Carpological Analysis of Two Endemic Italian Species: Pimpinella anisoides and Pimpinella gussonei (Apiaceae)

This study aims to clarify the taxonomic doubts, which have varied over the centuries, on the only two endemic species of the genus Pimpinella growing in Italy: P. anisoides and P. gussonei. For this purpose, the main carpological characters of the two species were examined, analyzing the external morphological traits and their cross-sections. Fourteen morphological traits were identified, obtaining datasets for the two groups using 40 mericarps (20 per species). The obtained measurements were subjected to statistical analysis (MANOVA and PCA). Our results highlight that at least 10 of the 14 morphological traits analyzed support the distinction between P. anisoides and P. gussonei. In particular, the following carpological characters are very significant ways to distinguish between the two species: monocarp width and length (Mw, Ml), monocarp length from base to maximum width (Mm), stylopodium width and length (Sw, Sl), length/width ratio (l/w) and cross-section area (CSa). In particular, the fruit of P. anisoides is larger (Mw 1.61 ± 0.10 mm) than that of P. gussonei (Mw 1.27 ± 0.13 mm), the mericarps of the first species are longer (Ml 3.14 ± 0.32 vs. 2.26 ± 0.18 mm) and the cross-section area (CSa) of P. gussonei is larger (0.92 ± 0.19 mm) than that of P. anisoides (0.69 ± 0.12 mm). The results also highlight the importance of the morphological traits of the carpological structures for the specific discrimination of similar species. The findings of this study contribute to an evaluation of the taxonomic significance of this species within the genus Pimpinella, and also provide valuable information for the conservation of these two endemic species.


Introduction
The genus Pimpinella L. is one of the most complex and species-rich of the family Apiaceae Lindl. Despite studies by numerous authors, its taxonomy is still not fully resolved [1][2][3][4][5]. Fernández Prieto et al. [6], in their study of the Pimpinella species from Western Europe, pointed out that it is a polyphyletic group. The genus includes about 150-180 taxa distributed in temperate and subtropical regions of Eurasia and Africa, South America, and western North America [6][7][8][9].
In the taxonomic studies on this genus, various morphological characters were considered [5,7,9,10], focusing on the micromorphological and anatomical characteristics of the fruit [11][12][13][14][15][16][17][18]. Carpological characters have traditionally been used for a long time in the systematics of Apiaceae, being among the most accessible to researchers [1,3]. The fruit anatomy and morphology have considerable taxonomic and evolutionary importance and, together with the molecular phylogenetic analyses, allow defining a stable taxonomy and an evolutionary history of species and genera [17]. Molecular phylogenetic analyses have raised new questions and, in many cases, have initiated a review of generic boundaries [5,6]. Kljuykov et al. [18] highlighted the requirements of morphological analyses for  [35], attributed to Tragium gussonei (on the right) by Presl [36], Gussone [37], Bertoloni [39].
Caruel [40] considers both P. gussonei and P. anisoides to be synonyms of Apium anisoides. Fiori [41] considers P. gussonei to be a variety of P. anisoides, pointing out that the distinctive characters of the varieties are attributable to carpological differences.
The historical excursus shows that the taxonomic events related to Pimpinella gussonei and Pimpinella anisoides are very complex and still unresolved.
This study provides a first characterization and differentiation between these two endemic species with the analysis of the carpological traits. This distinction is also helpful for economic purposes to identify commonly marketed fruits and counteract adulteration

Carpological Analysis
In agreement with Cano et al. [48], fruit morphometry is a good tool for distinguishing similar species that differ in a few characters that are not always unique.
In the case of P. anisoides and P. gussonei, the schizocarp of both species is similar, oblong-ovoid in shape, with five grooves between five prominent, and more or less prominent dorsal ribs; it consists of two homomorphic mericarps, having a flat ventral surface, and is joined by a carpophore bifid to the middle, usually splitting when ripe [19].
In P. anisoides, the fruit is glabrous throughout, dark brown-black in color, having an average length of 3.14 (±0.59) mm and a width of 1.61 (±0.16) mm; the average length/width ratio is 1.94, and the distance between the base and the maximum width is 1.25 (±0.20) mm. The stylopodium has a length of 0.38 (±0.13) mm and a width of 0.47 (±0.14) mm. In P. gussonei, the fruit is pubescent due to numerous single hairs appressed to the dorsal part, the ventral part is glabrous or with a few scattered hairs and grey-black in color; on average it is 2.26 (±0.33) mm long and 1.27 (±0.27) mm wide, the average length/width ratio is 1.70, and the distance between the base and the maximum width is 0.91 (±0.19) mm. The stylopodium has a length of 0.30 (±0.07) mm and a width of 0.25 (±0.42) mm ( Figure 2; Table 1). The historical excursus shows that the taxonomic events related to Pimpinella gussonei and Pimpinella anisoides are very complex and still unresolved.
This study provides a first characterization and differentiation between these two endemic species with the analysis of the carpological traits. This distinction is also helpful for economic purposes to identify commonly marketed fruits and counteract adulteration

Carpological Analysis
In agreement with Cano et al. [48], fruit morphometry is a good tool for distinguishing similar species that differ in a few characters that are not always unique.
In the case of P. anisoides and P. gussonei, the schizocarp of both species is similar, oblong-ovoid in shape, with five grooves between five prominent, and more or less prominent dorsal ribs; it consists of two homomorphic mericarps, having a flat ventral surface, and is joined by a carpophore bifid to the middle, usually splitting when ripe [19].
In P. anisoides, the fruit is glabrous throughout, dark brown-black in color, having an average length of 3.14 (±0.59) mm and a width of 1.61 (±0.16) mm; the average length/width ratio is 1.94, and the distance between the base and the maximum width is 1.25 (±0.20) mm. The stylopodium has a length of 0.38 (±0.13) mm and a width of 0.47 (±0.14) mm. In P. gussonei, the fruit is pubescent due to numerous single hairs appressed to the dorsal part, the ventral part is glabrous or with a few scattered hairs and grey-black in color; on average it is 2.26 (±0.33) mm long and 1.27 (±0.27) mm wide, the average length/width ratio is 1.70, and the distance between the base and the maximum width is 0.91 (±0.19) mm. The stylopodium has a length of 0.30 (±0.07) mm and a width of 0.25 (±0.42) mm ( Figure 2; Table 1).   The cross-section of the mericarps in both species is similar and shows morphological characteristics according to Laface et al. [20]; the dimensions of the different characters examined differ.

Statistical Analysis
The results of the multivariate analysis of the characters are summarized in Table 1 and shown in the boxplots in Figure 4. The MANOVA multivariate analysis used to define the significance of the analyzed characters, shows that Mw, Ml, Mm, Sw, Sl, l/w and CSa, are highly significant (p < 0.001), while CSw, CVw and CV w/t are not very significant (p < 0.05), and the characters CVt, CCSl and CS w/t are not significant. The F values also show that the two examined species display different morphological traits, The characters found to be non-significant, CSt, CVt, CCSl and CS w/t, have values of F < 1. The boxplots (Figure 4) allow us to visualize the centre and distribution of the data, and highlight some outliers for the characters Sw, CVw and CV w/t. The cross-section of the mericarps in both species is similar and shows morpholog characteristics according to Laface et al. [20]; the dimensions of the different charac examined differ.

Statistical Analysis
The results of the multivariate analysis of the characters are summarized in Tab and shown in the boxplots in Figure 4. The MANOVA multivariate analysis used to de the significance of the analyzed characters, shows that Mw, Ml, Mm, Sw, Sl, l/w and are highly significant (p < 0.001), while CSw, CVw and CV w/t are not very significan < 0.05), and the characters CVt, CCSl and CS w/t are not significant. The  4) allow us to visualize the centre and distribution of the data, and highlight some out for the characters Sw, CVw and CV w/t. The PCA ( Figure 5) clearly separates the fruits of P. gussonei and P. anisoides, showing a strong distinction between the carpological characters of the two species. In addition, the PCA shows that the first five characters with highest significance (Mw 76% variance, Ml 12% variance, Mm 4% variance, Sw 4% variance), explain 96% of the total variance. The biplot ( Figure 5) also shows that some characters (Ml, Mm, Sw, Mw, l/w) contribute better to the characterization of P. anisoides than P. gussonei. On the whole, the characters concerning the external morphology of the fruit allow better distinction between the two species than the characters concerning the section of the fruit.  Brig. and Pimpinella gussonei (C.Presl) Bertol. Character abbreviations: fruit width (Mw); fruit length (Ml); length from the lower end to the point of maximum width (Mm); stylopodium width and length (Sw, Sl); length/width ratio (l/w); area per section (CSa); commissural vittae width (CVw); section width (CSw); commissural vittae width/thickness ratio (CVw/t). The terminology of fruit parts and section is in accordance with Kljuykov et al. [19], Akalın et al. [9], Yeşil et al. [18] The PCA ( Figure 5) clearly separates the fruits of P. gussonei and P. anisoides, showing a strong distinction between the carpological characters of the two species. In addition, the PCA shows that the first five characters with highest significance (Mw 76% variance, Ml 12% variance, Mm 4% variance, Sw 4% variance), explain 96% of the total variance. The biplot ( Figure 5) also shows that some characters (Ml, Mm, Sw, Mw, l/w) contribute better to the characterization of P. anisoides than P. gussonei. On the whole, the characters concerning the external morphology of the fruit allow better distinction between the two species than the characters concerning the section of the fruit. ; stylopodium width and length (Sw, Sl); length/width ratio (l/w); area per section (CSa); commissural vittae width (CVw); section width (CSw); commissural vittae width/thickness ratio (CVw/t). The terminology of fruit parts and section is in accordance with Kljuykov et al. [19], Akalın et al. [9], Yeşil et al. [18].
The obtained results highlight that the two species have similar carpological characteristics but have different size characters (Figure 4). The fruit of P. anisoides is larger (Mw 1.61 ± 0.10 mm) than that of P. gussonei (Mw 1.27 ± 0.13 mm); in addition, the mericarps of the first species are longer (Ml 3.14 ± 0.32 vs. 2.26 ± 0.18 mm). The stylopodium of P. anisoides is wider (Sw 0.47 ± 0.06 mm) than that of P. gussonei (0.25 ± 0.19 mm). The cross-section area (CSa) of P. gussonei is larger (0.92 ± 0.19 mm) than that of P. anisoides (0.69 ± 0.12 mm). The commissural vittae width/thickness ratio (CV w/t) is 3.25 ± 0.84 in P. anisoides and 2.73 ± 0.45 in P. gussonei, showing a more compressed shape in the mericarp of the first species. Overall, the mericarps of P. anisoides appear slightly more oblong (l/w-length/width ratio = 1.94) than those of P. gussonei (l/w-length/width ratio = 1.70). In agreement with Gussone [37], all the samples of P. gussonei fruit are grey tomentose, whereas those of P. anisoides are glabrous throughout.
In addition, the carpological characters of P. anisoides and P. gussonei were compared with those of other species of the genus Pimpinella growing in Italy (Table 2).  Table 1). The relative contribution of each variable is shown in green.
The obtained results highlight that the two species have similar carpological characteristics but have different size characters (Figure 4). The fruit of P. anisoides is larger (Mw 1.61 ± 0.10 mm) than that of P. gussonei (Mw 1.27 ± 0.13 mm); in addition, the mericarps of the first species are longer (Ml 3.14 ± 0.32 vs. 2.26 ± 0.18 mm). The stylopodium of P. anisoides is wider (Sw 0.47 ± 0.06 mm) than that of P. gussonei (0.25 ± 0.19 mm). The cross-section area (CSa) of P. gussonei is larger (0.92 ± 0.19 mm) than that of P. anisoides (0.69 ± 0.12 mm). The commissural vittae width/thickness ratio (CV w/t) is 3.25 ± 0.84 in P. anisoides and 2.73 ± 0.45 in P. gussonei, showing a more compressed shape in the mericarp of the first species. Overall, the mericarps of P. anisoides appear slightly more oblong (l/w-length/width ratio =1.94) than those of P. gussonei (l/w-length/width ratio = 1.70). In agreement with Gussone [37], all the samples of P. gussonei fruit are grey tomentose, whereas those of P. anisoides are glabrous throughout.
In addition, the carpological characters of P. anisoides and P. gussonei were compared with those of other species of the genus Pimpinella growing in Italy (Table 2). The comparison in Table 2 shows that P. gussonei and P. anisoides are the only two natives to be of aromatic interest, apart from P. anisum of the species introduced for cultivation. Both species have oblong-ovoid fruits, similar to those of P. saxifraga, but this is not aromatic. The fruit size of P. anisoides is similar to that of P. major, which, however,  Table 1). The relative contribution of each variable is shown in green. The comparison in Table 2 shows that P. gussonei and P. anisoides are the only two natives to be of aromatic interest, apart from P. anisum of the species introduced for cultivation. Both species have oblong-ovoid fruits, similar to those of P. saxifraga, but this is not aromatic. The fruit size of P. anisoides is similar to that of P. major, which, however, has ovoid fruits. P. gussonei is similar in size to P. peregrina, which, however, has elliptic and hispid fruits.
Traditionally, the Pimpinella genus differs from related genera of the Apiaceae family due to a series of characteristics that are mainly of carpological type. The fruits, no longer than 5 mm, are slightly longer than wide, ovoid-oblong to subglobose, laterally compressed, with inconspicuous filiform primary ridges [29,42]. The phylogenetic analysis conducted by Fernández Prieto et al. [6] shows that in the Pimpinella genus, it is not possible to identify sections as proposed by de Candolle [49] and Wolff [1]. On the other hand, Fernández Prieto et al. [6] separate some species formerly included in the Pimpinella genus, attributing them to related genera: Spiroceratium H.Wolff and Parapimpinella Fern. Prieto et al.

Fruit Collection and Morphometric Data
The analysed fruits were collected from different localities: P. anisoides in Calabria in the Presila Catanzarese near Decollatura (CZ) and P. gussonei in Sicily near Gibilmanna (PA) ( Figure 6).

Fruit Collection and Morphometric Data
The analysed fruits were collected from different localities: P. anisoides in Calabria in the Presila Catanzarese near Decollatura (CZ) and P. gussonei in Sicily near Gibilmanna (PA) (Figure 6). The samples were collected in August 2022 during the period of maximum fruit filling, the collection was randomized within the sampled population for both species. At the same time, herbarium samples of the two species were collected and stored at the herbarium of the Mediterranean University of Reggio Calabria (REGGIO) for those of P. anisoides and at the herbarium of the University of Catania (CAT) for those of P. gussonei (acronyms follow Thiers [50]). The two species are very similar, in fact they are scapose hemicryptophytes, with enlarged woody root, erect stems, up to 80 cm tall, tripinnatifid leaves with lanceolate final segments, usually provided with 1-2 teeth, umbel-shaped inflorescence without involucre and involucel with 5-6 rays, flowers with white petals. The fruits were measured, in all their parts, by means of a stereomicroscope equipped The samples were collected in August 2022 during the period of maximum fruit filling, the collection was randomized within the sampled population for both species. At the same time, herbarium samples of the two species were collected and stored at the herbarium of the Mediterranean University of Reggio Calabria (REGGIO) for those of P. anisoides and at the herbarium of the University of Catania (CAT) for those of P. gussonei (acronyms follow Thiers [50]). The two species are very similar, in fact they are scapose hemicryptophytes, with enlarged woody root, erect stems, up to 80 cm tall, tripinnatifid leaves with lanceolate final segments, usually provided with 1-2 teeth, umbel-shaped inflorescence without involucre and involucel with 5-6 rays, flowers with white petals. The fruits were measured, in all their parts, by means of a stereomicroscope equipped with an Invenio 5SII HD camera connected to a computer where, by means of the DeltaPix inSight© software, it was possible to reconstruct the image using a multifocal system, and to determine the character measurements of the fruit. Morphometric analyses were carried out on a sample of 20 fruits and 20 cross-sections. The fruits measured were those that were found on a first macroscopic analysis to be perfectly ripe, well-formed and free of defects, caused by mechanical damage or insects. The following were measured in detail: fruit width (Mw), fruit length (Ml), length from the lower end to the point of maximum width (Mm), stylopodium width and length (Sw, Sl) and the length/width ratio (l/w). Fruit cross-sections were also observed to define their micromorphology, and the following were measured in detail: section width and thickness (CSw, CSt) and their ratio (CSw/t), commissural vittae width and thickness (CVw, CVt) and their ratio (CVw/t), commissure width (CCSl). The area per section (CSa) was also measured. The terminology of fruit parts and section is in accordance with Kljuykov et al. [19], Akalın et al. [9], Yeşil et al. [18]. Information of the carpological characters of the other species of the Pimpinella genus occurring in Italy is taken from Pignatti et al. 2018, Yeşil et al. [18] and Tutin et al. [42].

Statistical Analysis
The data from the measurements were collected and analysed in a matrix in Ex-cel©2019, the mean and standard deviation of the 14 characters examined were also calculated. To define the significance of the data, statistical differences were obtained by MANOVA multivariate analysis of the 14 characters (dependent variable) measured in both species (independent variable). SPSS ® 23.0 software (SPSS Inc., Chicago, IL, USA) was used for this purpose. Quantitative raw data were subjected to Principal Component Analysis (PCA) with a covariance matrix based on morphological features found significant by the multivariate MANOVA analysis. The statistical significance of the Principal Components (PCs) was analysed by the Broken Stick method, using Past 4.3 ® software. [51,52]. Boxplots were carried out with Excel© 2023 using only those characters found significant by the multivariate MANOVA analysis. In each boxplot, the centre and distribution of the data are highlighted.

Conclusions
This study contributes to the initial characterization of and differentiation between P. anisoides and P. gussonei, two often confused species that locally have a certain economic importance for the fruits used to flavor foods and drinks. The carpological analysis of the two species endemic to Italy revealed differences in size and morphological and anatomical fruit characteristics. Overall, the results allow a simple distinction of the two species in agreement with Presl [36], Gussone [37,38] and Bertoloni [39], and highlight the importance of carpological characters for the taxonomy of the Pimpinella genus. However, in order to determine the relationship between the two endemic Italian Pimpinella species more precisely, extensive studies are still needed, including the analysis of the morphological characteristics of the vegetative and reproductive organisms as well as DNA sequences.