Extensive hybridization in Ranunculus section Batrachium (Ranunculaceae) in rivers of two postglacial landscapes of East Europe
Variation, taxonomy and distribution
In the studied Lithuanian rivers, R. aquatilis, R. fluitans, R. kauffmannii, sterile hybrids R. aquatilis × R. circinatus, R. circinatus × R. fluitans, R. circinatus × R. kauffmannii and two complex hybrids R. aquatilis ? × R. circinatus × R. fluitans were found in fast-flowing habitats, and R. circinatus was recorded in still waters (Fig. 1). Such taxa composition differs from the previously known diversity recorded for this group in Lithuania15,41,42,43,44.
Ranunculus aquatilis from the Tatula River previously was regarded as R. pseudofluitans (Syme) Newbould ex Baker et Foggitt41,43. It differs from typical usually heterophyllous R. aquatilis by the absence of laminar leaves and closely resembles R. trichophyllus but it has somewhat intermediate deeply lunate to almost circular nectar pits. The studied plants have almost the same ITS characteristics as R. aquatilis samples from Croatia and Poland but share the plastid DNA of haplotypes A (haplotype of the R. peltatus group sensu Koutecký et al.5) together with all our R. fluitans, R. circinatus × R. fluitans and the single Polish R. aquatilis sample11. Ranunculus aquatilis from the Ūla River was also previosly reported as R. pseudofluitans or R. penicillatus (Dumort.) Bab. s.l. The Ūla River morphotype is closer to typical heterophyllous R. aquatilis by its ability to develop laminar leaves and by having cup-shaped nectar pits. These plants have similar ITS sequences to the R. aquatilis sample from the Czech Republic, i.e. additive polymorphism pattern due to the presence of R. circinatus-like ITS copy in combination with R. trichophyllus B or even R. peltatus-like copy. They shared the plastid DNA of the rpl32-trnL haplotype D and petL-psbE haplotype C (haplotype of the R. trichophyllus group sensu Koutecký et al.5) together with one Lithuanian R. kauffmannii according to the rpl32-trnL marker or with all our R. trichophyllus-like samples according to the petL-psbE marker as reported for some R. aquatilis lineages from Central Europe5,11.
In their study Koutecký et al.5 demonstrated that normally R. aquatilis has the additive ITS pattern and different variants of plastid haplotypes. Samples similar to our homophyllous plants (without polymorphic ITS) they5 treated as R. trichophyllus lineage B1 with plastid haplotype of the R. peltatus group (our A haplotypes). In our study all R. trichophyllus-like samples (our R. kauffmannii and additionally included different R. trichophyllus lineages) possess ITS ribotypes corresponding to the other R. trichophyllus lineage A by Koutecký et al.5 and shared plastid haplotypes of the R. trichophyllus group (our C and D haplotypes). It appears that these remote A (R. trichophyllus-like) and B (R. aquatilis-like) lineages of R. trichophyllus represents different species, as Koutecký et al.5 have also concluded. Therefore, it is clear that Central and West European R. aquatilis-R. trichophyllus B complex needs special attention as does as all R. trichophyllus A-like forms in the Holarctic context.
Ranunculus circinatus is a common species in Lithuania, but rarely occurs in flowing river habitats, therefore it was found in only one studied river locality. These samples have the same or very similar ITS and plastid marker sequences as the recently published Central European ones5,30.
The occurrence of Ranunculus fluitans in our study was confirmed in only one locality (Nemunas River) together with the hybrid R. circinatus × R. fluitans, despite the previous assumption that this species is common in Lithuanian rivers. According to molecular data, our samples of R. fluitans are identical to those recently published for Central Europe5 and Poland30.
Ranunculus kauffmannii was not previously reported in Lithuania. It had been regarded as R. pseudofluitans or R. penicillatus in Lithuania41,43. Ranunculus kauffmannii is represented by lineages that inherited the rpl32-trnL haplotype C characteristic for eastern European R. trichophyllus-like forms with capillary leaves only and the rpl32-trnL haplotype D characteristic for more western and northern R. peltatus-like and R. trichophyllus-like forms with capillary and laminar leaves. The first lineage of R. kauffmannii was found in northeastern Lithuania (Šventoji River), and the second type was found in the western (Viešvilė River) part.
We discovered a wide distribution of hybrids in Lithuanian rivers (see below). We found the hybrid R. circinatus × R. fluitans in almost all localities where R. fluitans was previously reported. In this study, this hybrid was observed in 12 of 21 studied rivers, in which it was found in 11 cases without R. fluitans. In only one locality these species and hybrid co-occurred. In all localities, the hybrid plants were sterile, and R. fluitans acted as a maternal parent. In all previous publications, the unusual features of local R. “fluitans”, its sterility and puberulent receptacles, were specifically noted15,41,43,44.
The closely related complex hybrid plants to R. circinatus × R. fluitans were found in the Grūda, Mera and Merkys rivers. Morphologically they differ from R. circinatus × R. fluitans by more dissected capillary leaves with finer segments, smaller flowers with wider petals and cup-shaped nectar-pits resembling river R. aquatilis-like or R. kauffmannii-like plants. These plants have ITS that clearly combine copies inherited from R. circinatus and R. fluitans with additional additive polymorphisms not characteristic of both species. These polymorphisms could appear from some additional hybridization. For plants from the Grūda and Merkys rivers, this assumption was supported by plastid markers belonging to rpl32-trnL haplotype D and petL-psbE haplotype C, which are characteristic of western and northern R. peltatus-like and R. trichophyllus-like forms with capillary and laminar leaves, and haplotypes shared with R. aquatilis distributed in the Merkys River basin (the Ūla River), which could have acted as a maternal parent. Ranunculus aquatilis has been pollinated by R. circinatus × R. fluitans which are unable to produce fruits but may contribute pollen. A similar pattern was recently found by Koutecký et al.5 for Central European plants and treated by them as tetraploid R. penicillatus B. Plants from the Mera River possess plastid markers of haplotypes A shared with R. fluitans as well as some lineages of R. aquatilis and R. peltatus. These hybrid plants have the ITS sequences clearly influenced by the R. aquatilis lineage (with R. peltatus-like copy), therefore it is most likely that R. aquatilis with haplotypes A was a maternal species in this hybrid too, as in the above mentioned example.
Another hybrid discovered in Lithuania is R. aquatilis × R. circinatus. These sterile plants occurred only in one locality in the southeastern part of Lithuania (Gauja River). They originated from the crossing of the R. aquatilis lineage which was found not far away, in the Ūla River of the Merkys River basin. Plants from the Gauja River are morphologically and genetically similar to plants from the Ūla River. But while having very similar hybrid ITS and plastid DNA, the former are sterile and the latter are fertile. The Ūla River R. aquatilis possess polymorphic hybrid ITS due to combination of R. circinatus-like and possibly R. peltatus-like copies, therefore they already inherited R. circinatus-like copy. Additional hybridization with R. circinatus gave more prominent peaks of this species on the ITS electrophoregrams from the Gauja River samples. Ranunculus aquatilis acted as a maternal parent in the formation of this hybrid. There is another explanation for the similarity of these plants. The Gauja River plants could originate from a crossing of R. peltatus and R. circinatus and gave the sterile triploid (2n = 3x = 24) hybrid. In a previous study15, we demonstrated that the Ūla River plants have 2n = 6x = 48, therefore they could evolve from this triploid hybrid in the result of the whole genome duplication. But we can get the pros and cons for this explanation only by counting chromosome or estimation genome size, as in the case of the hybrid plants from the Skroblus River15 of the same basin.
In previous morphological and ISSR analyses41 based on specimens from some of the same river localities, samples were split into two groups. This result corresponds well with the present data on morphology (Table 1) and molecular marker variation (Figs. 2, 3, 4, Supplementary data: Tables S1—S4). The first group consists of R. fluitans, R. circinatus × R. fluitans and the complex hybrid R. aquatilis ? × R. circinatus × R. fluitans, which are concentrated in the Neris River basin, and the second group consists of R. aquatilis, R. aquatilis × R. circinatus and the complex hybrid R. aquatilis ? × R. circinatus × R. fluitans, which are concentrated in the Merkys River basin and adjacent territory.
In the studied Central European Russian rivers, Ranunuculus kauffmannii and the sterile hybrid R. circinatus × R. kauffmannii were found in fast-flowing parts, while R. circinatus and the hybrid R. circinatus × R. kauffmannii were collected in still waters (Fig. 1). Such taxa composition differs slightly from the previously reported diversity for the group in the area45,46.
Ranunculus circinatus is a common species in Central European Russia, but avoids flowing waters, therefore in the studied rivers it was found in four localities. According to molecular data our samples of R. circinatus more variable than those recently published for Central Europe5 and Poland30.
Ranunculus kauffmannii is a common species in Central European Russia that is characteristic of fast-flowing river habitats. In this region, R. kauffmannii is represented by only one lineage that inherited the rpl32-trnL haplotype C characteristic for eastern European R. trichophyllus-like forms with capillary leaves only. No other variants have been found in the studied area. In the closely situated Ild and Sutka rivers, some samples contained a long copy of ITS, which has a duplication of CCCCA in position 401—405. These plants occur together with plants without such duplication in ITS in the same river and locality. In the studied rivers, R. kauffmannii was found in 12 localities.
We discovered a wide distribution of hybrids in rivers of the Upper Volga basin (see below). We found the hybrid R. circinatus × R. kauffmannii in only one locality of one river (Uleima River) together with R. kauffmannii, where R. kauffmannii and R. circinatus grew close to each other, upstream and downstream respectively. In two localities (Urdoma, Vostra rivers), only hybrids occurred. We also found hybrids with R. circinatus in two localities (Korozhechna and Shizhegda rivers). In all localities, the hybrid plants were sterile, and R. kauffmannii acted as a maternal parent. According to hybrid ITS variation hybrids in each locality share ITS copies of local or closely related populations of parental R. circinatus and R. kauffmannii. In the studied rivers, we have not found R. circinatus or its hybrids inherited an ITS ribotype identical to sample MZ338333 from relic postglacial Plescheevo Lake.
The hybrid R. circinatus × R. kauffmannii was previously reported from rivers in the Upper Volga region as R. circinatus × R. trichophyllus45. Moreover, according to the herbarium collection in the IBIW, a significant portion of river specimens identified as R. trichophyllus could actually be R. circinatus × R. kauffmannii, especially from the rivers where R. circinatus and R. kauffmannii co-occurred.
We may conclude that the Batrachium diversity differs in the rivers of Lithuania and Central European Russia despite similarity of morphotypes. In the former area, the West and Central European forms R. aquatilis, R. fluitans and their hybrids are dominant and represented by a larger number of taxa (4 species and 3 hybrids) whereas in the latter, the East European species R. kauffmannii and its hybrid are the only dominant forms with less total diversity (2 species and 1 hybrid). Althought both areas share R. circinatus and R. kauffmannii, the former is common and abundant in both areas, while R. kauffmannii become rare westward.
The rpl32-trnL region proved to be a highly variable marker with good resolution in Batrachium, therefore it is deservedly used for phylogenetic studies of the section5,11,15. We found that the petL-psbE region has significantly less resolution, and we do not recommend it for further studies although it was succesfully used in some previous works11.
Rivers as “evolutionary incubator”
The important role played by hybridization in increasing the taxonomic diversity, speciation and evolution of Batrachium has been reported and discussed in many publications3,4,5,6,7,8,9,10,11,12,13. However, molecular tools for hybrid identification have been applied in few papers4,5,10,11,12,13. Among these papers, only three4,5,30 reported both the existence of hybrids and also their frequency among the studied samples. For example, Prančl et al.4, used flow cytometry and chromosome number data and evaluated 16 cytotypes of hybrid origin (seven identified and the rest unidentified) for Central Europe. In a subsequent study completed by genetic data and with extended data set, Koutecký et al.5 detected four more unidentified hybrids. Both studies determinated that not less than 15% of the individuals were hybrids. In Poland, Gebler et al.30 reported hybrid R. circinatus × R. fluitans for 10 rivers of 49 rivers studied (> 20%), and 13 this hybrid clones were found among 58 populations studied (> 22%).
We demonstrated that in Lithuanian rivers, among 66 studied Batrachium individuals, 18 represent species from six localities, and 48 represent hybrids from 16 localities; in only one locality species and its hybrid co-occurred. Therefore, hybrids make up 73% of individuals from 76% of localities, and only hybrids occupied 71% of localities.
In Central European Russian rivers, among 36 studied Batrachium individuals, 23 represent species from 16 localities, and 13 represent hybrids from six localities; in three localities, species and hybrids co-occurred. Therefore, hybrids make up 36% of individuals found in 32% of localities. In 16% of localities only hybrids were found.
These data are in accordance with the results by Prančl et al.4 and Koutecký et al.5, and in fact demonstrate a much wider distribution and larger role of Batrachium hybrids in river ecosystems. We should note that we summarized only sterile hybrids, while in rivers in fast-flowing habitats hexaploid R. aquatilis and tetraploid R. kauffmannii occur in Lithuania, and tetraploid R. kauffmannii 47 are the most common and abundant in the Upper Volga basin, which originated in the result of crossing of different species lineages.
The wide distribution and abundance of Batrachium hybrids and polyploids in the rivers of two distant areas suggest that rivers and streams play a role as “evolutionary incubators” for such newly arising hybrids and polyploids4.
We propose several explanations for this fact based on the studied rivers. First, rivers are favorable for hybrids and allopolyploids as they are “open environments”18,48, because in newly created habitats, which are frequently developing in river ecosystems, hybrids have a good chance to originate and survive. This is true for Batrachium diversity in Europe1,4,5,12,13,16,30,49. We have seen this also on the example of Potamogeton and Stuckenia in the rivers of Lithuania and the Upper Volga region, where hybrids exhibit a significant diversity and abundance35,50,51, as well as in other areas21,33.
The second source of hybridization occurs when species grow near the edges of their distribution range and occupy specific habitats representing extralimital or marginal populations which often show reduced competitive ability. In this case, they may be vulnerable to being lost to hybridization with more common and abundant local and tolerant species18,20, especially in response to ongoing climate change52,53,54,55. Ranunculus fluitans is growing in Lithuania near the eastern edge of its range1 and has an ecological optimum in permanently flowing waters of relatively large rivers with stable pebble bottoms in nonlimestone areas3. In Lithuania, it grows in the Nemunas River and most likely in the Neris River basin in a similar but less optimal environment44.
The next, Alahuhta et al.62 found that current climate (especially mean annual temperature) was the main environmental driver of aquatic plant species richness in Europe and North America. In the North-West of European Russia, which is situated almost between studied areas, the mean annual temperature began to exceed the climatic norm by 0.9 to 1.2 °C per decade since 198963. Such a large increase of mean annual temperature may affect cold tolerant species53,54, i.e. river R. fluitans and R. kauffmannii. We have observed R. kauffmannii populations in several rivers in the Upper Volga region since late 1990s and can conclude that the number and area of populations has decreased more than two times. Climate change can promote the success of hybrids, not their origin, but rather their expansion.
The glaciation history of the landscapes is closely related to the previous two factors21. In postglacial areas of northern Europe, a number of sterile hybrids of aquatic vascular plants occur outside the modern ranges of parental species, e.g., Nuphar56, Potamogeton57,58 and Stuckenia36,59. The hybrids can survive better in postglacial areas compared to their parents since last glaciation by being more vigorous and adaptive. The studied rivers in Lithuania and the Upper Volga area were mostly covered with ice or were in the edge zone during the last Valdai (Würm, Weichselian) glaciation60. Therefore, the studied rivers predominantly run through a fluvioglacial landscape in the marginal zone of this glaciation. Wiegleb (pers. comm.) reported that the hybrid R. circinatus × R. fluitans is characteristic for glacial areas in Central Europe. There in the inflow and outflow regions of glacial lakes R. circinatus and R. fluitans can coexist despite different ecology. This was illustrated by Vollrath and Kohler49 in Bavaria and Gebler et al.30 in Poland in the Drawa River.
The last factor for hybridization is human impact18,20. All studied rivers are affected by pollution and eutrophication, the construction of dams and bridges, and the straightening of channels, which have severely changed the environmental characteristics of the rivers and destroyed the ecological barriers between species50,61. In West and Central Europe, the connection of formerly separate river catchments by canals, which occurred in most countries in the late 18th and throughout the nineteenth century, could served as a major trigger of hybridization. Such transformations of river ecosystems usually reduce the abundance of fast flowing water species (R. fluitans, R. kauffmannii) at the expense of still water species (R. circinatus). For example, Wiegleb [pers. comm.] reported that putative hybrid R. circinatus × R. fluitans occurs outside glacial deposits in highly modified water bodies in Great Britain, e.g., in the Stour River.
Therefore, we think that the wide distribution and abundance of Batrachium hybrids in the rivers of Lithuania and Central European Russia arose, first of all, due to the specificity of rivers as an open-type ecosystems and then were intensified by transformations of river ecosystems resulting from human activities. Evidently, the postglacial character of the studied river landscapes and ongoing climate change also have significant effect.
Moreover, the hybrid genomes provide greater plasticity of plants and, ultimately, the possibility of rapid adaptation to variable river conditions. Thus, hybrids are able to grow in fast and quiet currents in different bottom sediments, which ensures greater ecological and biological benefits in comparison to the parental species.
Why only Ranunculus circinatus is a pollen donor?
Most hybrids of R. aquatilis, R. fluitans and R. kauffmannii had R. circinatus as a pollen donor; all hybrids originated from unidirectional crossing. The same pattern was found for Central European R. circinatus × R. fluitans5,30.
A similar case was reported by Prančl et al.4 for R. peltatus × R. trichophyllus, when unidirectional gene flow might be driven by a large difference in flower size: the large-flowered R. peltatus is more likely to be a pollen donor than the small-flowered R. trichophyllus. The small-flowered Utricularia tenuicaulis Miki served mostly as maternal species in hybridization with the large-flowered U. macrorhiza Leconte64. The same explanation could be applied in our case, but there was no difference between flower size in R. circinatus and R. aquatilis or R. fluitans, and the flowers of R. circinatus were slightly larger than the flowers of R. kauffmannii (Table 1). Moreover, all Batrachium species pollinated by insects and their hybrids can potentially originate in bidirectional crossing. Some additional factors should drive such a crossing direction.
We also suspect difference in the frequency of the parent species where more rare species are under strong introgression pressure by their more frequent neighbors65,66,67. In Lithuania, R. circinatus occurs more frequently and in more abundant populations than river R. aquatilis and R. fluitans. In the Upper Volga region, R. kauffmannii is as generally frequent and abundant species as R. circinatus, but in areas closely situated to the reservoirs of the Volga River cascade, the latter is clearly a more abundant species occupying large areas of the reservoir’s bays and shallow waters.
In Lithuania, the flowering period of R. aquatilis, R. fluitans and R. circinatus is usually similar. In the Upper Volga region, the flowering period of R. kauffmannii usually starts and finishes at least two weeks earlier (the beginning of June—the beginning of July) than R. circinatus (the middle of June to the beginning of August), and the flowering peaks of these species (the middle—the end of June and the end of June to first half of July, respectively) usually do not overlap each other (AB, EC observations). Therefore, R. kauffmannii is usually in full bloom and ready for pollination when R. circinatus only begins to flower and produce pollen, and not vice versa.
In addition, the asymmetric hybridization can be explained by self-compatibility mechanisms68, where a self-compatible species can be successfully crossed with pollen from a self-incompatible species, whereas the opposite configuration cannot succeed. In Lithuania, R. aquatilis and R. circinatus normally develop numerous fruits, but R. fluitans does this very rarely.
We have checked herbarium collections of R. kauffmannii and R. circinatus from the Upper Volga region in IBIW and found that almost all mature specimens of R. kauffmannii bear numerous fruits on almost all pedicels whereas only some mature specimens of R. circinatus have fruits on only some pedicels. Indirectly this may suggest that the former species is a self-compatible species and the latter is a self-incompatible species. Such asymmetric crossing was found for Utricularia tenuicaulis and U. macrorhiza64.
In the case of complex hybrids of Ranunculus aquatilis with R. circinatus × R. fluitans, more rare R. aquatilis has been pollinated by the more frequent and abundant R. circinatus × R. fluitans which is able to produce fertile pollen.
Therefore, different factors could determine the unidirectional crossing in different areas. Thus, frequency and abundance of populations in Lithuania with slightly more mild climate completed by the difference in flowering period and maybe a level of self-compatibility in the colder Upper Volga region could be the main explanations why almost always the rigid-leaved species R. circinatus acted as a pollen donor in case where parental species share common pollinators and have similar flower morphology.
Descriptions of new taxa
The taxonomy of Batrachium is not resolved yet because diversity of this groups consists of diploid species, autopolyploids, allopolyploids, cryptic species, primary hybrids and introgressants5. To avoid this complex pattern we should first resolve and describe well-defined diploid and polyploid species and clearly evidenced primary hybrids, which will be necessary for further resolution of more complex forms and solving old nomenclatural confusions.
Ranunculus × redundans A. A. Bobrov, Butkuvienė et Sinkevičienė nothosp. nov. (R. circinatus × R. fluitans)
Lithuania, Varėna Distr., Nemunas River upstream of Merkinė, rapid, flow edge with weak current, 54.148938° N, 24.18015°, 22.06.2020, Butkuvienė J., Sinkevičienė Z. (holotype: BILAS92889 sheet 1, BILAS92890 sheet 2). As paratypes we can designated all the rest specimens of the hybrid listed in Supplementary data: Table S1. For image of the holotype see Supplementary data: Fig. S1.
Only genetically tested plants were selected as holotype and paratypes.
Hybrid differs from R. circinatus by much longer and more flaccid capillary leaves with fewer terminal segments, flowers with narrower petals, and an almost glabrous or puberulent receptacle; from R. fluitans by shorter and more rigid leaves with more numerous terminal segments, flowers with wider petals, and puberulent receptacle; it is sterile, some flowers have rugose or deformed petals, pedicels are elongating and rapidly decaying just after flowering. Hybrid more resembles R. fluitans but hybrid plants are of dark green color not fresh to light green as is the species, and the hybrid usually develops short axillary side shoots in the late vegetation period, which are adnate to the stem or the adjacent petiole bearing small R. circinatus-like leaves.
Hybrid differs from morphologically similar forms such as R. penicillatus and R. schmalhausenii Luferov by absence of laminar and intermediate leaves with the middle lobe just slightly shorter than the lateral ones, fewer capillary leaf lamina divisions and number of segments, from R. pseudofluitans and R. vertumnus (C. D. K. Cook) Luferov by less number of capillary leaf lamina divisions and number of segments and almost glabrous receptacles.
See Table 1.
The taxon’s epithet refers to “niche equivalent” or “vicariant” because the hybrid closely resembles and actually mimics R. fluitans, and was previously regarded as R. fluitans.
This hybrid was first reported from southern Germany by Vollrath, Kohler46 but without a binominal name. It was confirmed for Germany based on genetic data by Koutecký et al.5 and for Poland by Gebler et al.30. It is possible that this hybrid is not rare in the area where both parental species occur (West, Central and western East Europe). For example, Wiegleb [pers. comm.] reported such a hybrid from Great Britain (the Stour River).
Wiegleb et al.1 listed this taxon under R. pseudofluitans following the British tradition. However, the type specimen of R. aquatilis var. pseudofluitans Syme (lectotype, BM000057127) is not this taxon but resembles the hybrid of R. fluitans with R. peltatus-like or R. trichophyllus-like forms without laminar leaves.
Ranunculus × absconditus A. A. Bobrov et Chemeris nothosp. nov. (R. circinatus × R. kauffmannii)
Russia, Yaroslavl Reg., Tutaev Distr., near Vypolzovo village, Urdoma River, downstream bridge, 57.941523° N, 39.495951° E, 23.06.2013, Belyakov E. A. (holotype: IBIW71591; isotypes (currently at IBIW, to be distributed to several other herbaria): IBIW71592–71605). As paratypes we can designated all the rest specimens of the hybrid listed in Supplementary data: Table S1. For image of the holotype see Supplementary data: Fig. S2.
Only genetically tested plants were selected as holotype, isotypes and paratypes.
Hybrid differs from R. circinatus by longer and more flaccid capillary leaves, and usually smaller flowers with narrower petals; from R. kauffmannii by shorter and more rigid leaves, and usually larger flowers with wider petals; it is sterile, some flowers can be underdeveloped with some rugose or deformed petals, pedicels are elongating and rapidly decaying just after flowering.
Hybrid differs from morphologically similar hybrid R. circinatus × R. trichophyllus by longer leaves and larger flowers, also it occurs only in river habitats or clearly connected to river in comparison to a wide range of stagnant water bodies characteristic of R. circinatus × R. trichophyllus.
See Table 1.
The taxon’s epithet refers to “hidden” because the hybrid resembles some stages of R. kauffmannii and some lineages of R. trichophyllus to which it was previously frequently but wrongly attributed, and it is difficult to recognize by morphological characters without molecular analysis.
This hybrid was already reported without description and binominal name from the Upper Volga, European Russia, and Poland (see1). It is possible that this hybrid is not rare in the area where both parental species occur (mainly, boreal part of East Europe).
Wiegleb et al.1 listed the similar hybrid R. circinatus × R. trichophyllus from several countries in West, Central and East Europe, where it was recognized based on morphological characters. Taking into account the situation with cryptic lineages of R. trichophyllus found by Koutecký et al.5 and discussed above, all these records needs evaluation using molecular tools.