Distribution of Тrifolium rubens in Ukraine and the current state of its populations in Zakarpattia

Trifolium rubens is a Central European forest-steppe plant species reaching its eastern distribution limit in Ukraine and listed in the Red Book of Ukraine (2009). Our study was carried out in 2016–2020 and aimed to analyze the distribution of T. rubens in Ukraine, to identify the composition of phytocoenoses with T. rubens, and to evaluate the current state of its populations in the Zakarpattia region of Ukraine. The distribution of this species was analyzed based on data obtained from the field surveys, literature, online resources, and herbaria. In total, 78 localities of T. rubens were analyzed. Trifolium rubens was found to be distributed in Zakarpattia, Lviv, Ivano-Frankivsk, Ternopil, Rivne, Khmelnytskyi, Chernivtsi, and Vinnytsia regions of Ukraine. Two of these regions (Khmelnytskyi and Chernivtsi) were not mentioned before in the Red Book of Ukraine. The presence of T. rubens in Volyn, Zhytomyr, Cherkasy, and Kyiv regions is doubtful and not confirmed by recent data. Most localities of this species are known from Zakarpattia and Lviv regions. Three new localities were found in the Beregovo district of the Zakarpattia region in the foothills of the Volcanic Carpathians. The floristic compositions of the plant communities and ontogenetic structure of T. rubens populations in Beregovo and Mukachevo districts of the Zakarpattia region were investigated. Investigated populations are isolated with a dispersed disposition of plants on large areas. Trifolium rubens occurs here in meadow-steppe and meadow areas, on hillsides, and also participate in mesoxerophilous secondary (semi-natural) shrub communities in anthropogenically transformed areas. Often such populations are situated along pathways, on forest edges and fallow lands, and associated with ecotones of Trifolio-Geranietea sanguinei class. All studied populations were incomplete with the domination of generative shoots.


Introduction
Trifolium rubens L. is a Central European forest-steppe plant species. It is also present in Southern Europe, including southern Spain, Italy, and Eastern Europe. Some species' findings were reported from Belarus (Meusel & Jäger, 1992). Also, a few localities are known from the Baltic region (Rašomavičius, 2007) and the south of Thrace (Meusel & Jäger, 1992).
In Ukraine, T. rubens is a rare species (Didukh, 2009), reaching its eastern distribution limits. It is mostly distributed in the right-bank part of Ukraine, where it occurs on dry meadows, meadow-steppes, forest edges, and growths among shrubs. Didukh (2009) indicated that T. rubens occurs in Zakarpattia and Volyno-Podilia of Ukraine only. However, the preliminary chorological research showed that it probably has a broader distribution range.
In the adjacent countries, T. rubens is associated with some more communities. For example, in Poland it is listed for the order Quercetalia pubescentis Br.-Bl. (1931) 1932(Matuszkiewicz, 2001. Trifolium rubens is also mentioned as an indicator of thermophilic Trifolio-Geranietea communities in Slovakia (Ružičková et al., 1996).
Usually, the populations of T. rubens in Ukraine are small, with sporadically distributed or joined in small groups (from a few individuals to several dozens) plants. These populations are mainly situated on the slopes of the southern and south-eastern exposures (Ralo, 2002;Onyshchenko & Andrienko 2012;Dmytrash, 2015;Dmytrash-Vatseba & Shumska, 2016;Konishchuk et al., 2017).
This study was aimed to clarify the distribution of T. rubens in Ukraine and its participation in plant communities. It also aimed to evaluate the current state of T. rubens populations in the Zakarpattia region of Ukraine.

Material and methods
The study was carried out in 2016-2020. Trifolium rubens distribution analysis was based on the data from the field surveys, literature, Internet resources, and examination of the herbarium vouchers from UU, KW, LWS, КWHA, CHER, MW, MNR, MSUD, UPU, SOF, UM, KWHU, KWU (see Thiers (2016) for acronyms), and KW-museum (herbarium of the National Museum of Natural History of the NAS of Ukraine, Kyiv).
Populations were studied in the Zakarpattia region in June (flowering period) and August (fruiting period). In particular, the populations in the low mountain range of the Beregovo volcanic hills and on the mountain Lovachka were investigated in the following localities: 1) Beregovo; 2) Zatyshne 3) Muzhievo, and 4) Mukachevo (Figs. 1 & 2).
Trifolium rubens is a herbaceous perennial polycarpic. Shoot system оf T. rubens is represented by elongated monocyclic shoots. The degree of development and number of shoots reflect individuals' state and prospects of seed reproduction in the population. Establishing the real age structure of T. rubens populations in natural habitats, as in some other perennial species, is difficult. It is impossible to do this without damaging plants and soil surface, especially in dense communities. Such damage is unacceptable for studying rare plant species. In this case, a ratio of the number of generative and vegetative shoots may be a key for population monitoring (Kagalo et al., 2012). Hence, in each locus, the shoots of four age stages: virginal, generative, subsenile, and senile (Gnatiuk et al., 2020), were registered and investigated.
In each population, the vegetation composition was evaluated following Braun-Blaquet (1964). The scientific names of the plants are provided according to POWO (2021), the names of the vegetation communities -according to Dubyna et al. (2019) аnd Solomakha (2008).

Distribution of Trifolium rubens in Ukraine
Basing on the analysis of herbarium material and published data, we have confirmed that T. rubens is distributed only in the right-bank part of Ukraine (Fig. 2 A). It is registered from Vinnytsia, Zakarpattia, Ivano-Frankivsk, Lviv, Rivne, Ternopil, Khmelnytsky, and Chernivtsi regions. Although according to the Red Book of Ukraine (Didukh, 2009), T. rubens is not mentioned for the Khmelnytsky and Chernivtsi regions. Most locations are known from the Zakarpattia and Lviv regions. The current presence of T. rubens in the Volyn, Zhytomyr, Cherkasy, and Kyiv regions is doubtful and/or has no recent confirmation. For example, there is no recent confirmation of the presence of T. rubens in the Kyiv region. The herbarium voucher "Zhytomyr region. Novograd-Volynskyi district, village Mala Tsvilya, June 13, 2000, Orlov" deposited at the KW was re-identified by Y.P. Didukh as T. medium L., and we agree with that new identification. The report of T. rubens from Uman (Visyulina, 1954) was not confirmed too -there are no herbarium vouchers from this region yet.

Populations of Trifolium rubens in Zakarpattia region
In the investigated areas, the fragments of oak forests and shrubs occurred. The slopes were partially terraced and planted with vineyards, gardens or used as arable fields or pastures.

Population 1. The population of T. rubens in
Beregovo (Figs. 1 & 2) was confined to a plant community with rich floristic composition and mosaic structure. In this population, five fragments (loci) were studied ( Table 1). The population was numerous, with about 300 plants. Monodominant fragments of the community were formed mainly by Саlamagrostis, Trifolium, Coronilla, and Rubus species. Plants of T. rubens occurred on the slopes of the western and north-western exposures, and the relatively flat upper part of the hills. Some individuals grew on the northern slope among trees and shrubs, and some -along the trail. The herb layer was 30-60 cm high and has 80-90 % of the projective cover. The shrub layer reaches up to 2 m high. Sometimes Quercus petraea Liebl., Populus tremula L., Castanea sativa Mill. overgrew between shrubs of Cornus sanguinea L., Prunus spinosa L., Chamaecytisus supinus (L.) Link, C. austriacus L., Rhamnus cathartica L., Rosa canina L. agg., and Rubus species (Table 2).
Shoots were concentrated in the groups (clumps) by 5-50. Each clump occupied an area of about 0.5-2.0 m 2 . The average density of shoots was 28.4 ± 1.3 per 1 m 2 . In particular, 5.2 ± 1.0 per 1 m 2 -for virginal shoots, 21.2 ± 1.4 per 1 m 2 -for generative, 0.4 ± 0.4 per 1 m 2for subsenile, and 3.6 ± 1.0 per 1 m 2 -for senile. Like all others, this population was incomplete. Shoots in the generative state predominated (Table 1). There were also some separate generative plants at a distance 20-25 m from each other. Neither seedlings nor juvenile plants were found.
Plants were scattered irregularly on the territory. Occasionally, T. rubens plants co-dominated there together with Rubus fruticosus L. agg., Coronilla varia L., Lotus corniculatus L., and Clinopodium vulgare L.

Population 2.
Near the village Zatyshne, T. rubens occurred on gentle slopes of southeastern and northern exposures with an inclination of about 20-30 ° among shrubs, along paths, and, sometimes, in the 10-15-meter ecotone of the oak forest. A rich floral composition characterized this community due to the penetration of species from adjacent plant communities with the formation of mosaic complexes and due to the lack of shading. The community consisted of deciduous shrubs with significant participation of xerothermic species. The shrub layer was up to 2 m high. The herb layer's height was 30-60 cm, and its projective cover reached 80-100 %. Calamagrostis epigejos (L.) Roth, C. arundinacea (L.) Roth, Trifolium rubens, T. medium, Melampyrum nemorosum, Clinopodium vulgare, Inula salicina L., and Vicia cracca L. predominated here ( Table 2).
Plants of T. rubens were represented by small dense clumps (1-3 m 2 ). In each clump were 2-8 generative individuals. An average density of shoots was 31 per 1 m 2 . In this population, only three loci were found with 41 shoots (Table 1).
Population 3. In T. rubens population near the village Muzhievo, two clumps formed by generative shoots were found. The population situated in the upper part of the gentle slope of the eastern exposure near thickets of shrubs with Robinia pseudoacacia L., Rosa canina agg., Rubus fruticosus agg., and Vitis vinifera L.   Hence, the density of virginal shoots in the studied populations varied from 1 to 8 per 1 m². The most abundant were generative shoots (17-68 per 1 m 2 on average). The number of subsenile shoots was 0-3 per 1 m 2 , and senile -0-2 per 1 m 2 (Fig. 3). The total number of shoots in the studied loci ranged from 41 to 165 per 1 m 2 .

Conclusions
Trifolium rubens is distributed only in the right-bank part of Ukraine. There are 78 confirmed localities of this species in Ukraine. The presence of T. rubens in Volyn, Zhytomyr, Cherkasy and Kyiv regions is doubtful and has no recent confirmations.
Studied populations in Zakarpattia region were associated with meadow-steppe areas with the participation of shrubs. They were often located on slopes and associated with xerophile semi-natural shrub communities in areas with anthropogenic impact. They were often situated along pathways, on forest edges, and associated with ecotones between forest or shrubs and herb communities from the class Trifolio-Geranietea sanguinei. In studied localities, T. rubens participated in communities with Rubus fruticosus agg., Сoronilla varia, Lotus corniculatus, Clinopodium vulgare, Trifolium medium, Melampуrum nemorosum, Inula salicіna, Vicia cracca, and Сalamagrostis species.
Apparently, metapopulational structure with the dispersed disposition of plants on the large areas is characteristic for the T. rubens populations. The distances between loci in populations varied from 10 to 100 m. All studied populations were incomplete with the advantage of generative shoots (17-68 shoots per 1 m 2 or 51.7-82.5 % from the total number of shoots). The absence of senile shoots evidences normal populations' conditions.