Anthyllis apennina (Fabaceae), a new species from central Apennine (Italy)

Abstract A new species of Anthyllis endemic to central Apennine growing in dry pastures on limestone in the montane belt, within Abruzzo and Lazio administrative regions (central Italy), is here described and illustrated and the IUCN assessment is proposed. This new species belongs to the morphologically very variable Anthyllis vulneraria species complex and it is close to A. pulchella (south-eastern Europe and Caucasus), but it can be clearly distinguished by its smaller flowers, mainly light yellow-coloured, bracts longer than flowers, calyx pink-coloured (usually only at apex) and size of cauline leaves and leaflets. Furthermore, the name A. pulchella is here lectotypified, by a second-step typification, on a specimen preserved at PAD.


Introduction
The genus Anthyllis L. belongs to subfamily Papilionoideae, tribe Loteae (Polhill 1981;Allan and Porter 2000;Degtjareva et al. 2012). The A. vulneraria species complex, distributed in Europe, Mediterranean Basin, Turkey, Caucasus, Iran, Eritrea and Ethiopia, is morphologically very variable and several infraspecific taxa, many of them frequently regarded as independent species, have been recognised (e.g. Cullen 1968Cullen , 1976Akeroyd 1988;Roskov et al. 2006;Rola 2012;POWO 2019). Roskov et al. (2006) re-ported from Europe and Mediterranean countries 29 infraspecific taxa. Further taxa are quoted by Lampinen (1990), Benedí (2000), Greuter and Raus (2005), Puidet et al. (2005), Tison and Foucault (2014) and Conti et al. (2016). With regards to Italy, the A. vulneraria complex is represented by 15 subspecies, of which two are endemic and one is alien Galasso et al. 2018). On the other hand, Pignatti et al. (2017Pignatti et al. ( , 2019 recognise the taxa belonging to this group as species and quoted for Italy 13 species (two alien), one subspecies and five hybrids. In Italy, according to our field observations, many taxa are clearly sympatric, growing in the same localities and habitat. These taxa are usually clearly distinct and, probably, to be considered as separate and independent species. The lack of nomenclatural, taxonomic and molecular studies regarding this species complex has led to unclear descriptions of taxa and to unworkable analytical keys. We believe that some taxa should be recognised at species rank as proposed by Pignatti et al. (2019), but further studies are still needed. During the fieldwork concerning the "Flora of Abruzzo" project, one of us (FC) came across a population of Anthyllis on Mt. Ocre showing peculiar morphological features. Later, we found other populations with plants characterized by the same features on Mt. Boragine and Selva Rotonda (Lazio, Reatini Mountains) during the annual field trip of the working group for Floristics, Systematics and Evolution of the Italian Botanical Society held in 2016 . Additional populations were discovered on the southern slopes of Pizzo Camarda and between S. Stefano di Sessanio and S. Colombo (Gran Sasso massif ), Vallone di Sevice and Piani di Pezza (Mt. Velino), Mt. Sirente, Mt. Calvo and in some localities of the National Park of Abruzzo, Lazio and Molise (Mt. Tricella, Colle Biferno, La Brecciosa). Plants of these populations are morphologically different from other known taxa within the A. vulneraria species complex, showing a peculiar combination of characters as very small flowers, mainly light yellow-coloured, and bracts longer than flowers. They are confused in the past with the close A. pulchella (Vis.) Beck (≡ A. vulneraria L. subsp. pulchella (Vis.) Bornm.), a species distributed in south-eastern Europe, Crimea and Caucasus (Cullen 1976;Akeroyd 1986;Dimopoulos et al. 2013;Barina et al. 2018;Bartolucci et al. 2018;Pignatti et al. 2019). Two other similar taxa, but clearly distinguished, are the endemic to central Apennine A. vulneraria subsp. nana (Ten.) Tammaro (Conti et al. 2016) and the western European A. vulneraria subsp. vulnerarioides (All.) Arcang. (Benedí 2000;Tison and Foucault 2014;Conti et al. 2016). Field investigations and an extensive morphological study on herbarium material, providing evidence about the species differentiation between A. pulchella and the Apennine populations, have been carried out. The results allowed us to describe the Apennine populations as a new species with the name A. apennina.

Material and methods
This study is based mainly on field surveys, on an extensive analysis of relevant literature and on examination of herbarium specimens (including nomenclatural types) preserved at APP, B, BM, GAP, P, PAD, TO (codes following Thiers 2020). A total of 125 specimens from these herbaria were studied. Ad hoc sampling campaigns in the central Apennines to better understand the distribution of the new species were carried out. The herbarium specimens of A. vulneraria subsp. vulnerarioides and A. vulneraria subsp. nana were studied for a preliminary morphological circumscription of the species, and not used for morphological analyses. A more detailed morphological comparison involved A. apennina and the closest species A. pulchella. Morphological observations and measurements of qualitative and quantitative characters, considered as diagnostic in Anthyllis (e.g., Cullen 1968Cullen , 1976Akeroyd 1988;Rola 2012), were analysed on dried specimens. The analyses were performed on 58 variables, including 40 quantitative continuous characters, 6 quantitative discrete characters, 2 ratios and 10 qualitative characters ( Table 1). The morphometric analyses were carried out on 37 selected specimens including A. apennina (21 specimens) and A. pulchella (16 specimens). Each flower was soaked in water for a few seconds before taking the measurements, using the maximum parameters. Calyx length include hairs protruding upper teeth. The calyx was sectioned longitudinally and then the width of its entire development was measured. Flower colour is based on living specimens because it was not possible to evaluate this character reliably from herbarium specimens. All morphological characters were observed with a Leica MZ16 stereomicroscope. The herbarium specimens used for the morphometric analyses are shown in Fig. 1 (produced using QGIS 3.16.4).
For each quantitative character, Shapiro-Wilks normality test was first used to determine their distribution, then an independent sample T-test, after logarithmic transformation, was carried out with SPSS version 25 (IBM 2017). Principal coordinate analysis (PCoA) and cluster analysis (UPGMA) were performed in PAST package version 4.03 (Hammer et al. 2001;Hammer 2020). Furthermore, the variability of the analysed morphological characters was described by standard statistical parameters (mean, standard deviation, minimum, maximum, 10 th and 90 th percentiles). Boxplots were built by means of SPSS version 25 (IBM 2017).  For the lectotype selection of the name A. pulchella, the protologue has been compared with original material and the most complete and informative specimen was selected (Art. 9.4, Turland et al. 2018 Diagnosis. Anthyllis apennina differs from A. pulchella by the bigger cauline leaves and leaflets, leaves evenly distributed along the flowering stem vs. concentrated in lower half, higher number of inflorescences 2-5(-10) vs. 1-2, smaller flowers with standard (7.9-)8.5-9.9(-10.3) vs. (10-)10.4-13.6(-13.8) mm long, longer bracts (12-)14-23(-26) vs. (5.8-)6.5-11(-12) mm long, longer than flowers, with longer lobes (10-)11-21.5(-24) vs. (3.2-)3.9-7.5(-9) mm long, narrowly triangular and acute to apiculate vs. more or less parallel-sided and obtuse and by the colour of flowers which are mainly light yellow or flushed with pink vs. purplish-pink or cream flushed with pink and the calyx which is mainly pink only in the upper part.
Etymology. Anthyllis apennina is named after the Apennine to which the species is endemic.
Habitat. Pastures in montane belt from 1200 to 1800 m a.s.l. Phenology. Flowering from the second half of April to the end of July, fruiting in June-July.
Distribution. Central Apennine in Lazio and Abruzzo (Fig. 1). Conservation status. Some of the populations of A. apennina occurred in a NAT-URA 2000 network within the Sites of Community Interest "IT7110208 Monte Calvo e Colle di Macchialunga", "IT7110206 Monte Sirente e Monte Velino", "IT710202 Gran Sasso" and within the Abruzzo, Lazio and Molise National Park, Gran Sasso and Laga Mountains National Park and Sirente-Velino Regional Park. The extent of occurrence (EOO) is 1788 km 2 calculated with the minimum convex hull polygon in QGIS and area of occupancy (AOO) is 52 km 2 calculated with a 2×2 km cell fixed grid. No pressures or threats are evidenced. According to IUCN Criteria (IUCN 2019), we propose to include A. apennina in the following category: Least Concern (LC).
Morphometric analysis. The principal coordinate analysis (PCoA, Fig. 4) shows on the first two axes (explained variance: 57.1% and 11.1%) a clear separation between A. apennina and A. pulchella. Concerning the intraspecific variation within A. pulchella, one separated specimen ("37" from Turkey; see Fig. 1 and specimens examined) can be recognized in the PCoA diagram. Cluster analysis (UPGMA, Fig. 5), shows two well-delimited clusters supporting a clear separation between the studied species. Also in the UPGMA the specimen "37" from Turkey is separated. Most of the quantitative morphometric characters evaluated showed significant differences between the two Anthyllis species (Table 2). The most relevant morphological characters differentiating the two species are summarized in Table 3 and shown in Fig. 6.