A new subspecies of Peucedanum officinale L. subsp. album (Apiaceae) from the eastern part of the Iberian Peninsula

Abstract We describe Peucedanum officinale L. subsp. album Martínez-Fort & Donat-Torres subsp. nov., in which we grouped the thermomediterranean populations scattered along the eastern part of the Iberian Peninsula. The characters that differentiate this new subspecies from other infraspecific taxa in Peucedanum officinale are its canaliculated leaflet, the inflorescences much branched and lack of dominant terminal umbels, the umbels are few rayed, sometimes sessile and lateral, the petals are white and the fruit pedicels short, the same or shorter in length than the fruit. We provide here a full description of the new subspecies based on herbarium specimens and field measurements, as well as providing dichotomous keys to the subspecies within P. officinale. In addition, we provide a comparison of the ITS sequences of nrDNA with the most closely related taxons.


Introduction
Peucedanum L. 1753 is one of the most complex genera in the Apiaceae family. Based on the morphological characteristics of the fruit, according to the traditional classification systems, it is characterised by a strong dorsal compression and winged side ribs as in Drude (1898). It is included within the Peucedaneae tribe, subtribe Ferulinae and has been defined as Peucedanum sensu amplissimo. In addition, this genus is broadly represented by 29 species in European flora (Tutin 1968), of which 10 are present in Iberian flora (Guillén and Laínz 2003). In the grouping of Pimenov and Leonov, Peucedanum sensu lato includes 100-120 scattered species throughout the Old World and the need for its reduction into natural groups has been proposed Leonov 1993, Downie et al. 2000).
On the other hand, phylogenetic studies, based on the ITS rDNA sequences, define "Peucedanum sensu stricto clade including taxa that are very similar with respect to their ITS sequences and they are very related in habit, sharing not only fruit characters but also vegetative features, like linear-filiform leaf lobes" and it has been regrouped in the Tribe Selineae Spreng (Spalik et al. 2004). Molecular phylogenetic studies confirm the separate taxonomic status of the other taxa which have been accepted as separate genera (Spalik et al. 2004, Winter et al. 2008, Downie et al. 2010. Therefore, the genus is now reduced to a few species and the type of the genus is Peucedanum officinale L. The morphological differences between some populations from the central and eastern coastal parts of the Iberian Peninsula have long since been evidenced with the description and citation of several P. officinale subspecies (Boissier 1844, Willkomm and Lange 1880, Cadevall i Diars 1919-1923, Sennen 1913).
The first distinction from the Iberian populations in P. officinale was made with the description of P. stenocarpum Boiss. & Reut. ex Boiss., 1844 (Boissier 1844), which were grouped under this name, the populations being located in the centre of the Peninsula, characterised by the number of floral scapes (4 to 5), long leaf divisions of 3-4 inches in length (7.6-10.6 cm) and fructiferous raylets that triple the length of fruits, which are elliptic-ovate.
Subsequently, Willkomm and Lange (1880) cited P. officinale L. var. italicum (Mill.) DC. in Lam. &DC 1805 andP. paniculatum Loisel. (1807) in the Peninsula and these were added to encompass the dispersed populations in the central, northern and north-easterly parts of the Peninsula. Finally, these populations were assigned to P. stenocarpum (Cadevall i Diars 1919-1923. Another name proposed was P. stenocarpum var. catalaunicum Pau in Sennen (1913) (nom. in sched.), but no description or herbarium specimens were provided. Reduron and Muckensturm (2008) gave a short Latin diagnosis but did not designate a lectotype.
Afterwards, P. stenocarpum was included as a subspecies of P. officinale (viz. P. officinale L. subsp. stenocarpum Font Quer 1950) and included all the populations in the eastern half of the Peninsula. Its morphological characteristics were summarised as: extended (up to 15 cm) and very narrow (from 0.3 to 2 mm) leaf segments, with between 10 and 64 umbel rays and a ratio of fructiferous pedicel length to fruit length from 0.5 to 1.5 (Font Quer 1954). Font Quer measured some of the populations that we have included in the present study and which have always been assigned the extreme value in the description of his study. Along with the subspecies officinale, they are the only two taxa in the group recognised in the Peninsula at this time. Peucedanum paniculatum and P. longifolium Waldst. & Kit. 1812 were reduced to subspecies of Peucedanum officinale due to their ternate leaves, which were divided into linear or linear-lanceolate segments by Frey (1989).
The last subspecies in the group, described in the Iberian Peninsula, Peucedanum officinale L. subsp. brachyradium García-Martín and Silvestre 1991 specifically in the province of Málaga (Spain), was indicated as an edapho-endemism on peridotites. That description derives from a population with only two individuals and is morphologically distinguished from the subspecies type and from P. stenocarpum by the characteristics of its inflorescence and by the dimensions of its fruit and pedicels. García Martín and Silvestre (1992) also suggested similarities with P. officinale subsp. longifolium.
The latest review undertaken of the genus for the Iberian Peninsula (Guillén and Laínz 2003) recognised only one species with two subspecies, viz. P. officinale subsp. officinale and P. officinale subsp. brachyradium. These authors included P. stenocarpum as a synonym of P. officinale subsp officinale, suggesting that there was variability and mixture within and between the populations.

Materials and methods
While conducting fieldwork during the previous years, we located the population of an umbelliferous species in eastern spurs of the Serra Grossa mountain range, which lies in the southern part of the Valencian province (eastern part of the Iberian Peninsula). This species is always found at the bottom and top of rocky areas, on the edges of paths, always in cracks in rocks and on rocky soil. It is characterised by possessing ternatisect leaves, with linear and canaliculated leaf divisions; inflorescences with sessile lateral umbels and with few rays; white-petalled flowers; elliptic fruits with a strong dorsal compression and prominent dorsal ribs and winged marginal ribs, borne on short pedicels. As a result, we assigned it to the genus Peucedanum sensu stricto.
To further identify this taxon and compare all of the subspecies of P. officinale throughout its area of distribution, we reviewed the herbarium specimens of the genus deposited in the VAL and MA herbaria. We studied all the bibliography about the P. officinale subspecies cited and described in the Iberian Peninsula and the monograph on the genus by Frey (1989). For this monograph, 2,500 herbarium sheets from all over Europe were consulted and measurements were taken from 200 of them. For the subspecies P. officinale subsp. brachyradium, the measurements were provided in the description article (García Martín and Silvestre 1992).
We noted that the population under investigation here had been identified to date as P. stenocarpum, widely considered as a synonym of P. officinale subsp. officinale, despite the fact that it has a series of morphological characters that are not accommodated within any of the subspecies of P. officinale ( Figures 1B-D). For its characterisation and comparison, measurements of the plants were taken and the morphological ratios calculated for habit, leaves, inflorescences and fruits (Table 2, Suppl. material 1: Figure S1). The specimens belong to the located population in Carcaixent, as well as other closer populations located all along the coastline of the Valencian region (in Benicasim, Tavernes de la Valldigna, Alzira, Xeresa) ( Figure 1A) with the same series of morphological characteristics. Hereafter we referred to this new studied subspecies as P. officinale subsp. album. To complete the results, we compared these populations with a population assigned to the subspecies type. We selected those populations located further inland, specifically between the provinces of Cuenca and Valencia, which correspond to P. officinale subsp. officinale. The series of populations, studied in the field, are provided in Table 1.
Measurements were taken in the field and in the laboratory and always carried out with fresh plants. We used a CD-20DCX digital vernier caliper and a metal tape measure and saved measurements on spreadsheets and databases. We created the figure of P. officinale subsp. album using the holotype VAL 223161 to scan, as well as from the photos taken in the field with a Canon EOS 550D camera of inflorescences and fruits. Other photos were taken with a Leica stereomicroscope MZ 9.5 and a Leica DFC 320 digital camera of the cross-sectional cuts of fruits and leaf details. We obtained the flowering and fructification data while conducting fieldwork and according to the date of the specimens and phenologies from the consulted herbarium sheets. All the other figures are photos of herbarium sheets and pictures of scanned herbarium sheets. We compared and statistically analysed the taken measurements with the Statgraphics Centurion XVI software. For morphological characters, we followed the standardised terminology of Kljuykov et al. (2004). For the taken measurements, we calculated the range obtained from their mean +/-standard deviation. The extreme values beyond this range are in brackets in the tables.
Taking measurements was limited by the small proportion of individuals which were flowering or under fructification in all the populations; indeed, flowering and fructification did not even exceed 4% of the individuals in the largest populations. In the specimens that presented no flowering, measurements were taken from the basal rosette leaves. In all, we took measurements from 31 specimens, of which 19 belonged to populations of the newly proposed taxon and 12 to the type subspecies. In all, 1,316 measurements and ratios were taken and calculated.

Revised herbarium material
The examined herbarium specimens' material at VAL and MA are listed below.

DNA extraction, amplification and sequencing
We made a genetic comparison of the ITS regions of ribosomal DNA between the populations of P. officinale subsp. officinale and P. officinale subsp. album, by extending the field sampling to one of the populations close to the classical location where P. stenocarpum had been described, El Escorial (Madrid) (Population Po7 in Figure 1 and Table  1). Using fragments of the basal leaves and shoots obtained from the seeds collected in the field, we extracted total DNA with the Plant DNA kit of Omega Bio-Tek, following the manufacturer's instructions. To amplify the ITS regions of ribosomal DNA, we used oligonucleotides ITS5 and ITS4 (White et al. 1990) and the MBL Taq Polymerase kit of Molecular Biology Laboratory SL. Sequencing was done by MACROGEN using these same universal primers. The extraction process of the DNA extracted from the leaves collected in the field was complicated by the presence of metabolites, but was much easier to perform on the shoots of germinated seeds. We extracted DNA and sequenced the ITS region of ribosomal DNA from four populations ( Table 3). The obtained sequences were aligned with CLUSTALW from Bioedit 7.2.5. (Hall 1999). We aligned the consensus sequence obtained with BLASTn (Altschul et al. 1990) to obtain the genus Peucedanum sequences, with which a final set of sufficiently long sequences was used to be able to compare them with our sequence (Suppl. material 2: Table S1).
The pairwise genetic distances between sequences were calculated with MEGA, version X (Kumar et al. 2018) with 10,000 bootstraps replicates and gamma distribution (shape parameter = 0.7). The employed model was TN93+G, available in MEGA and amongst the best models obtained previously with JmodelTest and the Bayesian Information Criterion (BIC) (Darriba et al. 2012).

Results and discussion
By taking the morphological differences observed in leaves, inflorescences and fruits, its habitat and distribution in the humid and sub-humid thermomediterranean bioclimatic types as a basis, we distinguished P. officinale subsp. album as a new subspecies, after its comparison with all the revised herbarium specimens and data provided in the bibliography (Table 4). Compared to the nominated subspecies, with which it has contact in its distribution, it is easily distinguished by the white colour of the petals (Figure 2A), its inflorescences without dominant umbels and umbels with scarce rays and sometimes sessile and with canaliculated leaflets. There is a difference in the subspecies type that possesses inflorescences with dominant umbels of a greater number of rays and with flat leaflets (Figs 4 A-B). P. officinale subsp. album can be further distinguished from subspecies paniculatum, which is restricted to the islands of Corsica and Sardinia, by its inflorescences, which are much more branched and paniculated, with many rayed umbels and fruit pedicels that are three times the length of the fruit. The subspecies brachyradium has very limited distribution being an edapho-endemic species on peridotite of the province of Malaga. It is distinguished by its greater bearing, inflorescence with more rays and fruit pedicels equal in length to the fruit. Regarding the genetic results obtained, the ITS sequences of the studied specimens that belong to the populations of Xeresa, Benicasim, Tuejar and El Escorial were identical to one another. The length of the obtained consensus sequence was 603 base pairs. It is deposited in GenBank http://www.ncbi.nlm.nih.gov/GenBank). The accession number is KP681852.
This sequence is identical to all the P. officinale sequences in GenBank and has a comparable length when using sets of sequences with both 319 bp and 640 bp (with gaps) (Suppl. material 2: Table S1). In comparison, the distance between P. officinale and the other species of the Peucedanum s.str. varied from 0.003 with P. gallicum Latour. and 0.211 with P. sandwicense Hillebr. The distance with P. gallicum is very small, however it is a species clearly accepted. As the distances are so small between near species, the analysis has not allowed separation genetically at the subspecific level. This supports the intraspecific range in P. officinale for the subspecies album.
The briefness and the small number of characteristics that have been analysed in the studies have meant that our determination of these populations was complicated. With the exhaustive comparison that we have carried out, we have been able to identify the characteristics (Table 4) that distinguish these coastal populations as another subspecies. In many of the descriptions and determination keys, leaf morphology was stressed: the length, width and angle of the last-order leaf divisions; and some fruit characters or the number of umbel rays were also used to classify the different subspecies. According to the measurements and observations made in the field, we do not consider the length and width of the last leaf division to display good characters for distinction, as it could have already varied in the same population according to the environmental conditions where the plant grows.
Conversely, the leaflets shape characteristics of the last divisions; flat, folded-crested or canaliculated and the shape of inflorescences: with terminal, pedunculate umbels with 17-35 rays that are 30-85 mm long or sometimes with panicled inflorescences or with umbels, some of which are sessile and lateral, with 5-9 rays measuring 8-43 mm, were determining factors for separating the subspecies. Thus, the album, brachyradium and paniculatum subspecies share the combination of canaliculated leaflets and panicled inflorescences or with sessile umbels and fewer rays; as opposed to the officinale and longifolium subspecies, which have flat or grooved leaflets and inflorescences with terminal umbels and more rays.
These values obtained in the field have been used for the description of P. officinale subsp. album (Table 5). Of them all, we obtained statistically significant differences with a 95% confidence level between the two subspecies album and officinale in the following characters (Table 6). These parameters are the number of rays of the umbels and umbellules (raylets), the length of the fruit and its peduncle and the proportion in the leaves between length and width and angles of separation between the last leaf divisions (leaflets).
Distribution and habitat. It is dispersed in the thermomediterranean sub-humid bioclima (Rivas-Martínez 2004) on rocky soils of limestone and sandstone at the base  (180) * With more than 30 types of data. Differences obtained between the means with an ANOVA analysis; as normality or homoscedasticity was not met, the result was confirmed by the Kruskal Wallis test. ** With more than 30 data. Differences obtained between the means with an ANOVA analysis. *** With fewer than 30 data. If normality and homoscedasticity were met, the Mann-Whitney W (Wilcoxon) test was used.   and crest of cliffs and rocky slopes. Contacting with wet fringes of Rubio longifoliae-Quercetum rotundifolie Costa, Peris and Figuerola (Costa et al. 1983), together with ash and arbutus. With abundant populations on the coastal and southern foothills of the Iberian system (Serra de les Agulles) and on the northern coastal foothills of the external Prebaetic system (Serra Grossa) (Figure 3). Phenology. Flowering July to October. Paratypes. SPAIN. Valencia: Alzira. Serra de la Murta, 30SYJ2934; on rocky outcrops, 25-IX-2011; J. Martínez-Fort (VAL 223162!, Figure 1B; Valencia: Corbera de Alzira, 30SYJ23; J. Borja (VAL 154996!; Figure 1C-D); Valencia: Favara, Serra de Corbera, 30SYJ33, 5-IX-1986;G. Mateo & al. (VAL 117826!).