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Allium ducissae (A. subgen. Polyprason, Amaryllidaceae) a New Species from the Central Apennines (Italy)

In this paper, Allium ducissae (the LSID for the name Allium ducissae is: 77254606-1) is described as a new species based on morphological and molecular analyses, and its taxonomic relationships are discussed. It grows in crevices on calcareous rocks, rocky slopes and grassy ledges in the subalpine belt, within two regional protected areas in the Lazio and Abruzzo administrative regions (Central Apennines, Italy). Previously, these populations were attributed to A. strictum, a species described from Siberia, belonging to A. sect. Reticulatobulbosa. The new species is distinct from A. strictum in the morphology of vegetative and reproductive structures. Indeed, it is close to A. palentinum, an endemic species to Cantabrian Mountains (NW Spain). Both molecular and morphological data support the recognition of the Allium populations coming from the Central Apennines as a new species. Allium ducissae can be clearly distinguished from A. palentinum by longer and wider tepals, longer filaments, tooth of inner filament, flower pedicels, spathe appendage, and smaller seeds. Moreover, seed testa micro-sculptures revealed slight differences between A. ducissae and A. palentinum. Chromosome counts showed that A. ducissae is diploid with 2n = 16 chromosomes, as already known for A. palentinum. Molecular analyses support the affiliation of A. ducissae and A. palentinum to A. sect. Falcatifolia, contrary to what is known for the latter species, usually included in A. sect. Daghestanica. Finally, the IUCN assessment for the newly described species is proposed and briefly discussed.


Introduction
The genus Allium L. (Amaryllidaceae) is one of the richest and largest monocotyledonous genera, comprising about 1000 species [1,2]. It is distributed in temperate, semi-arid and arid regions of the Northern Hemisphere, with very rare exceptions [3,4]. The center of diversity of the genus extends from the Mediterranean basin to Central Asia [5]. Based on molecular phylogenetic studies, the genus Allium has been divided into 15 subgenera and 72 sections [4], which have been further classified into three evolutionary lineages. The classification of the genus is continuously revised and updated (e.g., [6][7][8]). With regards to Italy, Allium is represented in 78 taxa (species and subspecies), of which twenty-five are endemic and nine are alien species [9,10].

Plant Material and Morphological Analyses
This study is based mainly on field surveys, on an extensive analysis of relevant literature, and on the examination of herbarium specimens preserved at APP, BCN, MA, PI, and SALA (herbarium codes according to [30]) (Supplementary File S1). Morphological observations and measurements of 21 quantitative characters, considered as diagnostic in Allium, were analyzed on fresh material and dried herbarium specimens (Table 1). A total of 52 individuals of the new species ("DUC") from three localities in the Central Apennines (Lazio: Mt. Morrone and Murolungo; Abruzzo: Mt. Rozza), and 45 of A. palentinum ("PAL") from several localities in the Cantabrian Mountains (NE Spain), were analyzed. For each quantitative character, the Shapiro-Wilks normality test was first used to determine their distribution, then an independent sample t-test, after logarithmic transformation were carried out with SPSS ver. 25 software (IBM Corp., Armonk, NY, USA) [31]. Principal coordinate analysis (PCoA) was performed in PAST package ver. 4.04 software (Natural History Museum, Oslo, Norway) [32,33]. Some samples with missing data were not included in the principal coordinate analysis. Furthermore, the variability of the analyzed morphological characters was described by standard statistical parameters (mean, standard deviation, minimum, maximum, 10th and 90th percentiles). Boxplots were built through SPSS version 25. All morphological characters of dried material were observed with a Leica MZ16 stereoscopic microscope.

Chromosome Count
Chromosomes of the new species were counted from two individuals (on three metaphase plates) collected in the field on Mt. Morrone in loc. Fonte La Vena (July 2021) and then cultivated at the Floristic Research Centre of the Apennines (accession number 584/21). A voucher specimen has been deposited in the Herbarium Apenninicum (APP No. 66113). Squash preparations were made on ovules collected from living plants. Ovules were pretreated with a 0.4% colchicine solution for 4 h at room temperature and then fixed in Carnoy's solution for 45 min. Then, they were hydrolyzed in 1 N HCl solution for 6 min at 60 • C and stained with leucobasic fuchsine. Finally, they were squashed on clean glass slides with one drop of 45% acetic acid, before examination under a Leica DM750 light microscope for chromosome counting.

Scanning Electron Microscopic Analyses
Seed testa micro-morphology on mature and dry material (five seeds per species) was analyzed. For the new species the seeds were collected from cultivated plants (from Mt. Rozza, Central Italy, Abruzzo) at the Botanical Garden of Floristic Research Centre of the Apennine, for A. palentinum were collected from herbarium specimen SALA barcode 136402 (loc. Valle de Valverde, Castiglia and León, Northern Spain,). Before the analysis with scanning electron microscope Zeiss Gemini SEM 500 at an accelerating voltage of 7 kV, dried seeds were attached to carbon-coated aluminium sample blocks. Terminology of the seed coat sculpture follows Barthlott [34], and Baasanmunkh et al. [35].
Amplification reaction used a volume of 20 µL, with 5-9 ng of DNA template, 0.25 µM of each primer, and Phire Plant Direct PCR Master Mix (Thermo Fisher Scientific) according to the manufacturer's instructions. Annealing temperature (Ta) was equal to 55 • C for ITS and trnL (UAA) -trnF (GAA) IGS and 50 • C for the other two plastid IGS markers.
Phylogenetic analyses. After both a comparison of data in the literature (e.g., [25,39,40]) and our preliminary phylogenetic analyses, data sets from nrDNA and the combined cpDNA markers were analyzed separately. To know the systematic position of our new taxa, we selected from GenBank the Allium ITS accessions of almost the entire A. sect. Daghestanica (12 species for a total of 23 GenBank accessions) and some accessions representative of sect. Falcatifolia (3 species for a total of 10 GenBank accessions), sect. Oreiprason Hermann and sect. Reticulobulbosa (4 and 1 species, respectively, for a total of 10 GenBank accessions) (Supplementary Table S1). For the analysis of three plastid markers as a combined dataset, the accessions from GenBank were reduced in number to try to have the same specimen of the species used for the analysis of ITS, or at least that the plastid markers were always coming from the same specimen. Considering this and according to the ITS phylogeny results, 11 species (16 GenBank accessions) of A. sect. Daghestanica and 3 species (9 GenBank accessions) of A. sect. Falcatifolia were downloaded from GenBank (Supplementary Table  S1). Allium cyathophorum Bureau & Franch. [A. subgen. Cyathophora (Fritsch) Fritsch] was used as an outgroup for both analyses using both nuclear and plastid markers. The sequence accessions, herbarium code, locality information and literature references are listed in Supplementary Table S1.
The phylogenetic relationships were assessed both with Bayesian (BI) and maximum likelihood (ML) inference on a dataset comprising 44 and 26 previously published ITS and plastid concatenated sequences, respectively, plus eight new specimens (four of both A. ducissae sp. nov. and A. palentinum). The most likely substitution models for nuclear and plastid markers were computed with jModeltest ver. 2.1.10 software [48]. The better model according to the Akaike information criterion (AIC) was GTR + G for ITS marker, TVM + G for trnQ (UUG) -rps16 IGS, and rpL32-trnL (UAG) and TIM1 + G for trnL (UAA) -trnF (GAA) IGS.
For both nuclear and plastid datasets, MrBayes ver. 3.2.6 software [49] was used for BI and two runs of four Markov chains (three hot, one cold) were performed for 15,000,000 generations, sampling every 1500 generations, and discarding the first 10% and 19% as burn-in (ITS and plastid markers, respectively). Convergence diagnostics were checked with Tracer ver. 1.7.1 software [50]. According to the cpDNA markers the models GTR + G (trnL (UAA) -trnF (GAA) and rpL32-trnL (UAG) IGS) and GTR + I + G (trnQ (UUG) -rps16) were used, being closer to those models previously calculated with jModeltest. Finally, an ML inference was performed on both datasets using RaxML-NG via its web server portal (https://raxml-ng.vital-it.ch/#/; [51]; accessed date, 26 November 2021). Bootstrap analyses were carried out with an automatic number of replicates with a bootstopping cut-off of 0.03.      Etimology: Allium ducissae is named after the Duchessa mountains (in Latin, Montis Ducissae) where the species was discovered for the first time by Bruno Petriccione (under the name A. lineare, [11]).

Taxonomic Treatment
Chromosome number: a single population from "Mt. Morrone in loc. Fonte La Vena" (Lazio, Rieti province) of A. ducissae resulted in diploid with 2n = 16 + 2B chromosomes ( Figure 5). This chromosome count agrees with previous counts made for A. palentinum [52]. paucispecific primary habitat, such as crevices on calcareous rocks, rocky slopes, steppe grassland and grassy ledges, from 1800 up to 2130 m a.s.l (Figure 4)   Seed micromorphology: seeds of Allium ducissae showed irregularly polygonal testa cells with a broad, depressed and coarsely striated intercellular region. The anticlinal walls appeared depressed, straight to curved. The periclinal walls are flat, and densely granulate ( Figure 6A-C). The seeds of A. palentinum showed irregularly polygonal testa cells, with a broad, depressed intercellular region with obscure striation. The anticlinal walls appeared depressed, straight to curved. The periclinal walls are irregularly granulose (mainly on the margin), with a concave center ( Figure 6D-F).  Conservation status: Allium ducissae occurred in the NATURA 2000 network within the Sites of Community Interest "IT6020020 Monti della Duchessa" and "IT7110206 Monte Sirente e Monte Velino" in the Regional Natural Reserve "Montagne della Duchessa" (Lazio) and the Regional Park "Sirente Velino" (Abruzzo), respectively. Only the subpopulation of Mt. Orsello is not included in a protected area. The extent of occurrence (EOO) is 32.91 km 2 , calculated with minimum convex hull polygon in QGIS, and the area of occupancy (AOO) is 24 km 2 , calculated with a 2×2 km cell fixed grid. The main estimated threats include: global warming, which favors the arrival of more thermophilous competitive species; poor level of reproduction and regeneration, that leads to a decreasing population trend; restricted range, which causes a greater risk of extinction in the event of disease or pest attacks. The taxon occurs in one location and five subpopulations. The population is not declining and there are no extreme fluctuations. According to IUCN criteria [53], we propose to include A. ducissae in the following category: Near Threatened (NT).

Morphological Analyses
The principal coordinate analysis (PCoA, Figure 7) shows on the first two axes a clear distinction between A. ducissae and A. palentinum, and no overlapping areas among individuals were found. The most relevant morphological characters differentiating the two species are summarized in Table 2 and shown in Figure 8. The states of twelve characters (BW, SH, SAL, PFL, ITL, ITW, OTL, OTW, ITFL, OTFL, IFTL and SEL) show significant differences between the two species (p < 0.01). The geographic distribution of the herbarium specimens examined (Supplementary File S1) is shown in Figure 9. The map was created using the free and open source QGIS ver. 3.16.4. software [54].  Phenology: flowering from July to August, fruiting in September. Etimology: Allium ducissae is named after the Duchessa mountains (in Latin, Montis Ducissae) where the species was discovered for the first time by Bruno Petriccione (under the name A. lineare, [11]).
Chromosome number: a single population from "Mt. Morrone in loc. Fonte La Vena" (Lazio, Rieti province) of A. ducissae resulted in diploid with 2n = 16 + 2B chromosomes ( Figure 5). This chromosome count agrees with previous counts made for A. palentinum [52]. Seed micromorphology: seeds of Allium ducissae showed irregularly polygonal testa cells with a broad, depressed and coarsely striated intercellular region. The anticlinal walls appeared depressed, straight to curved. The periclinal walls are flat, and densely granulate ( Figure 6 A-C). The seeds of A. palentinum showed irregularly polygonal testa cells, with a broad, depressed intercellular region with obscure striation. The anticlinal walls appeared depressed, straight to curved. The periclinal walls are irregularly granulose (mainly on the margin), with a concave center (Figure 6 D-F). Conservation status: Allium ducissae occurred in the NATURA 2000 network within the Sites of Community Interest "IT6020020 Monti della Duchessa" and "IT7110206 Monte Sirente e Monte Velino" in the Regional Natural Reserve "Montagne della Duchessa" (Lazio) and the Regional Park "Sirente Velino" (Abruzzo), respectively. Only the subpopulation of Mt. Orsello is not included in a protected area. The extent of occurrence (EOO) is 32.91 km 2 , calculated with minimum convex hull polygon in QGIS, and the area of occupancy (AOO) is 24 km 2 , calculated with a 2×2 km cell fixed grid. The main estimated threats include: global warming, which favors the arrival of more thermophilous competitive species; poor level of reproduction and regeneration, that leads to a decreasing population trend; restricted range, which causes a greater risk of extinction in the event of disease or pest attacks. The taxon occurs in one location and five subpopulations. The population is not declining and there are no extreme fluctuations. According to IUCN criteria [53], we propose to include A. ducissae in the following category: Near Threatened (NT).
The phylogenetic trees derived from BI and ML analyses were topologically identical and the ML trees are only shown for both molecular markers (nrDNA and cpDNA) (Figures 10 and 11). The topology trees obtained were congruent with the literature data. (e.g., [25,39]) The posterior probabilities (PP) and bootstrap support (BS) showed high values in both analyses (Figures 10 and 11).
According to the ITS phylogenetic analysis (Figure 10), the accessions of A. ducissae sp. nov. and A. palentinum fit into a well-defined clade with other taxa belonging to A. sect. Falcatifolia N.Friesen; considering the tree topology, A. palentinum is close to A. ducissae. This datum was also confirmed with plastid data, as shown in Figure 11.

Discussion
Morphological and molecular analyses provide evidence that A. ducissae should be regarded as a new species, endemic to Abruzzo and Lazio (Central Apennines, Italy). The populations of A. ducissae were previously referred to as A. strictum [9,[11][12][13][14][15]20], a species morphologically different by having reticulate bulb tunics, leaves with a scabrousdenticulate margin, stamens usually slightly longer than perianth segments, inner filaments with a rounded tooth at the base, and subcapitate to capitate stigma [21]. Consequently, the occurrence of A. strictum from peninsular Italy should be excluded. This latter species is very rare in Italy, where it occurs only in the Alps, representing the western limit of its distribution area (e.g., [9,12,19,29]).
Allium ducissae is similar to A. palentinum, an endemic species of the Cantabrian Mountains (NW Spain), but it can be distinguished by several morphological characters, as shown in Table 1. It also showed slight differences in seed testa microsculptures, with densely granulated periclinal walls with a flat center (vs. mainly granulated at the margin with a concave center) and anticlinal walls with a coarsely striated intercellular region (vs. obscure striation).
Based on molecular analyses, both A. ducissae and A. palentinum belong to A. sect. Falcatifolia, contrary to what is known for the latter species, usually included in A. sect. Daghestanica [25]. Moreover, our results of seed testa micromorphology agreed with the findings for other species belonging to A. sect. Falcatifolia [35,55,56]. This latter section was originally described by Friesen [4] including only A. carolinianum DC. ex Redouté and A. platyspathum Schrenk; recently, however, this was newly circumscribed with the inclusion of several species [37,57,58]. Allium ducissae and A. palentinum are the westernmost representatives of this section, and the only ones present in Europe.
Allium ducissae joins the flora of the Central Apennines, an area with high plant species diversity, where there are the protected areas with the highest number of taxa in Europe and the Mediterranean Basin [59][60][61]. The Central Apennines are also known for being a large contingent of endemic taxa [62][63][64][65][66], some of which have recently been described or re-evaluated as, for example, Anthyllis apennina F.Conti & Bartolucci [67], Corydalis densiflora C.Presl subsp. apennina F. Conti [78], and Sedum aquilanum L.Gallo & F.Conti [79]. Endemics are the most vulnerable component of a flora, and many of the mentioned endemic taxa might become endangered or extinct due to their restricted area of distribution and strict ecological requirements. Systematic and taxonomic studies, and the phytogeographic analysis of endemic taxa are essential for setting conservation priorities and for driving in situ conservation measures and ex situ conservation activities [66,80,81].
The actual range of A. ducissae is restricted to the Velino massif, a territory in the Central Apennines with a distinctly continental climate. All the existing populations occur in paucispecific primary habitats, preferring northern exposures or other spots with long persistence of snow cover (microthermic conditions). The ecological features and the restricted distribution area could support the hypothesis that A. ducissae is a species differentiated in the Quaternary due to the fragmentation of the range of a widespread ancestral Tertiary taxon.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The datasets generated in the current study are available from the corresponding author on reasonable request.