Adonis fucensis (A. sect. Adonanthe, Ranunculaceae), a New Species from the Central Apennines (Italy)

Simple Summary Adonis sect. Adonanthe is characterized by species with strongly gibbous abaxial side of achenes, reticulate-venation on its surface, with short and recurved style and includes four series: ser. Amurenses, ser. Coeruleae, ser. Apenninae, ser. Vernales. In the Euro-Mediterranean area three species belonging to A. sect. Adonanthe are currently recognized: A. apennina (ser. Apenninae), A. volgensis (incl. A. transsilvanica; ser. Vernales), A. vernalis (ser. Vernales). In 2021 was discovered in the Central Apennines (Italy) a yellow-flowered Adonis population belonging to sect. Adonanthe similar to A. volgensis. Following an integrated taxonomic approach, we have shown that the newly discovered population should be regarded as a new species, named A. fucensis, endemic to Abruzzo (Central Apennines, Italy). Abstract Adonis fucensis is herein described as a new species based on morphological and molecular analyses. It is endemic to one locality of the Central Apennines between Amplero and Fucino plains within the NATURA 2000 network in the SAC IT7110205 (Central Italy). The only discovered population is composed of 65 individuals and is at risk of extinction. The conservation status assessment according to IUCN categories and criteria is proposed and discussed. The new species belongs to A. sect. Adonanthe and is morphologically similar to A. volgensis (incl. A. transsilvanica), a species distributed in Hungary, Romania, Bulgaria, and Turkey as well as eastward to SW Siberia and Central Asia. Adonis fucensis can be distinguished from A. volgensis by larger cauline leaves, pentagonal with lobes lanceolate, larger stipules with more lobes and teeth, and larger flowers. Finally, an analytical key to Adonis species belonging to sect. Adonanthe distributed in Europe is presented.


Introduction
The genus Adonis L. (Ranunculaceae) comprises 38 accepted, annual and perennial, species and subspecies, distributed in the northern hemisphere and native to Asia, Europe, northern Africa, and Mediterranean region [1]. According to Wang [2,3], based on a morphological study, the genus Adonis should be divided into two subgenera, six sections, and six series: subg. Adonis (divided into three sections and two series) and subg. Adonanthe (Spach) W.T.Wang (divided into three sections and four series). Recent molecular studies [4,5] do not fully support the taxonomic treatment based on morphological features proposed by Wang [2,3], whereas a phylogenetic classification has not yet been established. In Italy, the genus Adonis is represented by 10 taxa (species and subspecies): the annual and red-flowered A. annua L., A. flammea Jacq. (with two subspecies), A. aestivalis L. (with two subspecies), A. microcarpa DC., and the perennial and yellow-flowered A. distorta Ten.

Morphometric Analyses
A total of 18 morphological characters were selected and scored in 87 dried individuals belonging to A. volgensis (63) from Romania, Moldavia, Russia and to the new population from the Central Apennines (Italy), named A. fucensis (24). Two characters, i.e., height (H) and number of petals (NP), were scored for A. fucensis on the field. Among the morphological characters studied, 14 are quantitative, 1 is calculated ratio, and 3 are qualitative (Table 1). Samples with missing data were not included in the multivariate analysis (resulting dataset of 50 individuals × 18 variables). For each quantitative character an independent sample t-test was carried out with SPSS v25 software (IBM Corp., Armonk, NY, USA) [22]. A non-metric multidimensional scaling (NMDS) and Cluster Analysis (CA) using the average linkage method (UPGMA), were performed with PAST package v4.11 software (Natural History Museum, Oslo, Norway) [23]. The similarity matrix was calculated using the Gower coefficient, suitable for mixed data [24]. Furthermore, the variability of the analyzed morphological characters was described by standard statistical parameters (mean, standard deviation, minimum, maximum, and 25th and 75th percentiles). Boxplots were built through SPSS v25.

DNA Sequencing and AFLPseq Fingerprinting
Genomic DNA for both sequencing and genetic fingerprinting was extracted according to the CTAB DNA extraction protocol of Doyle and Dickson [25] and Doyle and Doyle [26]. Amplification of the two internal transcribed spacer regions (ITS1, ITS2) of the nuclear ribosomal repeat (nrDNA) was carried out with primers ITS-18SF [27] and ITS2 [28] for ITS1 and ITS-D [29] and ITS-SR [30] for ITS2, respectively. After purification of PCR amplicons with AmpliClean (Nimagen, Nijmegen, The Netherlands) magnetic beads, Sanger sequencing was carried out by a contract sequencing company (Macrogen Europe, Amsterdam, The Netherlands). Electropherograms were manually edited with CHROMAS v2.6.6 [31]; polymorphisms observed in accession A1251 were resolved manually and the two resulting sequences were independently included in the alignment together with sequences of other species of Adonis sect. Adonanthe and an outgroup sequence (from Trollius ranunculoides Hemsl.). We used PAUP* v4.0a169 [32] to calculate distances among the aligned sequences based on the Kimura-2-Parameter model and constructed a Neighbor-joining tree. A bootstrap analysis was performed with 1000 replicates.
The AFLPseq fingerprinting method has been proposed by [33] and combines the genome-complexity reducing AFLP approach [34] with the next-generation sequencing (NGS) of resulting AFLP bands using the Nanopore sequencer MinION from Oxford Nanopore Technologies (Oxford, UK). It provides sequence and single-nucleotide polymorphism (SNP) information for hundreds of anonymous loci from across the whole genome and could be used for both population genetic, phylogenetic, and species delimitation studies. It is suited for both well-preserved DNA from silica-gel dried leaf material and degraded DNA from herbarium specimens.
The present AFLPseq study comprised 12 Adonis accessions (Supplementary Table S1), either recently collected, silica-gel dried material (five accessions from Italy and Romania), or well-preserved herbarium material housed in the herbaria B and BRNO (seven accessions from Romania, the Russian Federation, and Kazakhstan). The accessions were selected (a) to cover large parts of the distribution range of A. volgensis and (b) to include only plant material in the fingerprinting procedure, for which extracts of unfragmented genomic DNA was expectable. The AFLPseq procedure followed the protocol given in [33] with the following modifications: in the restriction-ligation step, we used a double-digestion procedure with restriction enzymes MseI and EcoRI. After ligation of MseI and EcoRI adapters (MseI adapter: 5 -GACGATGAGTCCTGAG-3 + 5 -TACTCAGGACTCAT-3 ; EcoRI adapter: 5 -CTCGTAGACTGCGTACC-3 + 5 -AATTGGTACGCAGTCTAC-3 ), we continued with the AFLP genome-reduction protocol using primers with 1bp-overhangs (MseI-C: 5 -GATGAGTCCTGAGTAAC-3 ; EcoRI-A: 5 -GACTGCGTACCAATTCA) in the pre-selective amplification step and in the selective amplification step with additional 1bp-(EcoRI side) or 2bp-overhangs (MseI side), respectively. The two primers used in the latter amplification step, however, were additionally tailored to include Nanopore barcode adapter sequences at the 5 end of the primers (Mse_CTG_Nanopore_fw: 5 -TTTCTGTTGGTGCTGATATTGCGATGAGTCCTGAGTAACTG-3'; Eco_AA_Nanopore_rv: 5 -ACTTGCCTGTCGCTCTATCTTCGACTCCGTACCAATTCAA-3'), as suggested in the 'Ligation sequencing amplicons-PCR barcoding (SQK-LSK109 with EXP-PBC001)' protocol by Oxford Nanopore Technologies, substituting a subsequent ligation of the Nanopore barcode adapter with an additional barcoding PCR. To ensure specific binding with long and tailed primers, a two-step variation of the selective PCR was conducted (94 • C for 2 min; followed by 30 cycles of 94 • C for 20 s and 72 • C for 2 min; and a final step at 72 • C for 2 min). To every 2 µL of 1:10 diluted preselective PCR product, 5 µL Taq DNA Polymerase Master Mix RED, 0.25 µL of each 10 µM tailed selective primer, and 2.5 µL H2O were added. After the selective PCR, the length of the fragments ranged from 200-500 bp. All subsequent steps (Nanopore barcode PCR, sample multiplexing, size selection, preparation of Nanopore sequencing library) followed [33]. The resulting library was sequenced with the MinION using a Flongle flow cell. Read data processing, de novo locus assembly, identification of orthologous loci, and reference-based SNP calling with the SLANG pipeline, and the final calculation of frequency-sensitive SNP-based Nei distances followed the protocol described by [33]. Based on these pairwise distances both a phylogenetic network reconstruction using the Neighbor-joining method in SPLITSTREE v4.16.1 [35] and a principal co-ordinate analysis (PCoA) with a custom R v4.0.5 script using the 'phangorn' library to read the distance matrices and the 'ape' package to calculate and plot the PCoA was carried out.

Morphometric Analyses
The NMDS, performed with three dimensions, yielded an ordination with a stress value of 0.09224. The scatterplot shows on the first two axes a clear distinction between A. volgensis and A. fucensis, and no overlapping areas among individuals were found ( Figure 1). The UPGMA dendrogram ( Figure 2) yielded two well-defined clusters, one including all individuals of A. volgensis and the other all individuals of A. fucensis.
(SQK-LSK109 with EXP-PBC001)' protocol by Oxford Nanopore Technologies, substituting a subsequent ligation of the Nanopore barcode adapter with an additional barcoding PCR. To ensure specific binding with long and tailed primers, a two-step variation of the selective PCR was conducted (94 °C for 2 min; followed by 30 cycles of 94 °C for 20 s and 72 °C for 2 min; and a final step at 72 °C for 2 min). To every 2 µL of 1:10 diluted preselective PCR product, 5 µL Taq DNA Polymerase Master Mix RED, 0.25 µL of each 10 µM tailed selective primer, and 2.5 µL H2O were added. After the selective PCR, the length of the fragments ranged from 200-500 bp. All subsequent steps (Nanopore barcode PCR, sample multiplexing, size selection, preparation of Nanopore sequencing library) followed [33]. The resulting library was sequenced with the MinION using a Flongle flow cell. Read data processing, de novo locus assembly, identification of orthologous loci, and reference-based SNP calling with the SLANG pipeline, and the final calculation of frequency-sensitive SNP-based Nei distances followed the protocol described by [33]. Based on these pairwise distances both a phylogenetic network reconstruction using the Neighbor-joining method in SPLITSTREE v4.16.1 [35] and a principal co-ordinate analysis (PCoA) with a custom R v4.0.5 script using the 'phangorn' library to read the distance matrices and the 'ape' package to calculate and plot the PCoA was carried out.

Morphometric Analyses
The NMDS, performed with three dimensions, yielded an ordination with a stress value of 0.09224. The scatterplot shows on the first two axes a clear distinction between A. volgensis and A. fucensis, and no overlapping areas among individuals were found ( Figure  1). The UPGMA dendrogram (     Table 2. The states of 13 characters (H, MLL, MLW, NMLN,  ATL, LMW, LWB, LMW/ LWB, SL, SW, NSL, CD, SLD, SWD, PLD, and PWD) show significant differences between the two species (p < 0.01). Boxplots of relevant characters are showed in Figure 4.

nrDNA Sequence Variation
The Neighbor-joining tree based on Kimura-2-parameter distances among nrDNA ITS sequences of 15 Adonis accessions is shown in Figure 5. The central Italian Adonis fucensis accession (A1252) is found being closely related with A. volgensis and A. vernalis in the monophyletic group of A. sect. Adonanthe ser. Vernales. As also found by a more comprehensive phylogenetic analysis of section Adonanthe performed by [4], series Amurenses did not form a monophyletic group.

nrDNA Sequence Variation
The Neighbor-joining tree based on Kimura-2-parameter distances among nrDNA ITS sequences of 15 Adonis accessions is shown in Figure 5. The central Italian Adonis fucensis accession (A1252) is found being closely related with A. volgensis and A. vernalis in the monophyletic group of A. sect. Adonanthe ser. Vernales. As also found by a more comprehensive phylogenetic analysis of section Adonanthe performed by [4], series Amurenses did not form a monophyletic group.

AFLPseq Fingerprinting
In total, 731,698 reads and 243.72 Mbp were sequenced for the 12 Adonis accessions. After read preprocessing, 592,432 reads with lengths between 10 bp and 614 bp passed the Q5 quality filter. With the SLANG pipeline (cluster thresholds optimized to values of 0.85 and 0.95 for the first and second cluster step, respectively), 486 orthologous loci were inferred, containing 2,944 SNPs. After calculation of pairwise Nei distances, the resulting Neighbor-joining tree (Supplementary File S2) and the PCoA plot were received ( Figure  6). While in the first, the Adonis accession from the Central Apennines (A1252) is con-

AFLPseq Fingerprinting
In total, 731,698 reads and 243.72 Mbp were sequenced for the 12 Adonis accessions. After read preprocessing, 592,432 reads with lengths between 10 bp and 614 bp passed the Q5 quality filter. With the SLANG pipeline (cluster thresholds optimized to values of 0.85 and 0.95 for the first and second cluster step, respectively), 486 orthologous loci were inferred, containing 2944 SNPs. After calculation of pairwise Nei distances, the resulting Neighbor-joining tree (Supplementary File S2) and the PCoA plot were received ( Figure 6). While in the first, the Adonis accession from the Central Apennines (A1252) is connected with the remaining A. volgensis representatives without any exceptionally longer branch than the other accessions, the PCoA plot demonstrates the clear separation between the two taxa; with accessions of the latter on the left and accession A1252 on the right side of principal co-ordinate PCo axis 1, which account for 20.8% of the total variation in the data set. Additionally, PCo axis 2 (accounting for additional 15.0% of the total variation) shows a clear geographical separation within A. volgensis, with accessions of this species from Romania (sometimes considered as being an independent species, A. transsilvanica) on the positive and accessions from Russia and Kazakhstan on the negative side of the axis. An additional result of the analysis is worth mentioning in methodological respects: accessions A1251, A1273, and A1274 are very similar to each other in spite of the fact that the three probes come from the same locality (Romania, Constanta, Cotu Văii), but were recently collected as silica-gel dried leaf material (the latter two) or as an herbarium specimen (A1251) twenty years ago. This observation adds to the trustworthiness of the AFLPseq protocol and the comparability of differently preserved DNAs in terms of sequence information retrieved through this process.

Discussion
Morphological and molecular analyses provide evidence that A. fucensis should be regarded as a new species, endemic to Abruzzo (Central Apennines, Italy). It is similar to A. volgensis, a typical plant of the E-European and Asiatic steppes, distributed in Hungary, Romania, Bulgaria, and Turkey, as well as eastward to SW Siberia and Central Asia, but it can be distinguished by several quantitative and qualitative morphological characters, An additional result of the analysis is worth mentioning in methodological respects: accessions A1251, A1273, and A1274 are very similar to each other in spite of the fact that the three probes come from the same locality (Romania, Constanta, Cotu Văii), but were recently collected as silica-gel dried leaf material (the latter two) or as an herbarium specimen (A1251) twenty years ago. This observation adds to the trustworthiness of the AFLPseq protocol and the comparability of differently preserved DNAs in terms of sequence information retrieved through this process.

Discussion
Morphological and molecular analyses provide evidence that A. fucensis should be regarded as a new species, endemic to Abruzzo (Central Apennines, Italy). It is similar to A. volgensis, a typical plant of the E-European and Asiatic steppes, distributed in Hungary, Romania, Bulgaria, and Turkey, as well as eastward to SW Siberia and Central Asia, but it can be distinguished by several quantitative and qualitative morphological characters, as shown in Table 2. The new species lives in shrub-steppe habitat in contact environments between bushes dominated by Prunus spinosa L. subsp. spinosa and steppe grasslands with the presence of Festuca valesiaca Schleich. ex Gaudin subsp. valesiaca. Abruzzo is the Italian administrative region with the highest number of taxa belonging to the genus Adonis, and also hosts the only Italian populations of the extremely rare steppe species A. vernalis.
The dry sub-continental climate of internal basins of the Central Italy, together with wild herbivore disturbance and prehistoric anthropogenic fires [36], may have reduced the post-glacial reforestation. Subsequently sheep grazing and the practice of transhumance, dating back to the 6th century BC or earlier in Abruzzo and widely practiced until the 1950s [37,38], has probably favored the spread of grasslands [39]. Around Amplero, close to the locality of A. fucensis, lies an archaeological site inhabited since the VI century B.C. The area hosted, from the Bronze age until Medieval times and beyond, important shepherd settlements and was located on transhumance routes [40]. These causes explain the persistence of steppe species in the internal areas of the Central Apennines.
Alternatively, a recent study on the phylogeography of the closely related A. vernalis [5] revealed that this plant species expanded its range from SE Europe into the Euro-Siberian steppe, with a Spanish population of the species being the earliest-diverging lineage. Whether members of our present study group parallel this migration pattern and A. fucensis constitutes the earliest-diverging remnant of an eastwards expanding A. volgensis could be hypothesized here but must await a much denser sampling of the latter species. Due to the restricted number of accessions analyzed in the present contribution, the biogeographical history of A. fucensis and A. volgensis remain unresolved.
Adonis fucensis is a very rare species, consisting of a very small population of 65 individuals, assessed here as critically endangered. In the two years (2021-2022) in which we were able to study the population we observed that although plants have a large number of flowers, they produce few fruits (we have observed many abortive achenes), and its survival is probably related to vegetative reproduction with consequent loss of genetic diversity. It will be absolutely necessary to undertake a dialogue with the National Park of Abruzzo, Lazio, and Molise to plan correct in situ and ex situ conservation strategies, to try to save this new species from extinction.
Etymology: Adonis fucensis is named after Fucino Plain located nearby to the north and affected by the presence of the third largest Italian lake drained in 1878.
Phenology: Flowering from March to April; fruiting from May to June. Distribution: Endemic to one locality of Abruzzo (Central Italy) within the SAC IT7110205 "Parco Nazionale d'Abruzzo". The species grows in a small flat clearing on the slopes of Mt. Annamunna, between Amplero and Fucino plains (Supplementary File S1).
Conservation status: Adonis fucensis is known only by one location (locus classicus) where, during 2021, we counted only 65 individuals (genets). It is located within the NATURA 2000 network in the SAC IT7110205 "Parco Nazionale d'Abruzzo". The area of occupancy (AOO) is 4 km 2 (cell grid 2 × 2 km), calculated with GeoCAT (Geospatial Conservation Assessment Tool) software (http://geocat.kew.org/about (accessed on 10 October 2022)) [44]. We observed pressure due to the grazing of wild animals (especially wild boars that dig up single plants). Observing the aerial photos of the 1980s it is evident how in the A. fucensis habitat the shrub and tree vegetation increased by reducing the surface of the pastures probably due to a decrease in grazing by livestock. The natural succession of vegetation is a pressure and a threat for the population of A. fucensis. It is not possible to be certain of the decline of the species even if it is reasonable to assume that it was more common in the past. According to IUCN criterion B2ab(iii) [45], the species is assessed as Critically Endangered (CR).
Key to Adonis Species Belonging to sect. Adonanthe Distributed in Europe