New diploid species in the Ranunculus auricomus complex (Ranunculaceae) from W and SE Europe

Abstract: The Euro-Siberian complex of Ranunculus auricomus (Ranunculaceae) consists of about 800 mainly apomictic and polyploid taxa. So far, only four sexual species have been described (R. carpaticola Soó, R. cassubicifolius W. Koch, R. notabilis Hörandl & Gutermann and R. marsicus Guss. & Ten.). With the exception of R. marsicus and rare autotetraploids of R. cassubicifolius, sexuality seems to be linked to diploidy. Using flow cytometric ploidy estimation, flow cytometric seed screen and pollen quality, six new diploid sexual species have been revealed in C France, C and SE Slovenia and Croatia: Ranunculus austroslovenicus Dunkel, R. calapius Dunkel, R. cebennensis Dunkel, R. mediocompositus Dunkel, R. peracris Dunkel and R. subcarniolicus Dunkel. Here, they are described and depicted in detail. Sexual reproduction has been verified for R. austroslovenicus, R. cebennensis, R. subcarniolicus and also for the previously described R. envalirensis Grau and R. flabellifolius Heuff. ex Rchb. Citation: Dunkel F. G., Gregor T. & Paule J. 2018: New diploid species in the Ranunculus auricomus complex (Ranunculaceae) from W and SE Europe. – Willdenowia 48: 227–257. doi: https://doi.org/10.3372/wi.48.48205 Version of record first published online on 24 August 2018 ahead of inclusion in August 2018 issue.

Due to lack of clear boundaries between the main morphotypes, Marklund's concept of four distinct species (Ranunculus auricomus L., R. cassubicus L., R. fallax (Wimm. & Grab.) Slob. and R. monophyllus Ovcz.) with numerous subspecies (subspecies apomicticae) has been abandoned in recent years (Marklund 1961(Marklund , 1965Ericsson 1992Ericsson , 2001Hörandl 1998;Hörandl & Gutermann 1998a). Without presenting monophyletic clades (Hörandl & al. 2009), but for practical reasons, the complex is still subdivided into two morphological collective groups: the "R. auricomus group" (thereto R. notabilis, R. envalirensis) and the "R. cassubicus group" (thereto R. carpaticola, R. cassubicifolius) (Hörandl & al. 2005). They differ strikingly in size and leaf shape (Lohwasser 2001) as well as in relative genome size of diploids (Paule & al. 2018). The R. cassubicus group has basal sheaths without leaf blades (cataphylla), large undivided basal leaves and normally broad cauline leaves with a toothed margin. The R. auricomus group is generally smaller, lacks basal sheaths and presents heterophyllous leaves. Depending on the individual taxon, basal leaves vary in shape: they can be deeply divided, while the upper stem leaves are linear with predominantly entire segments. Using isozyme data, the separation of sexual diploids from these two groups has been estimated at 900 000 years ago (Hörandl 2004).
For the understanding of the evolution of this variable apomictic species complex, detailed knowledge of the distribution and identity of diploid sexual taxa is crucial. In nutrition-poor humid meadows and moist forests of the Slovenian Prealps, as well as in floodplain forests close to Karlovac, Croatia, the first author found five new diploid morphotypes. Additionally, the diploid plants, already found by Nogler in the French Massif Central (1982) were categorized as Ranunculus auricomus and were not attributed to any of the known species. Here, we describe new-ly discovered morphotypes as new species and compare them with previously known diploids from adjacent areas.

Material and methods
Material was collected in France, Slovenia and Croatia from 2007 to 2017. For morphological analysis 10 -30 plants of each site were investigated. One to eight plants of each taxon were transplanted and cultivated in the experimental garden (Karlstadt, Germany) and two to ten plants were collected as herbarium specimens for further investigation. Additionally, specimens from the herbaria Dresden (DR), Geneva (G), Göttingen (GOET), Jena (JE), Ljubljana (LJU), Munich (M) and Zurich (Z + ZT) were examined. All specimens collected are deposited in M and partly in other public herbaria as well as in the private herbarium of F. G. Dunkel ("Du"; collection number and herbarium number are identical). Collection details of transplanted plants are given in Table 1.
Geographical reference is given by coordinates or defined grid fields in the form of quadrants of topographic maps. A quadrant encounters a latitude of 3' and a longitude of 5' and refers to the number of the official topographic maps of Germany with extrapolation to adjacent countries. For floristic purposes this reference system is widely used in Central Europe (e.g. Jogan 2001).
Pollen quality was determined by carmine acetic staining according to Hörandl & al. (1997;see Fig. 4: 203). Two hundred pollen grains per specimen were investigated.
For determination of the reproduction mode, several single mature seeds or 5 -10 pooled seeds per plant were analysed by means of flow cytometric seed screen (FCSS) according to Matzk & al. (2000) using the two-step Otto protocol as outlined by Dobeš & al. (2013). For five out of the eight studied species, ripe fruits were available in cultivation (Table 1). The measurements were carried out both with and without the internal standard (Pisum sativum) in order to determine also the ploidy of the embryo. The reproduction mode was assessed based on expected fluorescence ratios of embryo and endosperm nuclei as reviewed for a pseudogamous apomictic system (Dobeš & al. 2013) considering the threshold of 1.65 for discrimination between sexual and apomictic seeds (Schinkel & al. 2017).
Species concept, definition of characters and depiction follow Hörandl & Gutermann (1998a) and Dun-  kel (2010). All data sheets (Fig. 2,4,6,8,10,12,14) present the most important and specific characters of a species: the basal leaf sequence from the initial leaves (normally no. 1 and 2), the spring leaves evolving during the flowering period (no. 3 -5) and the final leaves developing during the fruiting period (no. 6 and 7). The small letters next to each basal leaf denote the individuum of a population. Furthermore, the lowermost cauline or stem leaf with its specific number and form of segments is characteristic. Values of the largest segment are indicated in the description. In all diploid (and sexual) species the flowers consist of five (rarely six or seven) well-developed petals. Finally, form and hairiness of the receptacle is illustrated at the bottom of the right column of the data sheet.

Results and Discussion
Twenty-three transplanted individuals were investigated by flow cytometry, 2 -7 per newly described species. All studied accessions revealed the same sample/standard fluorescence ratio class as the chromosome counted individual Du-30442 (2n = 16) (Table 1). Hence, all six novel morphotypes as well as Ranunculus envalirensis and R. flabellifolius are considered diploid. Interestingly, all newly studied accessions fall into the relative genome size range (i.e. sample / standard fluorescence ratio) of the "R. auricomus group" as published previously (Paule & al. 2018).
Fluorescence ratios of embryo and endosperm nuclei for seven studied individuals ranged from 1.48 to 1.63 (mean 1.53) and measured embryos were revealed to be diploid. Hence two individuals of Ranunculus austroslovenicus sp. nov., R. cebennensis sp. nov. as well as one individual of R. envalirensis, R. subcarniolicus sp. nov. and R. flabellifolius can be considered for sexual embryo development and meiotic origin of female gametophyte (Table 1).
Staining by carmine acetic revealed 87.7 -97.6 % well-developed pollen in seven studied individuals, representing all novel morphotypes. Pollen quality is considered in line with regular meiotic (i.e. sexual) pollen development if the pollen stainability is above 80 % (Hörandl & al. 1997).
Species characteristics of members of the Ranunculus auricomus complex are best demonstrated by comparative illustrations, especially of the basal leaf cycle and the cauline leaves, which are illustrated in Fig. 2 -15. Detailed descriptions of the new species are below.
Distribution -From the Planinsko Polje SW of Ljubljana to the Kočevsko region in SE Slovenia, and adjacent Croatia (Fig. 1).
Etymology -The epithet refers to the main distribution in S Slovenia.
Taxonomy -Although the basal leaf sequence is heterophyllous, the round undivided leaves are prominent. The lateral segments of leaf no. 1, 2, 5 -7 are undivided, oc-casionally the first lateral incision amounts up to 60 %. In general, 2 -4(or 5) basal leaves are undivided, the blade at the base is mostly narrow-angled. In contrast to the most species of the complex, the third and not the fourth or fifth basal leaf is mostly divided.
Similar species are Ranunculus glechomoides (Markl.) Ericsson, R. subglechmoides Dunkel and R. suborbicularis Dunkel. Ranunculus austroslovenicus is readily distinguished by complete flowers with five petals; the basal apertures of the final leaves of R. glechomoides and R. suborbicularis are closed, the basis even overlapping in contrast to an angle of 20 -60° in R. austroslovenicus.
The form of the middle lobes of the spring leaves is variable but smaller, only deltoid to broadly deltoid and not spatulate as in Ranunculus subglechomoides. For R. austroslovenicus, sexual reproduction is suggested (Table 1).
Distribution -Known only from the type locality near Karlovac in the alluvial zone of the river Kulpa (Fig. 1).
Ecology -Softwood floodplain forest together with Fritillaria sp. and Leucojum aestivum L., deciduous forest.
Etymology -The epithet derives from Calapius, the Latin name for the river Kolpa, which is situated close to the type locality.
Taxonomy -The sequence of the basal leaves is heterophyllous and characterized by three different types of basal leaves. The initial leaves (no. 1 -3) possess a broadly deltoid to spatulate middle lobe, and the leaf edge has obtuse crenate teeth or it is only crenate. The spring leaves (no. 4 and 5) present often stalked middle lobes in narrowly deltoid to lanceolate form with deep and wide lateral incisions. In contrast, the lateral segments of the final leaves (no. 6 and 7) are undivided. In the last leaf of the cycle the main incision is reduced to at most 65 % or even lacking. In robust plants, sometimes intermediate leaves (Lohwasser 2001) are developed, obviously between basal leaves no. 4 and 5 of the cycle. They have a dissected blade by the main incision and three mostly deep lateral incisions, resembling the basal leaf of Ranunculus acris L.

DNA-ploidy -2x
Populations of the Ranunculus auricomus complex are rather common in the Auvergne (Antonetti & al. 2006). Therefore, indications of the occurrence are rather preliminary. Specimens originating from Ardèche and Lozère were not sufficient for secure determination, but the occurrence in these departments is probable. On the other hand, at the Margeride (Chapelle-Laurent) only tetraploid plants were found morphologically rather similar to R. marsicus.
Etymology -The epithet derives from the Gaulish name "Cebenna". It signifies "round mountain" or "back of a mountain". The Cévennes are the SE part of the Massif Central.
Ranunculus cebennensis represents a strongly heterophyllous leaf cycle. Unique to the species is the fourth basal leaf: deeply divided in a pedate manner with a second lateral incision (Fig. 6). In contrast to R. envalirensis, the lateral segments of the fifth to seven basal leaf of R. cebennensis are almost undivided and similar to R. marsicus.

Ecology -Subalpine meadows.
Etymology -The epithet refers to the type locality at Puerto de Envalira.
Taxonomy -Ranunculus envalirensis represents a species consisting of plants with a more divided blade of the basal leaves and a tendency to develop more than two enrichment shoots with intermediate leaves presenting deep incisions of middle and lateral lobes (Fig. 8). In contrast, within populations of R. envalirensis, plants with less divided blades of the basal leaves sometimes occur. These are characterized by the main incisions being up to 85 % instead of 100 % and mostly lacking lateral incisions (Fig. 8 [leaves h -i], 9). This demonstrates a broader morphological variability than in almost any apomictic taxon.
Etymology -The epithet refers to the median morphological position between Ranunculus austroslovenicus and R. peracris.
Distribution -A Slovenian endemic scattered from C Slovenia around Ljubljana to SE Slovenia around Novo Mesto (Fig. 1).
Etymology -The epithet alludes to the strongly divided basal leaves, which are reminiscent of the basal leaves of Ranunculus acris L.
Taxonomy -Ranunculus peracris is characterized by a strongly divided blade with a leaf edge of obtuse crenate teeth. Normally, five basal leaves are dissected, i.e. divided completely, by the main incision. The middle lobe is deeply trifid with incisions up to 55 %, the basal leaves no. 3 -6 possess at least a third lateral incision. Occasionally, the basal leaf blades are less divided but still with obtuse leaf segments or lacinulae. Species with such strongly divided basal leaf blades are especially found in Scandinavia, e.g. R. acidotus (Markl.) Ericsson, R. defectus (Markl.) Ericsson or R. rotundidens (Julin) Ericsson, but they differ by incomplete flowers, acute stem leaf segments and/or less incised middle lobes of the basal spring leaves (Julin 1965(Julin , 1980Marklund 1961Marklund , 1965. Similarly as in Ranunculus subcarniolicus, R. peracris is conspicuous by an early flowering period at the beginning of April. Willdenowia 48 -2018   Fig. 1, 14, 15, Table 1.
Distribution -From C Slovenia S of Ljubljana to SE Slovenia around Šentjernej, S border at the N Velebit, region of Lika, close to Gospić in Croatia (Fig. 1).
Etymology -The epithet refers to Lower Carniola, the SE part of the historical Carniola region.
Taxonomy -Ranunculus subcarniolicus possesses a rather homophyllous cycle, it is characterized by basal leaves with a V-shaped to wide-angled base, the fourth basal leaf is often even truncate at the base. The main incision is, with the exception of the fourth leaf, wideangled, up to 30°. In contrast to the other diploid species, the lowest cauline leaf consists of five oblanceolate segments and resembles the R. palaeoeuganeus group but with a completely different ecology (Dunkel 2011). The carpellophores are rather long (0.1 -0.25 mm). At the type locality, a nutrient-poor humid meadow, Ranunculus subcarniolicus grows together with two other taxa of the R. auricomus complex. There, R. subcarniolicus is easily recognizable not only by its small size but also by its earlier flowering period at the beginning of April.  Fig. 1, 16, 17, Table 1.
Ranunculus flabellifolius is a conspicuous taxon already described in the Flora germanica excursoria by Heuffel in 1832 (Reichenbach 1830(Reichenbach -1833. It is unmistakable by its fan-shaped cauline leaves. We can confirm R. flabellifolius as a diploid species (Milošević & al. 1977). Our results of FCSS suggest a sexual mode of reproduction for the species.
Although specimens of Ranunculus flabellifolius are scattered throughout all larger European herbaria, R. flabellifolius itself seems to be restricted to a few places in SW Romania and adjacent Serbia (Nyárády 1933;Josifovič 1977) (Fig. 1). With the exception of the single population outside Romania, all collections date from the 19 th century up to 1914. On a specimen label (B 10 0348680), W. Seymann annotated: "Banatus. In silvis montis "Kasan" prope pagum Plavisevicza. Locus unicus!".
The population of Ranunculus flabellifolius at Mount Simion was refound by the first author. Here the species hybridizes readily with other species of the Ranunculus auricomus complex. Such transitional forms were already mentioned by Reichenbach: "planta speciosa […] cuius forte cum R. auricomo hybrida proles" (1832: 732). Therefore, as far as no molecular biological data are available, R. flabellifolius should still be taxonomically included in the R. auricomus complex.