Enhancing In Situ Conservation of Crop Wild Relatives for Food and Agriculture in Lithuania

Abstract

The crop and crop wild relative (CWR) checklist of Lithuania was created containing 2630 taxa. The checklist comprises 1384 native taxa including archaeophytes and 1246 neophytes. In total, 699 taxa (26.6%) are defined for food and forage use. A list of 144 CWR priority species with 135 native species and archaeophytes and 9 naturalized species was generated. In total, 53 genera of food and forage species belonging to 15 families are represented by the priority CWR. Two approaches for CWR genetic reserve selection have been employed in this study: (1) CWR-targeted evaluation of preselected sites, including Natura 2000 sites, national protected areas, and other effective area-based conservation measures (OECMs), such as ancient hillfort sites and ecological protection zones of water bodies; (2) analysis of large georeferenced plant databases. Forty-five potential genetic reserve sites have been selected by the first approach covering 83 species or 57.6% of the national CWR priority list. With the second approach, the in situ CWR National Inventory database has been created by combining data from the Database of EU habitat mapping in Lithuania (BIGIS), Herbarium Database of the Nature Research Centre (BILAS), Lithuanian Vegetation Database (EU-LT-001), and Global Biodiversity Information Facility (GBIF). Hotspot analysis of CWR species richness and number of observations suggested that higher CWR diversity is more likely to be found in protected areas. However, Shannon diversity and Shannon equitability indices showed that the areas outside of the protected areas are also suitable for CWR genetic reserve establishment.

1. Introduction

As stated in the Plant Genetic Resources Strategy for Europe [1], crop wild relative (CWR) genetic resources native to Europe are related to the many socio-economically important crops cultivated in the region and in other parts of the world (food, fodder, forage, beverage, food additive, oil, biofuel, biomass, medicinal, ornamental) and contain a wide pool of evolving genetic diversity, not duplicated in the crop itself, and that is of potential value for crop improvement. Some wild species are harvested from the wild for direct use as food or feed and constitute a potential source for further domestication and creation of new crops. This statement is in congruence with the International Treaty on Plant Genetic Resources for Food and Agriculture [2], stating that contracting parties shall “promote in situ conservation of wild crop relatives and wild plants for food production, including in protected areas”.

Although Lithuania is not among the European hotspots of floristic richness, it has numerous CWRs as well as wild harvested plants, such as forage grasses (Poa, Festuca, Agrostis, Phleum, Alopecurus, etc.) and legumes (Trifolium, Vicia, Lathyrus, Medicago, Lotus, etc.), medicinal and aromatic plants (Hypericum, Mentha, Thymus, Origanum, Angelica, etc.), and fruit and berry species (Vaccinium, Fragaria, Ribes, Rubus, Malus, Pyrus, Prunus, Corylus, etc.), with some of which not only being harvested from the wild but also used for domestication and development of new cultivars. The most recent examples of the latter are bog cranberry (Vaccinium oxycoccos L.) and guelder rose (Viburnum opulus L.) with five and three new cultivars, respectively, released at the Botanical Garden of Vytautas Magnus University, Kaunas, Lithuania, and distinguished by enriched contents of fruit bioactive substances and the amount of yield [3,4]. Wild garlic, or ramson (Allium ursinum L.), is another example of the wild harvested plants being increasingly collected from the wild for food as well as cultivated in amateur gardens. These and other native food plant species have been contributing to the healthy diets of local people through generations and thus have been naturally adapted by their organisms. It is particularly important to consider this currently when globalization is conditioning a wide-spread consumption of imported food products and fast foods. Many wild forage species, predominantly from Poaceae and Fabaceae, are also used as CWRs and thus present valuable indigenous genetic resources to be conserved to ensure their sustainable use in the future.

It is generally admitted that the in situ conservation of CWRs is most feasible within the existing network of protected areas [5]. However, it depends on the development of the protected area network, including total area coverage and categories of protected areas established. In Lithuania, the protected areas of conservation protection priority (strict reserves, reserves, and heritage objects) and complex protected areas (public parks—national and regional parks, as well as biosphere monitoring territories—biosphere reserves and biosphere polygons) are the most important ones and collectively referred to as specially protected areas. They cover 1,152,793 hectares or 17.65% of the country’s territory [6]. The areas of the European ecological network Natura 2000 [7], including those established under the Habitats Directive, are largely integrated into the system of the nationally protected areas. The major questions to be answered are (1) whether the existing network of protected areas adequately covers the distribution of the priority CWRs in the country and (2) whether there is a need to establish any additional protected areas for CWR conservation.

Thus, in the context of food security concern combined with climate change, the aim of this paper is to enhance the in situ conservation of priority CWR species for food and agriculture through the analysis of their distribution across the country and selection of the most appropriate wild populations (MAWPs) as potential sites for the establishment of genetic reserves with a focus on the existing protected areas. It is expected that this effort will facilitate systematic conservation planning of the target species in the country and contribute to the development of the European CWR genetic reserves network. So far, there was no comprehensive research on CWR richness and species distribution patterns in Lithuania on a nationwide scale. We hypothesized that protected areas contain higher CWR diversity than unprotected areas.

2. Materials and Methods

2.1. National CWR Checklist

A floristic approach was used to prepare the national CWR checklist as described by [8]. The checklist of the flora of Lithuania was compiled by using the following sources: Dictionary of Plant Names [9], Vascular Plants of Lithuania [10], Flora of the Baltic Countries [11,12,13], Orchids (Orchidaceae) of Lithuania [14], Red Data Book of Lithuania [15] and the Euro + Med PlantBase [16]. The taxonomic harmonization was carried out by referring to the International Plant Name Index [17] and Plants of the World online [18].

2.2. Prioritization of CWR Species

Prioritization of CWR species was carried out with a focus on plant genetic resources for food and agriculture (in a wide sense), with the native species and archaeophytes given top priority. Some close relatives of food crops, such as Lactuca serriola L., Malus toringo (Siebold) de Vriese, Prunus cerasifera Ehrh., Armoracia rusticana G.Gaertn., B.Mey. and Scherb., Avena fatua L., and Raphanus raphanistrum L., were excluded from the priority species list due to their invasive or weedy behavior. Prickly lettuce (L. serriola) is a particularly rapidly spreading species that invades different natural plant communities, while cherry plum (P. cerasifera) poses a threat by hybridization with the native species P. spinosa (Rašomavičius, pers. comm.; [19,20]. Wild oat (A. fatua) and wild radish (R. raphanistrum) are listed among the worst agricultural weeds. The other criteria used are as follows:

–

Annex I. List of crops covered under the multilateral system of the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA);

–

List of global priority crop wild relative genera;

–

Lithuanian national plant variety lists;

–

Wild species relationships to crops;

–

Socio-economic and cultural importance, including use of traditional species;

–

Threat status of species.

All CWR taxa falling within the genera listed in the Annex 1 of the International Treaty on Plant Genetic Resources for Food and Agriculture [2] as well as in the list of global priority crop wild relative genera [21] were selected as priority ones.

Lithuanian national plant variety lists (e.g., [22]) were used as proxy indicators of crop importance for the respective consideration of their wild relatives. Similarly, the determination of socio-economic and cultural importance was based on well-established plant cultivation and wild harvesting traditions.

CWR relatedness to crops according to the gene pool [23] and taxon group concepts [24] was determined by using the data available at the Germplasm Resources Information Network [25], Nordic Priority CWR Dataset [26], Flora of the Baltic Countries [11,12,13], Wikispecies [27], World Plants [28], and scientific articles on separate genera or species.

Threat status of the target species was cited from the Red Data Book of Lithuania [15], which provided assessment results based on IUCN criteria [29].

The CWR checklist and inventory data template [30] was used to facilitate structural data arrangement and management.

Several data sources from other European countries, i.e., Nordic countries [26,31,32] Czech Republic [33], and the Netherlands [34] were used, and we also consulted some crop breeders from the Department of Grass Breeding at the Lithuanian Research Centre for Agriculture and Forestry to find the best solutions for CWR prioritization.

2.3. Selection of Sites for Genetic Reserves and Hotspot Analysis

Two approaches of site selection for CWR genetic reserves were employed: (1) evaluation of preselected sites on state-owned land like ancient hillforts (which are state-protected archeological objects), sites of community importance (SCI, Natura 2000 network), and national protected areas including ecological protection zones of water bodies; (2) application of several large georeferenced plant databases, both national (BIGIS and EU-LT-001) and international (the Global Biodiversity Information Facility—GBIF), for distribution and hotspot analysis of priority CWR species in 4 × 4 km grid cells. Shannon’s diversity index [35] was employed for CWR diversity evaluation in grid cells by using the following formula:

H = −Σ [(n/N) × ln(n/N)]

(1)

where:

• Σ—a Greek symbol for sum;
• H—Shannon diversity index;
• n—number of observations of a given CWR species within a grid cell;
• N—number of observations of all CWR species within a grid cell;
• ln—natural logarithm.

The same formula in a simplified version [36] could be presented as follows:

H = −Σp_i × ln(p_i)

(2)

where p_i is the proportion of the entire community made up of species i.

A derivative of the Shannon diversity index, the Shannon equitability index [36], is a way to measure the evenness of species proportions in a community. It is calculated as follows:

E_H = H/ln(S)

(3)

where:

• E_H—Shannon equitability index;
• H—Shannon diversity index;
• S—total number of unique species.

The cover-abundance of each CWR species was estimated by using the Braun–Blanquet scale [37]. However, the presence/absence data were used only to avoid complication of the data analysis. QGIS software v. 3.34.5 [38] was used for the mapping of CWR distribution, hotspot analysis, and location of potential genetic reserve sites.

2.4. Data Sources

Several georeferenced plant databases were used. BIGIS (Institute of Botany GIS database) contains the countrywide species distribution data related to the inventory of the EU’s important natural habitats. BILAS is the major Lithuanian herbarium database maintained by the Institute of Botany of the Nature Research Centre. EU-LT-001, the vegetation database of Lithuania, is a part of the European Vegetation Archive [39]. The Global Biodiversity Information Facility (GBIF), which contains citizen science data, was also used.

3. Results and Discussion

3.1. Checklist Description

The crop and CWR checklist of Lithuania contains a total of 2630 taxa, including species (and microspecies), subspecies, varieties, and hybrids (see Supplementary Materials, Table S1). The checklist is comprised of 1384 native taxa (incl. archaeophytes) and of 1246 neophytes, of which 905 are used in cultivation. The major categories of use are food, forage, medicinal, technical, and forestry species. In the checklist, 471 taxa (17.9% of the checklist) are food crops, or their wild relatives, and 399 taxa (15.2%) are forage/fodder crops or their wild relatives. A total of 699 taxa (26.6%) are defined as food and forage use. Additionally, 841 taxa (32%) are of medicinal use, and 1396 taxa (53.1%) are of other use. Furthermore, 178 taxa are related to those included in Annex I of the ITPGRFA [2]. The checklist also includes 169 taxa that are under the legal national protection [40]. In addition, the checklist covers 16 taxa (incl. 13 native ones) from Annex II, 1 taxon from Annex IV, and 10 taxa (incl. 8 natives) from Annex V of the [41] on the conservation of natural habitats and of wild fauna and flora. Moreover, the checklist contains 22 species that have been declared invasive in Lithuania and 34 species that are considered potentially invasive [20]. In general, the above-mentioned numbers are consistent with those of the other European countries, particularly in Scandinavia. For example, of the 2276 crop and CWR species recorded in Norway, 2084 species (92%) are included in the agricultural and horticultural crop group [42].

3.2. Prioritization of the Checklist

In total, 53 genera of food (including culinary herbs, aromatic plants, and berries) and forage/fodder species were selected, belonging to 15 families. A list of 144 CWR priority species with 135 native species (including archaeophytes) and 9 naturalized ones (mostly those, escaped from cultivation) was generated. In total, 74 species (51.4%) are representatives of the genera listed in Annex 1 of the ITPGRFA and those of the 92 global priority CWRs [21]. The current CWR priority list is shorter by 20% compared to the previous version of 180 species [43], which included a portion of non-food CWR species and is consistent with the Swedish priority list, containing 121 CWR taxa [32]. The summary of the prioritized CWR inventory by botanical families is presented in

Table 1. Summary of the prioritized Lithuanian CWR inventory by families.

Family	Genera	Species	Species %	Genera with Numbers of Species
Poaceae	19	47	32.6	Agrostis (5), Alopecurus (4), Anthoxanthum (3), Arrhenatherum (1), Avenula (1), Briza (1), Bromus (1), Cynosurus (1), Dactylis (1), Deschampsia (2), Elymus (1), Festuca (8), Glyceria (4), Helictochloa (1), Leymus (1), Lolium (1), Phalaris (1), Phleum (2), Poa (8)
Fabaceae	11	46	31.9	Anthyllis (1), Astragalus (3), Lathyrus (7), Lotus (2), Medicago (2), Melilotus (2), Onobrychis (2), Ononis (1), Securigera (1), Trifolium (14), Vicia (11)
Rosaceae	5	16	11.1	Fragaria (3), Malus (2), Prunus (3), Pyrus (2), Rubus (6)
Lamiaceae	3	6	4.2	Mentha (3), Origanum (1), Thymus (2)
Brassicaceae	2	5	3.5	Barbarea (2), Rorippa (3)
Amaryllidaceae	1	6	4.2	Allium (6)
Ericaceae	1	5	3.5	Vaccinium (5)
Apiaceae	4	4	2.8	Angelica (1), Carum (1), Daucus (1), Pastinaca (1)
Grossulariaceae	1	3	2.1	Ribes (3)
Asparagaceae	1	1	0.7	Asparagus (1)
Asteraceae	1	1	0.7	Cichorium (1)
Betulaceae	1	1	0.7	Corylus (1)
Cannabaceae	1	1	0.7	Humulus (1)
Papaveraceae	1	1	0.7	Papaver (1)
Elaeagnaceae	1	1	0.7	Hippophae (1)
Total: 15	53	144	100

(see Supplementary Materials for the full CWR priority list, Table S2).

As seen from Table 1, nearly 2/3 (64.5%) of the prioritized species are members of two families, Poaceae (47 species) and Fabaceae (46 species). The third richest CWR family is Rosaceae with 16 species. These three families make up 75.7% of the total CWR priority list.

Analysis of the priority CWRs by their use categories showed that 88 species (61.1%) could be attributed to the CWRs of forage/fodder crops and 58 species (40.3%) to those of the food crops with only 2 species, Daucus carota and Vicia lathyroides, overlapping both categories (

Table 2. Summary of the prioritized Lithuanian CWR inventory by use categories.

Use Category	Families with Numbers of Genera/Species	Number of Species *	Percent of Priority CWR List *
Forage/fodder	Fabaceae 11/46 Poaceae 16/41 Apiaceae (1/1)	88	61.1
Food	Amaryllidaceae (1/6) Apiaceae (4/4) Asparagaceae (1/1) Asteraceae (1/1) Betulaceae (1/1) Brassicaceae (2/5) Cannabaceae (1/1) Elaeagnaceae (1/1) Ericaceae (1/5) Fabaceae 1/1 Grossulariaceae (1/3) Lamiaceae (3/6) Papaveraceae (1/1) Poaceae (3/6) Rosaceae (5/16)	58	40.3

* Includes two overlapping species, Daucus carota L. and Vicia lathyroides L.

).

Regarding legal protection, 17 CWR priority species (11.8%) are protected by the Order of the Minister of Environment (2024). These are evaluated according to the IUCN categories and criteria at the national level (

Table 3. CWR priority species under the legal national protection with the regional IUCN assessments.

No.	Species	IUCN Category and Criteria *
1	Allium angulosum L.	EN B1ab(ii,iii) + 2ab(ii,iii)
2	Allium scorodoprasum L.	VU A4ac
3	Allium vineale L.	EN B2ab(iii,iv,v)
4	Alopecurus arundinaceus Poir.	VU D2
5	Astragalus danicus Retz.	NT B2b(iii); B1b(iii)
6	Festuca altissima All.	DD
7	Festuca psammophila (Čelak.) R. M. Fritsch	EN B1ab(ii,iii,v) + 2ab(ii,iii,v)
8	Glyceria lithuanica (Gorski) Gorski	VU B1ab(iii) + 2ab(iii)
9	Helictochloa pratensis (L.) Romero Zarco	VU D2
10	Lathyrus laevigatus (Waldst. & Kit.) Gren.	NT B2
11	Lathyrus pisiformis L.	EN B1ab(iv) + 2ab(iv)
12	Poa remota Forselles	NT B2
13	Prunus spinosa L.	VU B1ab(ii,iii,v) + 2ab(ii,iii,v)
14	Trifolium lupinaster L.	EN B2b(iii)c(iv)
15	Trifolium rubens L.	EN B2ab(i,ii,iii,iv)
16	Vicia lathyroides L.	EN B2b(iii)c(ii)
17	Vicia pisiformis L.	NT B1 + 2

* Source: [15].

).

Based on tentative observations, most of the rest of CWR priority species fall into the IUCN category LC (Least Concern). However, a detailed assessment of at least some species, like Lathyrus palustris, Mentha longifolia, Onobrychis arenaria, and Vaccinium microcarpum, is needed to improve their conservation planning.

Analysis of the priority CWR relatedness to crops revealed that 75 species (52.1%) represent the closest relationships with their respective crops: 62 species are within the primary gene pool (GP1) and 13 species are within taxon group one (TG1) to their respective crops (see Supplementary Materials, Table S2). Twenty-four CWR species fall into the second closest group to crops (GP2, TG2, and TG3). And the most distant group (GP3 and TG4) comprises 45 CWR species. As lack of data on genetic relatedness is quite evident, the taxon group concept allows us to compensate this data gap, at least partly. However, the CWR of the most distant taxonomic group, TG4, should still be studied by molecular methods to better reveal their use possibilities in breeding. This does not apply when wild species are being introduced into cultivation themselves.

3.3. Selection of Sites for Genetic Reserves

By using the method of preselected site evaluation, 45 potential CWR genetic reserve sites were identified. Out of these, 29 sites were established in 2022 through 2023. The rest of the sites were originally identified in 2011–2021 [44], with the oldest ones inventoried repeatedly. These 45 sites contain 83 CWR priority species (57.6%) with a total number of 748 occurrence records. The suggested sizes of the sites vary from 0.22 to 23.40 ha, with a total area of 177.61 ha (see Supplementary Materials, Table S3). A weak correlation has been established between site area and number of CWR species (R = 0.298, p-value = 0.046). Although it has been reported [45] that locality area and connectivity between similar localities in conservation planning best conserve both species and intrapopulation genetic diversity, some other factors, such as different land use category, different land ownership, and the limited size of protected areas (e.g., protected hillfort sites usually are small), prevent us from following this recommendation more closely.

Within the 45 sites, the most frequent species inventoried were Dactylis glomerata (33 sites), Vicia cracca (32 sites), Corylus avellana (28 sites), Phleum pratense (26 sites), Prunus padus (26 sites), Rubus idaeus (24 sites), Thymus pulegioides (23 sites), Fragaria viridis (22 sites), Poa angustifolia (21 sites), Fragaria vesca (20 sites), and Trifolium medium (20 sites). The least represented populations were those of Allium angulosum, A. scorodoprasum, A. vineale, Asparagus officinalis, Hippophae rhamnoides, Mentha aquatica, Onobrychis viciifolia, Poa trivialis, Rubus nessensis, R. plicatus, Trifolium campestre, T. hybridum, Vaccinium myrtillus, V. oxycoccos, V. vitis-idaea, Vicia pisiformis, and V. tetrasperma, each occurring in one single site out of the 45 sites investigated. We have grouped the distribution of the 83 CWR species across the 45 sites into five frequency groups (

Table 4. Distribution of 83 CWR priority species across 45 potential CWR genetic reserve sites.

Frequency Group	No. of CWR Occurrence Sites	No. of CWR Species	Percent of CWR Priority List
1	1–4	36	25.0
2	5–9	20	13.9
3	10–14	3	2.1
4	15–19	13	9.0
5	≥20	11	7.6
Total species in 45 sites		83	57.6
Full priority list		144	100.0
Total records in 45 sites	748

). As seen from Table 4, the required minimum of 5 populations, as proposed by [5], has not yet been met for 36 CWR species, while a minimum of 10 populations, as suggested by [46] for relatively widespread species, has not been met for 20 more species.

The selected 45 potential CWR genetic reserve sites were mapped with QGIS, showing that they represent all four climate regions and 7 of the 10 subregions of the country (Figure 1) and are in different national protected areas and/or Natura 2000 sites of Community importance (SCIs) (Supplementary Materials, Table S3). As seen from Figure 1, only three climatic subregions (A1, A2, and B5) are not represented by the current approach, while three subregions (C7, D8, and D9) are the least represented ones.

Advantages of the first approach, the evaluation of the preselected sites, include well-studied concrete locations, usually in a protected area, state-owned land status, and relatively easily distinguishable site boundaries, which are more manageable small sites. This is in line with the concept of the so-called micro-reserves initiated in Spain for rare species conservation [48], which could also be applied in the selection of the most appropriate wild populations (MAWPs), a key object in in situ conservation, by treating the MAWPs as analogues of rare species. In such small reserves, the target species are relatively highly concentrated per site area, i.e., 16.6 species per 3.9 ha on average (see Supplementary Materials, Table S3) compared to 17.8 species per grid cell 4 × 4 km in protected areas (

Table 5. Total and mean (per grid cell 4 × 4 km) numbers of CWR species, numbers of CWR species observations, and mean values of Shannon diversity and Shannon equitability indices inside and outside of protected areas (± is followed by standard deviation; different letters indicate significant differences at p = 0.001).

Area Category	No. of Unique CWR Species		No. of CWR Species Observations		Shannon Diversity Index (H)	Shannon Equit-ability Index EH = H/ln(S)
	Total (S)	Mean per Cell	Total	Mean per Cell	Mean per Cell	Mean per Cell
Inside PAs	138	17.8 ± 10.6 a	190,106	82.6 ± 83.6 a	2.53 ± 0.67 a	0.513
Outside PAs	140	13.8 ± 9.5 c	111,222	50.9 ± 61.3 c	2.41 ± 0.74 b	0.488
Total country	140	16.0 ± 10.3 b	301,328	67.9 ± 75.7 b	2.48 ± 0.71 ab	0.502

). It has been reported that small reserves allow for advantage to be taken of the conservation opportunities provided by cultural sites, sacred natural sites, and other faith-based sites in otherwise transformed landscapes [49]. And that is the case in the current study, as 30 potential CWR reserve sites were identified in the ancient hillfort sites. These, along with the water protection zones, may be attributed to the other effective area-based conservation measures (OECMs) as defined by the Convention on Biological Diversity in Decision 14/8 [50] as they deliver the effective in situ conservation of biodiversity, including CWRs, regardless of their primary objectives. From a legal point of view, it is easier to establish small reserves outside of protected areas than large ones. However, the coverage of all target species, let alone their distinctive populations, by such preselected small sites may require a high number of small reserves. This leads to the need for a more comprehensive approach and the application of large georeferenced plant databases.

By applying the second approach, the in situ CWR National Inventory database has been created by combining target taxa occurrence data from four major datasets: (1) Database of EU habitat mapping in Lithuania (BIGIS); (2) Database of Herbarium of Nature Research Centre (BILAS); (3) Lithuanian Vegetation Database (EU-LT-001); and (4) Global Biodiversity Information Facility (GBIF). Most of the recent data are contained in BIGIS and GBIF (

Table 6. The numbers of occurrences of CWR priority species ¹ in four databases by data oldness.

Data Collected	BIGIS	BILAS	EU-LT-001	GBIF 2	Total 3
Before 2010	0	7673	19	21	7713
After 2010	284,264	9	2155	7187	293,615
Total	284,264	7682	2174	7208	301,328

¹ The numbers refer to 140 out of 144 CWR priority species. No data on Barbarea stricta, Lathyrus pisiformis, Onobrychis arenaria, or Vaccinium microcarpum are presented. A lack of distribution data of the four CWR priority species have been attributed to the species identification issues (Barbarea stricta vs. B. vulgaris and Vaccinium microcarpum vs. V. oxycoccos) and rarity of species (Lathyrus pisiformis and Onobrychis arenaria). ² GBIF.org (12 July 2023) GBIF Occurrence Download https://doi.org/10.15468/dl.y43bqn. ³ The most up-to-date total is 293,615, which includes multiple occurrences of the same species per grid cell 4 × 4 km. If counting only distinct species records per grid cell, the total number of occurrences is 68,686.

). The compiled database can run in both Microsoft Access and QGIS formats.

Hotspot analysis of priority CWR occurrences performed with QGIS in 4 × 4 km grid cells showed that the highest numbers of the target species occurrences are in the north-western and south-eastern parts of the country (Figure 2), where most of the potential genetic reserve sites (green dots) are identified as well (see also Figure 1 for details).

Further, to facilitate conservation actions, we have focused on CWR distribution in protected areas (PAs). These incorporate European Union PAs, i.e., Natura 2000 network, dedicated to the protection of habitats, and national PAs, i.e., national parks, regional parks, strict nature reserves, and other nature reserves. It was established that 1882 grid cells at least partly overlap with the Natura 2000 sites and 1941 grid cells overlap with national protected areas. As most of the national PAs overlap with Natura 2000 sites, we analyzed CWR distribution in the grid cells inside both the EU and national PAs. The most species-rich grid cells (up to 60 species per cell) were identified in the PAs in Vilnius County, Southeast Lithuania, including Aukštadvaris and Neris Regional Parks (Figure 3). This is, at least partly, due to diverse ecogeographic conditions, and these sites are valuable in situ reserves for CWR use in research and education as they are easily accessible by researchers and those who use iNaturalist or similar applications for sharing data of species observations.

If compared with species richness, the mapping of species observations across the country presented a quite different mosaic of grid cells (Figure 4). A similar pattern was observed when the protected areas were analyzed separately, revealing the observation-richest grid cells in Žemaitija National Park, Salantai Regional Park, Nemuno Delta Regional Park, and Pagramantis Regional Park (all in Western Lithuania) (Figure 5).

The mean number of species per grid cell in PAs was 17.8, while outside of PAs it was 13.8 (Table 5). The Kruskal–Wallis test for equal medians showed that there was a significant difference between the area category medians (H = 147.5, p < 0.01). Post hoc pairwise comparisons using the Mann–Whitney method revealed that the medians differed significantly in all categories (Bonferroni corrected p < 0.001) with the highest number of species per cell in PAs. Regarding the number of observations per grid cell, significant differences were established between all three categories (Kruskal–Wallis test H = 202.8, p < 0.001; post hoc Mann–Whitney, Bonferroni corrected p < 0.001). These results suggest that higher CWR diversity is more likely to be found in PAs, where the in situ conservation of CWR should be focused on.

As the numbers of species and their occurrences taken separately provide an incomplete picture of CWR distribution, the Shannon diversity index (SDI), a combined indicator of species richness and abundance, was calculated for each grid cell. The results showed that the mean SDI in PAs was 2.53, while outside of PAs it was 2.41, suggesting statistically significant differences between the inside and outside of PAs (Kruskal–Wallis test for equal medians H = 17.32, p < 0.001; Table 5) (see also Supplementary Materials, Figures S1 and S2 for maps of species richness and the number of observations, respectively, outside of PAs, and Figures S3–S5 for SDI-based species mapping across the country, in its PAs and outside PAs, respectively). The Shannon equitability index was also calculated to find out how similar the abundances of different CWR species are in a grid cell with the advantage that it is easier to interpret, as its value ranges from 0 to 1, where 1 indicates complete evenness [36]. Differently from the above-mentioned results, this indicates that potential CWR in situ conservation sites can also be established outside of the PA network.

The obtained Shannon diversity index values could be interpreted as indicating moderate (2.50–2.99) and low (2.00–2.49) diversity [51]. Similarly, the Shannon equitability index shows moderate abundances of CWR species both inside and outside of protected areas.

4. Conclusions

A comprehensive and annotated national CWR checklist has been created for Lithuania for the first time. It serves as a baseline information resource for CWR prioritization for conservation planning and action. Most of the priority CWR (93.8%) are native species of Lithuanian flora, and 8.3% are threatened (IUCN regional evaluation categories EN and VU). In the current study, 56.9% of priority CWR species are related to crops based on the gene pool concept, and the rest (43.1%) stand for taxonomic group relationships. There are 62 CWR species within GP1 of their respective crops and 22 CWR species within TG1 and TG2 crops.

Two approaches towards CWR genetic reserve selection have been employed in this study which can complement each other: (1) CWR-targeted evaluation of preselected sites, like sites of community importance (SCI, Natura 2000 network) and national protected areas, as well as other effective area-based conservation measures (OECMs), such as ancient hillfort sites (which are state-protected archeological objects) and ecological protection zones of water bodies; all 45 potential genetic reserve sites have been selected in this way, covering 83 species or 57.6% of the national CWR priority list, and (2) analysis of large georeferenced plant databases for CWR species richness and number of observations; multiple hotspots of priority CWR species have been identified in this way, covering 140 species, or 97.2%, of the priority list. If the first approach evaluates all conditions at the place, the second approach is based solely on target species distribution and abundance. Hotspot analysis of CWR species richness and number of observations suggested that higher CWR diversity is more likely to be found in protected areas, which confirmed the initial hypothesis. However, Shannon diversity and Shannon equitability indices showed that the areas outside of the protected areas are also suitable for CWR genetic reserve establishment.

Among the main CWR conservation challenges regarding the current protected area network is that it covers not all habitat types needed to adequately conserve different CWR populations. Also, having no formal recognition for CWRs as such results in a hindrance to their active conservation by protected area managers.

It has been reported that area-based conservation efforts, which include both protected areas and other effective area-based conservation measures (OECMs), are likely to extend and diversify [52]. Thus, the current study is in congruence with this statement as the use of OECMs (ancient hillfort sites and river protection zones) contributed substantially to selecting the potential CWR genetic reserve sites, developing the national genetic reserve network and in situ CWR dataset inclusion into the European Catalogue EURISCO.

As the availability and accuracy of data on CWR distribution are of crucial importance, the creation of the national network of data providers should be seen as a must to effectively implement conservation efforts. Protected area managers, NGOs, farmers, and individual people are among those who should be part of the network.

The success of long-term in situ conservation efforts requires ongoing monitoring of CWR populations. For this purpose, some other CWR-related projects may effectively benefit, e.g., EU habitat monitoring, which is being carried out in Lithuania and covering 10% of each habitat type.

Regarding the limitations of this study, one of the major databases (BIGIS) used in this study covers only habitats of EU importance, leaving aside all the others. Also, a significant part of the data from the georeferenced databases is more than 10 years old, with updates becoming urgent. Considering that the changing environmental conditions are major factors affecting species occurrence, research in the near future should focus on both climate change effects and the impact of human activities on CWR habitats and species distribution.