Ranunculus dahlgreniae (section Batrachium , Ranunculaceae), a new species from Crete, Greece, with remarks on taxonomy and phylogenetic relations within the section

A new species, Ranunculus dahlgreniae , is described from a seasonal lake at Omalos Plateau, Lea Ori Mountains, western Crete, Greece. e heterophyllous species resembles R. saniculifolius, R. peltatus , and the Mediterranean forms of R. baudotii . It diﬀers from the aforementioned species by a combination of characters not found in any of them, i.e., 5–6 mm long petals, up to 2.2 mm long, glabrous achenes with a partly persistent style, a densely pubescent, in fruit slightly elongating receptacle, and intermediate leaves with rigid filiform apical segments, being divided into two or three cuneate, shortly petiolate leaflets. A key to all similar East Mediterranean taxa is presented. DNA analyses based on the sequencing of nuclear, ribosomal Internal Transcribed Spacer (ITS), and two chloroplast noncoding regions ( rpl 32 -trn L and psb E -pet L), complemented by the analysis of genome-wide polymorphism using double digest RAD Sequencing (ddRADseq) supported that Ranunculus dahlgreniae is a distinct lineage, clearly separated from R. peltatus, R. baudotii , and R. saniculifolius . e phylogeny based on ddRADseq resembles the topologies obtained from chloroplast and nrITS data but with increasing resolution and support of fine-scale relationships. Extensive sampling, including taxa from temperate Europe and the West Mediterranean area, as well as the application of reduced-representation sequencing, allowed to better understand the pattern of diversity in the section Batrachium .


Introduction
Ranunculus L. section Batrachium DC., hereaer referred to as Batrachium, is a monophyletic group of aquatic plants within the morphologically and ecologically diverse genus Ranunculus L., Ranunculaceae Juss. (Hörandl & Emadzade, 2012). In the latest worldwide taxonomic account of the section, 30 species were recognized (Wiegleb et al., 2017). e section is regarded as taxonomically challenging due to a limited number of taxonomically informative traits, considerable plasticity, hybridization, and polyploidization (summarized in Koutecký et al., 2022;Wiegleb et al., 2017). In earlier comprehensive taxonomic treatments of Batrachium (Cook, 1966;Pizarro, 1995;Tzvelev, 1998;Wang & Tamura, 2001;Whittemore, 1997), species delimitation was mainly based on extensive analysis of morphological characters, in some instances complemented by karyological, phytogeographic and ecological observations. However, due to differing views on the taxonomic importance of traits, species delimitations showed considerable divergence. A striking example is the differential use of the binominal 'R. penicillatus' in the monographic works of Cook (1966) and Pizarro (1995), respectively. Moreover, species boundaries are blurred by ongoing hybridization and introgression, which may not allow defining boundaries between newly created, mostly sterile hybrids and allopolyploid species originating from the same parentage.
e Mediterranean Basin hosts ca. 25,000 vascular plant species, of which about 5500 are endemic, rendering the region one of the major hot spots of global plant biodiversity (Lopez-Alvarado & Farris, 2022). e unique spatial patterns of the Mediterranean region in terms of a complex climatic and geological history, biotic interactions, migratory bird flyways, small-scale habitat heterogeneity, isolation effects on islands and mountain ranges, as well as thousands of years old anthropogenic pressure are considered driving forces of observed diversity (Nieto Feliner et al., 2023). Species of the section Batrachium mainly occurring in the northern hemisphere, show the highest diversity in Western Europe, followed by Eastern Europe, West Asia, and North Africa (Cook, 1966;Wiegleb et al., 2017). e Mediterranean region covers all these areas. us, we expected the region to be a rewarding research field for the study of taxonomic diversity and microevolution of the section. e taxonomic diversity of Mediterranean Batrachium is still poorly understood. Plants collected by early botanists in the region were regarded as conspecific with taxa described from temperate and boreal Europe, e.g., the heterophyllous taxa R. aquatilis L., R. peltatus Schrank, R. baudotii Godr., or R. penicillatus (Dumort.) Bab. More recently, Mediterranean plants were oen treated as subspecies or varieties of northern species, e.g., R. aquatilis var. marizi (Coutinho, 1939) and R. peltatus var. microcarpus (Meikle, 1959). Simultaneously, deviant Mediterranean morphotypes were partly regarded as species in their own right, e.g., R. saniculifolius (Viviani, 1824), R. fucoides and R. leontinensis (Freyn, 1880), R. macranthus (Lojacono-Pojero, 1889), and R. castamonuensis (Dönmez, 2002).
West Mediterranean (Iberian) Batrachium was summarized by Cook (1986), Valdés et al. (1987), Velayos (1988), Pizarro (1995), and Cirujano Bracamonte et al. (2014). A closer look at their respective classification approaches shows profound differences in taxa delimitation. For this reason, the chromosome counts of Diosdado et al. (1993) cannot be properly assigned to taxa we consider valid nowadays. Taxa described by Maire (1964) from North Africa do not correspond to any of the aforementioned Iberian treatments. For the Central Mediterranean region, no synthetic approach except Pignatti (2017) is available. e treatments of Istrian (Englmaier, 2014), Corsican (Jeanmonod & Naciri, 2021), Sardinian (Desfayes, 2008), and Sicilian Batrachium (Giardina et al., 2007) all adopted divergent species concepts. East Mediterranean Batrachium was studied by Meikle (1959: West Asia;1977: Cyprus), Cook (1965: Turkey), and Hand et al. (2011). In the 1990s, Batrachium diversity of the Aegean islands and continental Greece was comprehensively analyzed by Dahlgren (1991Dahlgren ( , 1992, Dahlgren and Svensson (1994), and Fiasson et al. (1997), including morphological, karyological, and phytochemical studies. Dahlgren's findings were the basis for the Batrachium treatment in Flora Hellenica (Dahlgren, 2002). Dahlgren's species concepts were strongly influenced by the circumstances typical for southern Sweden and Denmark, where she had worked before. In worldwide accounts (Cook, 1966;Wiegleb et al., 2017), Mediterranean Batrachium diversity was just touched upon in general terms. Several of the described and undescribed morphotypes known from herbaria remained unresolved. A striking example is R. fucoides Freyn, which was classified as 'incertae sedis' in both papers.
With the emergence of molecular techniques and a growing interest in integrative taxonomic approaches, the delimitation of Batrachium species started to become verified by Sanger sequencing of DNA markers (e.g., Bobrov et al., 2015Bobrov et al., , 2022Lansdown, 2007;Telford et al., 2011;Zalewska-Gałosz et al., 2015), and genome size estimates (Koutecký et al., 2022;Prančl et al., 2018). Phylogenies based on the molecular characters showed a complex reticulate pattern of Batrachium evolution (e.g., Bobrov et al., 2015;Gebler et al., 2022;Koutecký et al., 2022), and allowed confirmation of hypotheses on phylogenetic relations beyond morphological analyses. Application of selected, multicopy DNA markers turned out to be useful in species and hybrid identification, however, for some species complexes, e.g., R. aquatilis/R. trichophyllus, R. peltatus/R. penicillatus agg. or R. baudotii, remained still indecisive due to low resolution. In these cases, the genome size estimates obtained by flow cytometry (FCM) are helpful since the genome size is characteristic of the individual taxa (Koutecký et al., 2022;Prančl et al., 2018). e FCM method, however, demands fresh tissues, and, for tracing hybridization and polyploidization events, it must be complemented by classical chromosome counts.
Contradicting nomenclatural approaches and a partly unresolved position of several Batrachium taxa indicate that classification solely based on morphology can oen be misleading (Koutecký et al., 2022). Wiegleb et al. (2017) tried to develop an 'enlightened' morphological species concept for Batrachium. Species definitions were implicit and based on keys and species descriptions. Genetic and karyological information was used in addition if they supported morphological delimitation. e integration of molecular and morphological traits is nowadays more oen used for species delimitation. In line with this practice, Hong (2020) proposed a new 'gen-morph species concept' that is theoretically objective and practically operable.
Considering the above, in this study, we apply an integrative morphological and genetic approach to selected European and Mediterranean Batrachium taxa. For the first time in Batrachium studies, we make use of novel high-throughput sequencing techniques that have opened new perspectives in plant taxonomic studies (Hörandl & Appelhans, 2015) and combine them with "classic" genetic markers generated by Sanger sequencing. We applied one of the reducedrepresentation sequencing techniques, namely the ddRADseq method, which generates genome-wide polymorphism data (Andrews et al., 2016).
e main aim of the present paper is to describe a new species, Ranunculus dahlgreniae, from Crete (Greece) in terms of integrative taxonomy. In addition, we aim to elucidate the morpho-genetic divergence within polymorphic species, such as R. baudotii, by comparing Batrachium phylogeny based on Sanger-sequenced nuclear (ITS) and cpDNA with the newly generated ddRADseq phylogenomic data. Finally, we shortly outline taxonomic and nomenclatural problems resulting from our work for the future treatment of Mediterranean Batrachium taxa.

Plant material
Reference herbarium specimens of Batrachium taxa used for morphological and molecular studies are deposited in the Herbarium of the Institute of Botany, Jagiellonian University, Krakow (KRA). Plants collected in Denmark and Bavaria (Germany) in 2013 are deposited in the Oldenburger Landesmuseum für Natur und Mensch (LMO). Plant materials were collected in a responsible manner and exported in accordance with relevant permits and local laws.

Morphological evaluation
Morphological observations were made on fresh as well as herbarium material. Mediterranean specimens were collected during Mediterranean field trips to Croatia (2012Croatia ( , 2016Croatia ( , and 2018, Crete (2018), Spain (2021), and Portugal (2022). Measurements in the Description and in Table S2 refer to dried material. GW studied Batrachium specimens in B (Flora Hellenica collection, R. & E. Willing collection from Greece, Flora of Cyprus collection, personal collections R. Hand, N. Hadjikyriakou); GLM (incl. the Senckenberg Collection from FR); GOET (type material of R. fucoides), JE (material from Libya), LD (Gertrud Dahlgren collection, Flora Hellenica collection), LMO (own collection from Sardinia, J. Lambinon specimens from Corsica), MA (comprehensive Iberian collection), MAF (Iberian collection and private herbarium of J. Pizarro), STU (various Mediterranean specimens), and the private herbarium of B. Biel (Würzburg). JZG studied herbaria specimens in COI (Portuguese collection and Moritz Willkomm's Historical Herbarium), K, H, and FRU. Additionally, Batrachium specimens were traced in virtual herbaria, photo portals, databases, and literature sources. In the present paper, only images retrieved from GBIF were used for taxonomic decisions. Observations and capture of images were performed using a dissecting binocular microscope (Zeiss Stemi SV 11, Germany) that was equipped with a digital camera (Canon EOS 760D).

Genomic DNA extraction, PCR amplification, and Sanger sequencing
Fresh leaves of Batrachium individuals representing studied taxa were collected and stored in small plastic tubes filled with silica gel. Altogether, 191 Batrachium samples were analyzed at the molecular level; detailed information is summarized in Table S1.
Between 10 and 18 mg of dried plant material was used for DNA isolation. e plant tissue was ground to a fine powder using an MM 400 (Retsch) mixer mill and 3-mm tungsten beads. Total genomic DNA was extracted using the Plant Mini Kit (Qiagen), following the manufacturer's protocol. e nuclear ribosomal Internal Transcribed Spacer region (including ITS1, 5.8S, and ITS2) was amplified and directly sequenced as described in Ronikier (2010, 2012). Additionally, two non-coding plastid spacers (cpDNA): rpl32-trnL and psbE-petL, were investigated to detect potential differences in the haplotypes among taxa. Amplification of cpDNA regions as well as sequencing was performed as described in Zalewska-Gałosz et al. (2009. All sequences were submitted to GenBank (Table S1). Sequences were manually verified based on the forward reads or, in the case of intraindividual polymorphism, on the reads of both sequencing directions (forward and reverse). Alignments were prepared using BIOEDIT 7.0.5. (Hall, 1999). Polymorphic nucleotides in the ITS region were coded using IUPAC ambiguity codes. e two alignments, (1) ITS and (2) two concatenated regions of cpDNA, were trimmed according to the shortest sequence. Accessions that yielded identical ITS or cpDNA sequences were represented in the phylogenetic analyses by the single terminals, as described in Gebler et al. (2022), except for R. dahlgreniae. To obtain better clarity of phylogenetic visualization, intraspecies differences in sequences belonging to the same species, univocally, morphologically determined, and located in the same clade, were presented as a consensus sequence, using IUPAC code in polymorphic positions. Single ribotypes and haplotypes were identified by different numbers listed in Table S1. In cases where different ribotypes and haplotypes were observed within one species, it was specified with appropriate abbreviation (i.e., R. baudotii r1 cp1 and R. baudotii r3 cp3; Table S1). For R. dahlgreniae samples, the number of species-specific substitutions in the ITS region was calculated using the Fastachar program (Merckelbach & Borges, 2020).

Phylogenetic analyses based on the markers obtained in a Sanger sequencing
e ITS data were visualized as a phylogenetic network using SplitsTreeCE 6.0.0_alpha soware (Huson & Bryant, 2006). e Hamming Distances Ambiguous States method (Hamming, 1950) was used to obtain a distance matrix. Splits were computed using the NeighborNet algorithm (Bryant & Moulton, 2004) , 2012). e evolutionary distances were computed using the HKY substitution model, which was chosen based on the lowest BIC (Bayesian Information Criterion) score computed in MEGA X (Kumar et al., 2018). e analysis was performed using two independent runs with four Markov chain Monte Carlo (MCMC) chains running for 10 5 generations. Trees were sampled aer every 100th generation, and the first 25% of trees were discarded as the burn-in phase. e remaining trees were used for the construction of a 50% majority consensus tree. Ranunculus sceleratus L. was used as an outgroup, with three sequences acquired from GenBank: MW430773 (ITS), KC842129 (rpl32-trnL), and KC842059 (psbE-petL), trimmed to the required length prior analyses.

ddRADseq
RADseq libraries were prepared using the protocol (Suchan, 2020) and sequenced using Illumina HiSeq (150 bp singleend reads). Reads were demultiplexed with process_radtags from stacks 2.53 (Rochette et al., 2019). Further, analyses were performed with dDocent (Puritz et al., 2014) using mostly default parameters, except for the Minimum within individual coverage level to include a read for assembly (K1), which was set to 6 and the Minimum number of individuals a read must be present in to include for assembly (K2) set to 7. SNP calling was done with FreeBayes (Garrison & Marth, 2012) using the default parameters of the dDocent pipeline and assuming the diploidy of all samples. Genotypes with coverage below 5 or genotype quality below 20 phred were set to missing, and only the markers with less than 50% missing data were used in phylogenetic analysis. e pairwise genetic distances between samples were calculated using function dist.gene() from ape (Paradis et al., 2022) using the pairwise deletion option, and the Neighbor-Joining tree was constructed using function nj() in ape. e tree was rooted with R. sceleratus. e robustness of the obtained relationships was tested with 1000 bootstrap replicates with boot.phylo() from ape. e tree was graphically visualized in the FigTree program v.1.4.4 (http://tree.bio.ed.ac.uk), and aesthetically improved in CorelDraw 2021.

Molecular survey
Phylogenetic relations among Batrachium taxa were reconstructed in three ways, based on different sets of molecular markers: (1) Bayesian analysis based on two, concatenated cpDNA spacers: rpl32-trnL and psbE-petL, (2) the ITS Neigh-borNet network, and (3) Neighbor Joining based on genetic distances between individuals obtained from SNP genotypes of ddRADseq data. e alignment of two, concatenated chloroplast DNA spacers: rpl32-trnL and psbE-petL, was 1074 bp long. It contained 44 substitutions (28 of them potentially informative) and six 1-10 bp long indels. 23 haplotypes were distinguished among Batrachium samples based on the polymorphism detected (Table S1). e haplotype detected in R. dahlgreniae was very similar to haplotype cp3, which is widely distributed and shared by different Batrachium species, i.e.: R. aquatilis, R. fluitans, R. baudotii r3, R. peltatus, R. penicillatus A, R. saniculifolius, R. schmalhausenii, however, differed from cp3 by one mutational step in a position 865 of the alignment. In the phylogenetic tree based on plastid regions, four main, well-supported evolutionary groups were recognized ( Figure 1). e ITS alignment of the whole data set was 573 bp long. ese consisted of 43 substitutions (30 potentially informative) and two 1-bp long indels. Based on the polymorphism detected, 84 ITS ribotypes were distinguished (Table S1). e ITS sequence of R. dahlgreniae did not show any individual polymorphism. It was the most similar to the ribotypes of R. fluitans and R. baudotii r1 and r3 (but not r2). e R. dahlgreniae ribotype differed from the ribotype of R. fluitans by four substitutions (76th, 181st, 319th, 554th position of the alignment), from the ribotype of R. baudotii r1 by five substitutions (43rd, 48th, 181st, 192nd, 319th position of the alignment), and from the ribotype of R. baudotii r3 by three substitutions (86th, 181st, 319th position of the alignment). Adenine in positions 181 and 319 of the alignment was unique for R. dahlgreniae in comparison to ribotypes of the most similar species. Two species-specific substitutions were confirmed using the Fastachar program (Merckelbach & Borges, 2020). e phylogenetic ITS network showed five genetic groups comprised of more than one species. Additionally, two monospecific branches with the species of hybrid origin: R. aquatilis and R. penicillatus A were distinguished. ese branches were located between groups to which their parents belonged ( Figure 2). All species were spaced in a line with their taxonomic affiliations except for R. trichophyllus and R. baudotii. Six ribotypes of R. trichophyllus were distributed among three different groups. One of the groups comprised three R. trichophyllus ribotypes, although each of them had a different topology. ree ribotypes of R. baudotii were split between two groups ( Figure 2).
In ddRADseq, we obtained 8966 markers with less than half missing data, and the fraction of missing data per sample ranged from 0.06 to 0.64 (mean = 0.234, SD = 0.131).
3.1.1. Batrachium phylogeny based on three different molecular data sources e topologies of Bayesian analysis (plastid DNA regions), NeighborNet Network (nrITS), and Neighbor-Joining (dd-RADseq genotyping) were mostly congruent. Main genetic groups, marked in yellow, green, red, blue, and orange, were well-supported in all three analyses (Figures 1-3). Neighbor-Joining phylogenetic tree based on ddRADseq SNP data had the best resolution and turned out to be phylogenetically and taxonomically the most informative. In this tree, not only well-supported groups of taxa but also individual species were well-supported. e first phylogenetic group (yellow) was formed by R. circinatus, R. rionii, R. subrigidus, and R. trichophyllus r5. Depending on the analysis, all samples of R. trichophyllus r5 were ascribed to this group (ITS Network, NJ ddRADseq) or split between 'yellow' and 'blue' groups, reflecting the inheritance of different haplotypes (cpDNA Bayesian tree). In NJ ddRADseq phylogeny, the yellow group was divided into five clades. ree of them corresponded with the accepted and widely recognized species, additional two reflected genetic differentiation within R. trichophyllus r5. Samples from Croatia,  representing the Mediterranean R. trichophyllus (haplotype 10 and 11), are clearly separated from the individuals with haplotype 13, from Montenegro and Georgia (Figure 3; dark yellow squares; Table S1).
e second group (green) is formed by the West European species: R. ololeucos, R. hederaceus, R. omiophyllus, and R. tripartitus. is group is well-supported and was resolved in all three phylogenies. e next group (red) was less congruent among the three phylogenies. e red group comprised core species: R. fluitans, R. baudotii, R. saniculifolius, and R. dahlgreniae sp. nov. In the cpDNA Bayesian tree, additional taxa were included here, i.e., R. aquatilis, R. schmalhausenii, R. peltatus, R. penicillatus A (sensu lato Koutecký et al., 2022), and the samples representing R. trichophyllus r3 and r6 with haplotype cp3 (Figure 1; Table S1). In the ITS Network, R. baudotii r2 was placed together with R. trichophyllus r6 in the other split than the 'red' group ( Figure 2). In the NJ ddRADseq tree in the 'red' group, five clades were distinguished. Two of these reflected an intraspecific split in R. baudotii into two groups: the first comprised R. baudotii r1 occurring in Central and Northern Europe, and the second in which the Mediterranean R. baudotii r3 were grouped together with ribotype 2 from Central Europe. In this phylogeny within the 'red' group R. saniculifolius was resolved as a sister species to R. fluitans (Figure 3). e 'blue' group was the biggest. In the ddRADSeq tree, which has the highest resolution, the group was split into two subclades, comprising mainly homophyllous R. trichophylluslike plants (Figure 3; pale blue) and heterophyllous R. peltatus-like plants (Figure 3; dark blue). Both subclades were further subdivided into groups corresponding to individual taxa or intraspecific genotypes within R. trichophyllus, R. kauffmannii, and R. peltatus (discussed below). e 'blue' group in the ITS Network comprised the same taxa except for R. penicillatus A, which in the ITS network was separated into a monospecific group, placed between the 'blue' and  'red' groups reflecting its hydrogenous origin (Figure 2). e topology of the cpDNA Bayesian tree was in a great part congruent with the results described above, however, additionally, R. trichophyllus r5 samples from Croatia were included in the 'blue' group ( Figure 2).

Diagnosis (see
Ranunculus dahlgreniae is characterized by a combination of characters, which is not found in any other species within the section. ese are the short pedicels, the small flowers (otherwise only found in dwarf forms of related species), and the relatively large achenes (in majority > 2 mm), with partly persistent styles (Figure 4). e species differs from all other recognized species in at least two independent characteristics: from R. saniculifolius it differs by the presence of filiform apical segments in intermediate leaves, the lack of triangular or elongated nectar pits, the larger achenes ( Figure 4A,C), and the densely pubescent receptacle ( Figure 4B); from R. baudotii it differs by the sparse branching, the shape of the intermediate leaves, lacking parallel-margined lobes, the short, mostly straight pedicels, the larger and largely unwinged achenes, and the subglobose receptacle, elongating only slightly in fruit; from R. peltatus it differs by the smaller size, the lower number of final capillary segments in submerged leaves, the lack of pyriform nectar pits, and the glabrous achenes, and from R. aquatilis it differs by the smaller size, the more deeply dissected laminar leaves, the shape of the intermediate leaves lacking basal filiform segments, the smaller flowers, and the glabrous achenes from the immature state onwards.
With R. saniculifolius, R. dahlgreniae shares the frequent annual life form, the triangular lobes in intermediate leaves, and the slightly elongating receptacles. With R. baudotii, it shares the whitish fleshy stem and the combination of glabrous achenes and a pubescent receptacle. With R. peltatus, it shares the intermediate leaves with apical filiform segments and the tendency to form crenulate or incised petals. With R. aquatilis, it shares the short non-elongating pedicels and the sometimes circular nectar pits. Overall, R. dahlgreniae is most similar to R. saniculifolius, followed by R. baudotii and R. peltatus. With respect to generally high-rated characteristics, such as the hairiness of the receptacle and the glabrous achenes, it resembles R. baudotii the most. e species does not share any significant trait with other Eurasian laminarleaved species such as R. fucoides, R. tripartitus, R. ololeucos, R. schmalhausenii, and R. mongolicus (Nobis et al., 2017). Similarities with American laminar-leaved species such as R. lobbii and R. oblitus are of superficial nature.
Etymology: e species is named aer Gertrud Dahlgren (1931Dahlgren ( -2009, who pioneered Batrachium research in the eastern Mediterranean region. Habitat and ecology: e species inhabits freshwater bodies (astatic ponds) at an altitude between 1000 and 1200 m asl. e Omalos plateau is made of calcareous rock.

Discussion
e new species R. dahlgreniae has never been formally described before; thus, synonyms are not available. Its specific morphological features were either not recognized or not regarded as important. Even though R. dahlgreniae is most similar to R. saniculifolius, it was never included in descriptions of that taxon in comprehensive taxonomic treatments. Cook (1966) described R. saniculifolius based on a specimen from Cyprus as a species with few (2-5) large achenes (2.5 mm long) and a glabrous receptacle. Achene number and size were later corrected (Cook, 1967), but the description does not fit either. Pizarro (1995) Cook (1966), Pizarro (1995), and Wiegleb et al. (2017).
e species R. fucoides Freyn,in Lange & Willk.,Prodr. Fl. Hispan. 3(4): 912 (1880) was included as a subsp. of R. peltatus Schrank by Muňoz-Garmendia (1985) and synonymized with R. peltatus subsp. saniculifolius, as denominated by Cook (1984). is had enormous negative consequences on Batrachium taxonomy, as the name 'fucoides' now spread to areas where the taxon does not occur. We studied specimens of R. fucoides in MA and GOET. It is a distinctive species, characterized by the large flowers, the short rigid fleshy capillary leaves, and the long, mostly coiled pedicels. It was correctly separated from R. peltatus, R. baudotii, and R. saniculifolius at the subspecies level by Valdés et al. (1987, Fig. on p. 112), Diosdado et al. (1993, Fig. 274, p. 238) and Cirujano Bracamonte et al. (2014) and should be re-established as a separate species.
We could not fully solve the problem of the identity of R. saniculifolius. Even aer the separation of R. fucoides and R. dahlgreniae as separate species, the taxon remains Acta Societatis Botanicorum Poloniae / 2023 / Volume 92 / Article 167462 Publisher: Polish Botanical Society heterogeneous. Our samples, which were collected in the West Mediterranean region, are genetically related to R. fluitans and R. baudotii and showed big genetic heterogeneity (Figure 2). Even though the morphology of central and east Mediterranean plants is similar, additional studies will be necessary for the future to confirm their affiliation to the R. baudotii-R. fluitans clade.
We assume that the following unresolved taxa described from the region are extreme morphotypes of Ranunculus peltatus (see remarks of Parolly et al., 2007) and do not belong to the new species: R. kastamonuensis Dönmez (from Turkey) is a slender plant with a few large achenes; it is also found in northern Greece. R. peltatus var. microcarpus Meikle (from Cyprus, Meikle, 1977) is a slender plant with many small achenes; it is also found on Rhodes, Sicily, and in southern Spain, Portugal, and Morocco (see Pizarro, 1988). R. tripartitus G. Dahlgren non DC. (from Greece, Mykonos, also found in Milos and in central Greece) is a small hexaploid plant similar to R. peltatus, both with respect to morphology and karyotype (Dahlgren, 1991;Dahlgren & Svensson, 1994).
e phylogenetic relations within Batrachium are consistent with those obtained in the previous molecular studies (Bobrov et al., 2015;Koutecký et al., 2022;Prančl et al., 2018). However, the application of a new, high-throughput genome-wide analysis ddRADseq and sampling of taxa not included in the previous molecular studies allowed expanding the knowledge about phylogenetic relationships within the section and describing Ranunculus dahlgreniae, a species new to science.
Our study supports the hypothesis of Wiegleb et al. (2017) that Ranunculus ololeucos belongs to a West European clade comprising R. omiophyllus, R. hederaceus, and R. tripartitus (Figure 1, Figure 2 It has been known for a long time that R. baudotii is a complex species, as reflected by the common synonym R. confusus Godr. and R. marinus (Fr.) Fr. Wiegleb et al. (2017) assumed that there is a northern morphotype and a southern morphotype, with a slightly different stipule and achene shape. Moreover, the existence of inland forms with partly intermediate characters was mentioned, which was later confirmed by Koutecký et al. (2022). Genetic analysis showed that the current picture is more differentiated. e northern forms may go further southwards, both along bird flyways in inland areas and along the coasts. Morphologically inland forms rather resemble the southern form more than the northern one. is observation is well-supported by phylogeny based on ddRADseq data, where samples of R. baudotii from Croatia and from southern France were clustered with central European inland ribotype r3 (Figure 3). However, the southern form may not be homogenous but may be divided into a western form with larger flowers and an eastern form with smaller flowers. e exact distribution of the forms and their relationships must be studied in more detail in the future.
e molecular findings fully support our previous assumption that Mediterranean Batrachium may comprise more species than is currently known. From the combined studies, it became evident that the well-known Batrachium flora of Central Europe (Germany, Poland, Czech Republic) does not have many species in common with the East Mediterranean region. Only R. rionii, R. trichophyllus, and R. baudotii are regularly found in both regions. e latter two are represented by markedly different ribo-and haplotypes in the respective regions. is observation may hold for the Western Mediterranean region. Other 'good species' , associated with the Mediterranean and mentioned by Wiegleb et al. (2017) as uncertain are R. fucoides Freyn (see above), R. lutarius (Rev.) Bouvet, and R. pachycaulon (Nevski) Luferov. e status of R. sphaerospermus Boiss. et C.I. Blanche as a separate species is undisputed nowadays. Phylogenetically this species originates from the 'yellow' group, which can be hypothesized based on the ITS sequence (Hörandl & Emadzade, 2012). R. sphaerosphermus is closely related to Mediterranean R. trichophyllus r5. In the RADSeq tree, R. trichophyllus r5 was split into two clades ( Figure 3). Distinct evolutionary lineages within R. trichoplhyllus that are otherwise morphologically indistinguishable have been recognized before Koutecký et al., 2022;Prančl et al., 2018;Zalewska-Gałosz et al., 2015). Our research shows that in the Mediterranean area, there are further, phylogenetically distinct genotypes in this wide-range morphospecies (e.g. r5, Mediterranean r6). e factors shaping this variability, as well as its taxonomic implications, require further research.
New species have also been discovered elsewhere. Recently, R. oblitus was described from South America (Wiegleb et al., 2022), which is related to North American R. trichophyllus.
In that paper, another new species related to R. subrigidus W.B. Drew was suggested by genetic data but could not yet be morphologically verified due to a lack of well-preserved material. Furthermore, the so-called R. trichophyllus from New Zealand (Wiegleb et al., 2017) could be identified as an undescribed taxon close to R. baudotii (A.A. Bobrov, G. Wiegleb, P. Garnock-Jones, unpublished data). Overall, none of these newly described taxa can be regarded as 'cryptic species' in the sense of Prančl et al. (2018). ey may be called 'semi-cryptic' species. As soon as sufficient material is collected and analyzed, the case will become evident. We estimate that the total number of recognizable species in Batrachium is closer to 40 than 30, as assumed by Wiegleb et al. (2017).
Comparison of the Batrachium phylogeny resolved with different methods highlights the utility of reduced-representation sequencing for resolving phylogenetic relationships in non-model organisms with reticulation and recent divergence.

Supplementary material
e following supplementary material is available for this article: Table S1. Samples of Ranunculus section Batrachium included in the DNA studies.

Data availability
e Sanger sequences reported in this paper have been deposited in GenBank databases (www.ncbi.nlm.nih.gov/ genbank/). e datasets used and/or analyzed during the current study are available from the corresponding authors upon request. Herbarium specimens of R. dahlgreniae are preserved in KRA.