. It should be noted that the cited literature in Table 2 has been used as a guide to design the current treatments and ensure physiological and ecological relevance for the focal species. Seeds underwent cold stratification at 0 ± 1 °C for 3 months. Germination was monitored weekly. The treatment was selected based both on the literature review for the selected taxa but also for its simplicity and feasibility in common nursery settings.

Following viability testing and pretreatments, seeds were sown in Petri dishes filled with sterilized sand. Treatment application entailed four replicate samples of 50 seeds each per species. Germination tests were conducted in a controlled environment chamber under alternating temperature: 25 °C/15 °C D/N, 12 h light/12 h dark. The applied temperature regime (25 °C /15 °C) represents a range of environmental conditions occurring across Mediterranean habitats. These conditions may correspond to autumn temperatures in lowland areas, but also to late spring conditions in higher elevation sites. As the species studied were collected across an altitudinal gradient, from lowland to upland environments, the selected temperature regime represents the range of conditions found in their natural habitats. Untreated seeds served as controls to assess baseline germination potential and to evaluate the effectiveness of the pretreatment. Germination was recorded every 7 days for 12 weeks to assess germination dynamics and evaluate dormancy status [20]. When the germination test ended, the non-germinated seeds were dissected and empty seeds were removed. Germination percentages were corrected based on the total number of filled seeds per repetition.

2.4. Data Analysis

Germination data were checked for normality and homogeneity using Shapiro–Wilk and Levene tests, respectively, and were found to meet both assumptions. Consequently, the effects of pretreatment were tested via one-way ANOVA and differences in mean values were checked using Tukey’s HSD post hoc test at 5% significance level. The time in days for 50% of germination to be reached (T50) was also calculated using the “dplyr” library built under the R-Studio application (version 4.5) (Posit Software Inc., Boston, MA, USA).

3. Results

In the current work, 26 native plant species from northeastern Greece were subjected to germination tests after cold stratification.

Table 3. Seed germination dynamics (germination percentage at week 1 ± SE and cumulative germination at week 4 ± SE), germination rate (T50), total seed germination (±SE) and viability percentages of the tested species after cold stratification (CS) and control. Germination conditions in the growth chamber entailed alternating temperature of 25 °C/15 °C D/N, and photoperiod of 12 h light/12 h dark.

Species	Treatment	First Week Germination (% ± SE)	Fourth Week Germination (% ± SE)	Final Germination (% ± SE)	T50 (Days)	Embryo Viability (%)
Achillea crithmifolia	CS	20.04 ± 7.26	74.00 ± 2.79 a	74.00 ± 2.79 a	11.05 ± 1.55 a	85
	Control	0	24.00 ± 2.83 b	45.00 ± 1.91 b	27.91 ± 1.84 b
Achillea millefolium	CS	23.60 ± 4.35 a	70.36 ± 2.29 a	72.00 ± 1.68 a	9.70 ± 1.11 a	100
	Control	1.00 ± 1.04 b	7.00 ± 3.00 b	39.00 ± 3.41 b	36.84 ± 2.21 b
Achillea clypeolata	CS	55.78± 6.88 a	73.25 ± 1.37 a	73.25 ± 1.37 a	7.00 ± 0.03 a	95
	Control	3.77 ± 2.18 b	18.47 ± 5.36 b	41.75 ± 1.18 b	27.34 ± 5.17 b
Asphodeline lutea	CS	0	2.92 ± 1.90 b	58.00 ± 1.47 b	34.15 ± 1.67 a	90
	Control	0	65.00 ± 4.12 a	74.00 ± 4.16 a	25.01 ± 0.26 b
Armeria canescens	CS	0	69.00 ± 5.00 a	83.00 ± 4.72 a	15.80 ± 1.57 a	100
	Control	0	82.00 ± 3.82 a	88.00 ± 5.16 a	11.07 ± 0.23 a
Briza media	CS	7.55 ± 5.44	49.16 ± 4.86 a	68.50 ± 1.55 a	21.18 ± 1.91 b	91
	Control	0	23.02 ± 4.07 b	64.25 ± 0.85 a	30.39 ± 1.11 a
Campanula lingulata	CS	0	47.00 ± 4.72	22.00 ± 2.58 b	11.26 ± 0.34	100
	Control	0	61.00 ± 5.74	61.00 ± 5.74 a	15.26 ± 0.68
Campanula persicifolia	CS	0	0	78.00 ± 2.58 b	43.20 ± 1.97 a	100
	Control	0	95.00 ± 1.91	95.00 ± 1.91 a	13.04 ± 0.97 b
Campanula trachelium	CS	0	0	71.00 ± 4.43 a	36.71 ± 0.78 a	100
	Control	0	5.00 ± 2.51	10.00 ± 2.58 b	22.75 ± 3.03 b
Dianthus cruentus	CS	0	23.00 ± 2.51 b	56.00 ± 5.65 b	32.50 ± 3.85 a	95
	Control	2.00 ± 2.04	49.00 ± 8.38 a	74.00 ± 1.15 a	19.10 ± 3.43 b
Dianthus gracilis	CS	34.00 ± 6.21	66.00 ± 8.71 b	87.00 ± 3.00 b	21.37 ± 2.77 a	100
	Control	0	95.00 ± 1.00 a	98.00 ± 1.14 a	10.94 ± 0.27 b
Dianthus pinifolius	CS	68.00 ± 4.89 a	89.00 ± 0.91 a	89.00 ± 0.91 a	7.00 ± 0.02 b	92
	Control	20.10 ± 4.19 b	33.79 ± 7.79 b	50.50 ± 0.64 b	18.46 ± 6.62 a
Digitalis lanata	CS	0	0	89.00 ± 4.43 a	39.98 ± 0.74 a	96
	Control	0	79.00 ± 9.14	81.00 ± 6.19 a	14.46 ± 1.24 b
Digitalis viridiflora	CS	0	0	94.00 ± 2.58 a	40.73 ± 0.33 a	100
	Control	0	64.00 ± 4.00	74.00 ± 3.82 b	13.64 ± 1.02 b
Epilobium angustifolium	CS	0	3.00 ± 1.91 b	82.00 ± 2.58 a	51.70 ± 0.21 a	100
	Control	0	84.00 ± 2.82 a	84.00 ± 2.82 a	10.58 ± 0.05 b
Heracleum sphondylium	CS	0	0	63.00 ± 5.74 a	40.02 ± 0.47 a	91
	Control	0.83 ± 0.8 3	0.83 ± 0.83	4.50 ± 0.28 b	32.38 ± 9.08 a
Hypericum maculatum	CS	1.92 ± 1.92 b	28.87 ± 3.38 b	65.00 ± 1.87 b	29.51 ± 1.17 a	100
	Control	22.36 ± 2.07 a	59.93 ± 3.00 a	88.25 ± 1.93 a	15.81 ± 2.11 b
Hypericum olympicum	CS	0	0	66.00 ± 2.58 b	47.20 ± 0.75 a	100
	Control	0	41.00 ± 11.12	77.00 ± 2.51 a	27.58 ± 5.81 b
Hypericum perforatum	CS	0	0	60.00 ± 1.63 b	43.64 ± 1.06 a	99
	Control	0	82.00 ± 8.86	95.00 ± 3.00 a	19.69 ± 1.12 b
Pedicularis friderici-augusti	CS	0	0	53.00 ± 7.18 a	45.84 ± 0.34 a	90
	Control	0	0.5 ± 0.62	1.75 ± 0.47 b	31.50 ± 2.86 b
Potentilla recta	CS	0	1.00 ± 1.02 b	35.00 ± 6.19 b	38.33 ± 0.18 a	100
	Control	0	77.00 ± 9.29 a	93.00 ± 4.72 a	10.67 ± 0.13 b
Prunella vulgaris	CS	1.00 ± 1.00 b	4.25 ± 1.65 b	5.75 ± 0.85 b	17.50 ± 6.23 a	90
	Control	41.97 ± 6.56 a	66.78 ± 2.73 a	69.50 ± 1.04 a	7.00 ± 0.00 b
Pulsatilla halleri	CS	0	4.51 ± 0.51	5.50 ± 0.95 b	21.00 ± 3.20 a	80
	Control	12.61 ± 4.89	38.74 ± 0.74	52.00 ± 1.08 a	16.15 ± 2.19 a
Silene coronaria	CS	0	0	79.00 ± 3.41 a	39.34 ± 0.42 a	86
	Control	0	67.00 ± 8.38	73.00 ± 8.06 a	11.19 ± 0.16 b
Thymus thracicus	CS	0	11.76 ± 1.75 b	12.75 ± 1.25 b	17.94 ± 3.38 a	100
	Control	29.82 ± 3.40	62.99 ± 2.85 a	63.75 ± 2.13 a	10.79 ± 1.87 a
Thymus praecox	CS	1.00 ± 1.00 b	8.00 ± 3.26 b	15.00 ± 1.91 b	28.22 ± 3.43 a	100
	Control	39.80 ± 1.97 a	74.68 ± 3.54 a	86.00 ± 2.48 a	9.80 ± 1.83 b

Different letters indicate significant differences between treatments (Tukey HSD, p ≤ 0.05). The abbreviation CS stands for cold stratification treatment

presents the mean germination percentages per species, along with the results of the post hoc analysis (Tukey’s HSD, p < 0.05). The embryo viability for each species is also included. The viability test (TTZ) verified high embryo viability in all examined species. The lowest embryo viability was recorded for Pulsatillahalleri (80%), followed by Silene coronaria (86%) and Achillea crithmifolia (85%). On the contrary, most of the species, including Armeria canescens, Campanula lingulata, Campanula persicifolia, Campanula trachelium, Dianthus gracilis, Digitalis viridiflora, Epilobium angustifolium, Hypericum maculatum, Hypericum olympicum, Potentilla recta, Thymus thracicus and Thymus praecox, presented 100% embryo viability (Figure 1).

Early germination data (week 1 and week 4, Table 3) showed that germination in the control during the first week was generally low in most of the species. Several species presented increased germination percentages by week 4, indicating a gradual progression of germination over time. However, this increase varied between species, with some showing a significant increase between week 1 and week 4, while others presented limited changes. However, some species presented notable early germination in the first week in the control, including Prunella vulgaris (41.97%), Thymus praecox (39.80%), Thymus thracicus (29.82%), and Hypericum maculatum (22.36%), indicating rapid germination responses under favorable temperature conditions.

In contrast, most species in the control treatment showed little or no germination during the first week, with germination increasing progressively by week 4. This pattern was particularly evident in species such as Campanula persicifolia, Epilobium angustifolium, and Silene coronaria, where germination was negligible at week 1 but substantially higher at week 4 (Table 3). Overall, these results indicate considerable interspecific variation in early germination responses under control conditions, with some species exhibiting rapid germination and others showing delayed germination over time, suggesting the presence of physiological dormancy in part of the seed lot.

The highest final germination percentage in 14 of the 26 species was for seeds in the control, namely Asphodeline lutea (74.00%), Armeria canescens (88.00%), Campanula lingulata (61.00%), Campanula persicifolia (95.00%), Dianthus cruentus (74.00%), Dianthus gracilis (98.00%), Epilobium angustifolium (84.00%), Hypericum maculatum (88.25%), Hypericum olympicum (77.00%), Hypericum perforatum (95.00%), Potentilla recta (93.00%), Prunella vulgaris (69.50%), Pulsatilla halleri (52.00%), Thymus thracicus (63.75%) and T. praecox (86.00%) (Table 3, p < 0.05).

Cold stratification on the other hand improved germination in Achillea crithmifolia, A. millefolium and Achillea clypeolata (74.00%, 72.00% and 73.25%, respectively), Briza media (68.50%), Campanula trachelium (71.00%), Dianthus pinifolius (89%), Digitalis lanata and D. viridiflora (89.00% and 94.00%, respectively), Heracleum sphondylium (63%), Pedicularis friderici-augusti (53.00%) and Silene coronaria (79.00%). Particularly strong responses to stratification were observed in Heracleum sphondylium and Pedicularis friderici-augusti, where germination increased from 4.5% to 63% and from 1.75% to 53% respectively (Table 3, p < 0.05). No germination was observed during the cold stratification period, and germination occurred only after transfer to 25/15 °C temperature conditions.

For some species, seed germination percentages in the control were similar to those of cold stratified seeds, indicating that cold stratification was not necessary to overcome dormancy. This pattern was observed in Armeria canescens, Briza media, Digitalis lanata, Epilobium angustifolium and Silene coronaria, where no significant differences between treatments were detected (Table 3, p < 0.05).

However, in most species, the frequency of germinated seeds was lower than the frequency of viable seeds recorded in the TTZ test (Table 3).

4. Discussion

Seed dormancy is a key ecological mechanism regulating the timing of germination and ensuring seedling establishment under favorable environmental conditions. In Mediterranean ecosystems, dormancy release is typically associated with seasonal temperature variation, particularly after-ripening during summer and exposure to low winter temperatures [20,43,44]. Thus, in the current study design, seeds were placed in dry storage followed by cold stratification to replicate Mediterranean conditions. Additionally, in these environments, variation in temperature and moisture availability along altitudinal gradients may influence germination timing, which can occur either in autumn following the first rainfall events or later in spring under cooler conditions, depending on local climatic constraints [45].

Physiological dormancy is common among herbaceous species of Mediterranean ecosystems [20], which can be released following after-ripening during summer or exposure to low winter temperatures [46]. As all seeds in the current study were subjected to a period of dry storage prior to experimentation, the response of freshly collected seeds to cold stratification was not assessed. Therefore, it remains unclear whether an initial after-ripening phase is required before stratification. Future studies should address this aspect. The germination results in the present study indicated that most of the investigated species presented either weak physiological dormancy or no dormancy at all. For several species, germination percentages were high in the control suggesting either the absence of dormancy, or that dormancy release had already occurred during seed storage through 3 months of after-ripening. This behavior was observed in species such as Armeria canescens, Briza media, Digitalis lanata, Epilobium angustifolium and Silene coronaria. This seed behavior has been reported for many Mediterranean herbaceous species that germinate readily when favorable environmental conditions are encountered [20,21]. On the contrary, the delayed germination observed under control conditions, particularly during the first weeks, suggests that seeds were partially non-dormant [20]. The gradual increase in germination over time indicates a partial dormancy release under warm conditions (25/15 °C), supporting the presence of non-deep physiological dormancy in several species.

On the other hand, species such as Achillea crithmifolia, Achillea millefolium, Achillea clypeolata, Campanula trachelium, Dianthus pinifolius, Digitalis viridiflora, Heracleum sphondylium and Pedicularis friderici-augusti germinated after cold stratification, indicating the presence of physiological dormancy. Heracleum sphondylium have been previously shown to germinate under temperatures < 10 °C [36], whereas in the current study, successful seed germination was achieved under higher temperatures following cold stratification, providing evidence for broadening the temperature range for successful ex situ seed germination for this species. Cold stratification simulates winter environmental conditions and is known to promote germination by reducing levels of abscisic acid while increasing gibberellin levels [21]. However, dormancy seems to be relatively non-deep as high germination percentages were obtained after only 3 months of cold stratification. It should be noted that it remains unclear whether freshly collected seeds exhibit dormancy and whether the after-ripening period reduced the duration of cold stratification required for dormancy release since, as it is mentioned above, all seeds subjected to cold stratification had previously remained in dry storage for approximately 3 months (after-ripening). Therefore, it remains unclear whether freshly collected seeds exhibit dormancy and whether the after-ripening period reduced the duration of cold stratification required for dormancy release. In addition to the above, comparing germination under control and cold stratification gave key implications for the design of effective propagation protocols. In several species, germination was higher under control conditions, while cold stratification resulted in reduced or negligible germination. This pattern suggests that exposure of non-dormant seeds to low temperatures may inhibit germination or induce secondary dormancy in certain taxa, despite their high embryo viability [21].

The establishment of self-sustaining urban meadows has been recognized as a key objective in meadow creation projects [8]. In urban environments, plant communities with the ability to reproduce and regenerate naturally are particularly important as they may develop more stable vegetation over time and simultaneously, they require lower management inputs [6]. Based on the seed germination responses observed in the current study, the focal species can be broadly divided into two ecological groups. The first group includes species that benefit from cold stratification, presenting low germination under control and increased germination after exposure to low temperatures. These species are likely to be adapted to autumn sowing in potential urban meadow applications, with germination occurring after winter conditions (indicative examples: Achillea crithmifolia, A. millefolium, A. clypeolata, Campanula trachelium, Dianthus pinifolius, Digitalis viridiflora, Heracleum sphondylium and Pedicularis friderici-augusti). The second group includes species that show higher germination under control and reduced germination following cold stratification. These species are more suitable for spring sowing, as exposure to winter cold may negatively affect their germination performance (indicative examples: Asphodeline lutea, Campanula lingulata, Dianthus cruentus, Hypericum olympicum, H. perforatum, Potentilla recta, Prunella vulgaris, Pulsatilla halleri, Thymus thracicus and T. praecox). Such species in their ecosystems usually are under conditional dormancy resulting from non-optimal temperature conditions during the winter period [20,46]. Therefore, the selection of appropriate sowing periods should be based on species-specific seed germination behavior.

Differences in ecological requirements should also be considered when selecting species combinations for urban meadow establishment. Urban environments are often heterogeneous, including both open, sun-exposed areas and more shaded or sheltered sites. Using species with different ecological tolerances can help improve establishment success under these variable conditions.

Finally, the variation in functional and esthetic traits among the selected species may further support their use in urban meadow plantings by enhancing both ecological interactions and visual diversity; such considerations coupled with the germination protocols developed in this study may facilitate the wider use of native Mediterranean species in seed mixtures for urban meadows.

5. Conclusions

The present study reports the germination capacity of 26 native plant species from northeastern Greece within the context of sustainable utilization in urban meadow plantings. The results showed that most species presented either no seed dormancy or relatively shallow physiological dormancy, as high germination percentages were achieved in the control or in seeds receiving 3 months of after-ripening plus cold stratification. The results also indicate that dormancy release may occur under both cold stratification and prolonged exposure to warm conditions, as shown by the gradual increase in germination under control conditions. However, responses to cold stratification were species-specific. In several species, cold stratification enhanced germination, whereas in others it resulted in reduced germination compared to control conditions, suggesting that exposure to low temperatures may inhibit germination in certain taxa. These contrasting responses have important practical implications for propagation strategies. Species that respond positively to cold stratification are more suitable for autumn sowing, allowing germination after winter conditions. On the contrary, species that exhibit higher germination under control conditions and reduced performance after cold exposure should be sown in spring, avoiding potential inhibitory effects of low temperatures.

These characteristics indicate that the studied species can be propagated relatively easily under simple nursery conditions. In addition, the selected species present traits such as perennial life form, variation in plant height, diverse flower and foliage colors and different flowering periods, which may enhance both the visual quality and ecological value of urban meadow plantings. The relatively shallow dormancy observed in most species may facilitate natural regeneration from seed and contribute to the development of self-sustaining urban meadow communities. Such plant communities may support diverse pollinator assemblages and, indirectly, urban fauna that depend on insects as a food resource.