(Araceae), a Mediterranean endemic monotypic genus

Phytotaxa 328 (1): 053–066 http://www.mapress.com/j/pt/ Copyright © 2017 Magnolia Press

Article

ISSN 1179-3155 (print edition)

PHYTOTAXA

ISSN 1179-3163 (online edition)

https://doi.org/10.11646/phytotaxa.328.1.2

Morphological observations on the reproductive structures of Ambrosina (Araceae), a Mediterranean endemic monotypic genus ANNA GERACI1, PIERLUIGI CORTIS2, ROSARIO SCHICCHI3 & ANGELO TROIA1*

Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), Sezione di Botanica ed Ecologia Vegetale, Università degli Studi di Palermo, Via Archirafi 38, I-90123 Palermo, Italy; e-mail: [email protected] 2 Dipartimento di Scienze della Vita e dell’Ambiente, Sezione Botanica, Università degli Studi di Cagliari, Viale S. Ignazio 13, I-09123 Cagliari, Italy 3 Dipartimento di Scienze Agrarie, Alimentari e Forestali (SAAF), Università degli Studi di Palermo, Viale delle Scienze Ed. 4, I-90128 Palermo, Italy * author for correspondence 1

Abstract This research was carried out to better define the pollen, fruit and seed morphology of Ambrosina bassii, the only species of this rare Mediterranean endemic genus. Pollen traits were already known, but here we investigated on their variability in distant populations and in different morphotypes (‘varieties’). Fruit and seed traits were almost unknown, also because of the rarity of fruit-set in this species. The surface features of pollen, fruit and seed, as well as length, width and length/width ratio, were analysed by means of scanning electron microscopy (SEM). Comparative observations were made also on Arisarum vulgare, since Arisarum is the closest genus. The principal component analysis of the Ambrosina pollen traits highlighted a separation of the different varieties and populations. As regards the surface features, the ornamentation of Ambrosina pollen is predominantly striate while in A. vulgare is striate-foveolate. The fruit of Ambrosina is a dry globose capsule showing stomata, not observed in the A. vulgare fruit. The seed is strophiolated with a reticulate surface. The results are discussed in comparison with previous observations and with morphological evolutionary patterns in the Araceae. Key words: Ambrosineae, Arisareae, Arisarum, fruit, morphology, pollen, seed

Introduction Araceae comprise 118 genera and over 3400 species, with a subcosmopolitan distribution, mainly in tropical regions (Mayo et al. 1998, Boyce & Croat 2014). The family exibits an unusually wide range of variation in nearly all characters (Grayum 1986, Mayo et al. 1997). Large variations concern not only morphology and anatomy of vegetative organs, but also reproductive traits (inflorescences, embryology, fruits and seeds), palynology (including pollination biology), cytology (chromosome numbers range between 14 and 168), phytochemistry (a lot of different secondary metabolites), and ecological adaptations (Petersen 1989, Grayum 1992, Mayo et al. 1997, 1998, Gibernau 2011, Chartier et al. 2014). In almost every pollen morphological character, the Araceae show a range of variation comparable to what might be expected at the subclass rather than the family rank (Grayum 1986). The major contributors to the palynology of the family were Thanikaimoni (1969) and Grayum (1986, 1992). From a detailed description of the main features (made from 380 species representing 99 genera), the pollen grains appear sulcate, zonate, forate and mostly (about 73% of the genera) inaperturate (Grayum 1992). The pollen size ranges from 12 to over 100 µm; in 68% of the genera it is comprised between 25 and 50 µm and the shape of the grains is globose to ellipsoid. The exine architecture can be grouped within the following categories: foveolate or reticulate (the basic type in Araceae but very common in Monocots), scabrate, spinose, spinulose, fossulate, verrucate, gemmate (rarely), areolate, regulate or striate (Grayum 1986, 1992, Mayo et al. 1998). The seeds of Araceae are frequently enclosed in berries (juicy or drier and leathery) and are usually straight with smooth, rough, verrucose or costate testa. In the tribes Ambrosineae, Areae, Arisaemateae and Arisareae, most genera show a prominent fleshy strophiole (aril) (Mayo et al. 1997, 1998). Accepted by Manuel B. Crespo: 1 Oct. 2017; published: 14 Nov. 2017

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Few data have been reported about fruit, seed and fine structure of pollen of Ambrosina bassii Linnaeus (1763: 517), a species with a restricted distribution area (Killian 1929, Grayum 1992, Mayo et al. 1997, Halbritter 2005, Barabé et al. 2004, Quilichini et al. 2005, Geraci et al. 2009). It represents an isolated taxon, so that it is usually treated as the only species in that genus; in addition, Ambrosina Bassi (1763: 3) is traditionally placed in the monotypic tribe Ambrosineae (Mayo et al. 1997, Cabrera et al. 2008, Mansion et al. 2008). In the present study, we used scanning electron microscopy (SEM) to achieve a morphological characterisation of the reproductive structures of A. bassii; in particular, the observations were undertaken on pollen, fruit and seeds, and compared with the same structures of Arisarum vulgare Targioni Tozzetti (1810: 67). Arisarum Miller (1754: 121) is, in fact, the closest genus, usually placed in a different monotypic tribe, Arisareae (Mayo et al. 1997, Cabrera et al. 2008, Mansion et al. 2008), or more recently included in the same tribe together with Ambrosina (Cusimano et al. 2011, Nauheimer et al. 2012). Although some data on pollen morphology of Ambrosina are available (though sometimes unsatisfactory), our knowledge of fruit and seed structure is still quite meager, as in the whole family (Kulkarni et al. 1990): on the general importance of fruit and seed morphology to characterise genera and species, several recent papers have been published (e.g. respectively De Castro et al. 2015, Ghimire et al. 2015). Moreover in this work we have the opportunity to verify the geographical variability of pollen traits, sampling different disjunct populations, and the value and significance of several taxa, described as separate varieties on the basis of a remarkable leaf morphological variability (cf. Geraci et al. 2009, Ben Khalifa et al. 2017).

Material and Methods The species Ambrosina bassi, a perennial species with diploid chromosome number 2n = 22 (Vignoli 1939, Scrugli & Bocchieri 1976), is the only representative of the genus. Its range is limited to a few areas in the Central-Western Mediterranean, including peninsular Italy (not recently confirmed, cf. Geraci et al. 2009), Sicily, Sardinia, Corsica, Tunisia and Algeria. As already said, several taxa were described on the basis of a remarkable leaf morphological variability but they are currently considered at the varietal rank (cf. Geraci et al. 2009). The plant is a small herb with a rhizomatous tuber as a stem. It loses its leaves during the hot dry summer season. The inflorescence is unique within the entire family: solitary and lying horizontally on the ground, it bearing a single female flower in an upper (ventral) chamber and several male flowers in an internal (dorsal) chamber. The flowering time is unusual for the Mediterranean region because it starts in November and extends until March. The fruit-set appears extremely rare (Killian 1929, Geraci et al. 2009). Quilichini et al. (2005) observed in natural condition a fructification rate of 17% that increased until to 26% in manual pollination experiments. Its pollination biology is not well known; on the basis of available data few animals (insects or other small invertebrates such as mites) have been observed inside its flowers, and we do not know exactly who its pollinators are. Killian (1929), Vignoli (1939) and Mayo et al. (1997) hypothesise that the pollinators are extinct (see also Geraci et al. 2009). Arisarum vulgare is the type-species of the genus Arisarum, including other 2 species (Mayo et al. 1997). Like A. bassii, it is a seasonally dormant herb, with tetraploid chromosome number 2n = 56 (Garbari 1967), with a tuber or slender rhizome as a stem, and widespread around the Mediterranean Sea from Morocco to Caucasus (Euro+Med 2006–2017). A single leaflike bract (spathe) forms a purplish-brown or olive-green striped tube, with an open upper part helmet or hood-shaped curved forward; it encloses a fleshy greenish club-like spike (spadix) bent forward, protruding from the tube and bearing at the bottom minute unisexual flowers. The ca. 20−40 male flowers are located above the three to six female ones (Koach & Galil 1986). Like in Ambrosina, the flowering period extends from November through March. Study sites and sampling The study was conducted on A. bassii plants collected from Sardinia, Corsica, Tunisia, and Sicily, and on plants of A. vulgare collected in Sicily (Table 1). In Sicily we could collect plants ascribable to 4 different taxa of A. bassii, namely: var. bassii (= var. ‘typica’), var. angustifolia Gussone (1844: 594), var. maculata (Ucria 1793: 256) Parlatore (1852: 231), and var. reticulata (Tineo 1827: 276) Parlatore (1852: 232); as regards the other populations the plants were identified as var. bassii. Populations will be named hereafter according to the ‘population codes’ (Table 1). From 5 to 8 inflorescences per population were sampled, dried, opened and kept at room temperature. Afterwards, the pollen was sprinkled on a surface. The pollen was further dried at room temperature for 6−8 h and put into brown glass bottles 54 • Phytotaxa 328 (1) © 2017 Magnolia Press

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with silica gel. Samples were stored in a refrigerator at +4 °C until examination. Fruits were collected in June in plants from Sicily, and the seeds stored at room temperature. Table 1. Details of sampled populations of Ambrosina bassii and Arisarum vulgare. Vouchers of all the populations are deposited in the Herbarium Mediterraneum Panormitanum (PAL). Taxon Ambrosina bassii var. bassii Ambrosina bassii var. angustifolia Ambrosina bassii var. maculata Ambrosina bassii var. reticulata Ambrosina bassii Ambrosina bassii Ambrosina bassii Arisarum vulgare

Pop. Code Sic-Typ Sic-Ang Sic-Mac Sic-Ret Sardinia Corsica Tunisia Aris

Locality S. Martino delle Scale (Palermo) Mazara del Vallo (Trapani) Monte Gallo (Palermo) Mazara del Vallo (Trapani) Sarroch (Cagliari) Near Bocca d’Aresia, N of Bonifacio NW Tunisia Near Palermo

Region Sicily Sicily Sicily Sicily Sardinia Corsica Tunisia Sicily

SEM analysis For SEM evaluation, properly dried pollen and seeds were placed on the double-sided transparent tape on the surface of the polished aluminum stub. The samples on the stub were coated with gold (Au) layer 225 Å thick (for two minutes in sputter). The pollen grains were studied in a LEO 420 SEM Microscope at 10–15 KV and photographed 2000−7700× magnification for the whole grain and for the exine sculpturing patterns. Seeds and fruits were observed and photographed 62−962× magnification. Pollen morphological characterisation The ornamented outer pollen wall of the studied species was observed in the dry condition, which gives the best information about the pollen grains at anthesis (Hesse et al. 2009). For each population, 25−30 pollen grains were considered for morphological characterisation. Because it was impossible to detect the polar axis in the inaperturate pollen grains of Ambrosina and Arisarum, pollen grain size is given as length (L, largest “polar” diameter) and width (W, smallest ‘equatorial’ diameter). The ratio L/W is given as an indication of pollen grain shape. The following traits were also considered: number of striae along the ‘equatorial’ axis (NS), thickness of striae (TS), number of vertical ribs (according to the terminology of Grayum 1992 – those same structures are named ‘rugulae’ in Hesse et al. 2009) in 2 µm (NR), thickness of vertical ribs (TR), distance between vertical ribs (DR), in addition to general shape and exine sculpturing in the ‘polar’ and ‘equatorial’ view (morphological terms according to Hesse et al. 2009, Grayum 1992). The measurements were made in micrometers (μm). The pollen shape was described following Erdtman (1943). The significance of variation among different populations for each trait was initially tested using a one-way analysis of variance (ANOVA), after Barlett’s test of homogeneity of variance. Then, two multivariate procedures were used for data comparison (without L/W ratio due to autocorrelation with L and W variables). In order to evaluate the contribution of each parameter to discriminate different populations, a principal component analysis (PCA) was carried out by computing eigenvalues and eigenvectors from a Pearson correlation matrix. Morphological relationships among populations were also analysed by an agglomerative cluster analysis (CAG) based on similarity matrix and performed using the Ward’s method and the Euclidean distance coefficient. The results are summarised in form of a dendrogram. ANOVA, PCA and clustering were performed using PAST software (Hammer et al. 2001).

Results Pollen morphology Pollen of all populations of Ambrosina can be described as inaperturate and striate (Fig. 1A–N). The mean value of length is 50.11 μm, ranging from 46.8 in Sic-Mac to 51.45 in Tunisian population; the width is comprised between 19.84 (Sardinia) and 23.98 μm (Tunisia). Its shape is oblong–ellipsoid with L/W ratio (in dried conditions) greater than 2 (Table 2). The exine sculpture is striate, composed by narrow striae forming a parallel pattern in ‘equatorial’ region; in the “polar” zones the pattern is verrucate with several small foramina more evident in pollen from Tunisia, Sardinia, and Corsica (Fig. 1A–N). The surface of the striae is psilate with vertical ribs and reticulate striae walls (in particular in Sardinian pollen). The number of the striae along the equatorial axis is comprised between 17.71 (Tunisia) and 23.4 (Corsica), while their thickness ranges from 0.70 in Corsica to 1.13 in Sic-Mac (Table 2). Reproductive structures of Ambrosina (Araceae)

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Table 2. Descriptive statistics of some morphometric pollen traits analysed in seven populations of Ambrosina bassii and one population of Arisarum vulgare (pop. codes as in Table 1). Reported values for each character are mean ± standard deviation, and minimum–maximum values. Each abbreviation is explained in the title of each column, except VR = Vertical Ribs. L, W, TS, TR and DR are in μm. Length Width Ratio n° striae thickness striae n° VR/ 2 µm thickness VR Distance VR (L) (W) (L/W) (NS) (TS) (NR) (TR) (DR) 50.78 ± 1.24 21.74 ± 0.46 2.34 ± 0.03 21.43 ± 1.51 0.78 ± 0.11 3.75 ± 0.45 0.28 ± 0.02 0.38 ± 0.04 Sic-Typ 49.2−52.1 21.31−22.1 2.31−2.36 20−23 0.65−0.91 3–4 0.25–0.3 0.32–0.414 50.46 ± 0.64 21.43 ± 0.47 2.36 ± 0.06 20.57 ± 1.13 0.80 ± 0.11 3.92 ± 0.29 0.24 ± 0.03 0.39 ± 0.08 Sic-Ang 49.86–51.2 21–22.1 2.37–2.32 20–23 0.65–0.91 3–4 0.22–0.28 0.33–0.54 49.42 ± 0.29 22.85 ± 0.57 2.16 ± 0.07 21 ± 1.41 0.73 ± 0.19 3.33 ± 0.49 0.37 ± 0.07 0.43 ± 0.08 Sic-Ret 49.28–49.86 22–23.14 2.13–2.27 20–24 0.65–1.04 3–4 0.27–0.45 0.32–0.54 46.8 ± 0.86 22.48 ± 0.99 2.09 ± 0.12 20.25 ± 0.5 1.13 ± 0.17 3.33 ± 0.49 0.32 ± 0.04 0.31 ± 0.04 Sic-Mac 45.8–47.9 21.31–23.32 1.96–2.19 20–21 0.91–1.3 3–4 0.27–0.36 0.22–0.36 51.45 ± 4.12 23.99 ± 1.59 2.14 ± 0.09 17.71 ± 0.75 0.97 ± 0.21 4.34 ± 0.49 0.32 ± 0.04 0.25 ± 0.05 Tunisia 46.75–56.67 21.31–26.67 2.01–2.29 17–19 0.78–1.3 4–5 0.27–0.36 0.18–0.32 51.12 ± 1.67 19.84 ± 0.98 2.58 ± 0.07 21 ± 0.89 0.74 ± 0.11 4±0 0.25 ± 0.06 0.35 ± 0.05 Sardinia 49.8–53.28 19.35–21.31 2.50–2.58 20–22 0.65–0.91 4–4 0.18–0.36 0.27–0.42 50.78 ± 1.34 23.03 ± 0.22 2.20 ± 0.05 23.4 ± 1.14 0.70 ± 0.23 4.58 ± 0.51 0.28 ± 0.02 0.27 ± 0.05 Corsica 49–52 22.74–23.2 2.15–2.26 22–25 0.52–1.04 4–5 0.25–0.3 0.21–0.35 42.21 ± 1.58 16.93 ± 1.62 2.51 ± 0.18 19.2 ± 0.84 1.02 ± 0.29 Arisarum (absent) (absent) (absent) 40–45 15–19 2.30–2.80 18–20 0.80–1.50

Figure 1 A–H. SEM micrographs of pollen grains of Ambrosina bassii (A–N) and Arisarum vulgare (O–P). A–B) A. bassii from Tunisia; C–D); A. bassii from Sardinia; E–F) A. bassii from Corsica; G–H) A. bassii var. bassii (Sicily).

Reproductive structures of Ambrosina (Araceae)

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Figure 1 I–P. SEM micrographs of pollen grains of Ambrosina bassii (A–N) and Arisarum vulgare (O–P). I–J) A. bassii var. angustifolia (Sicily); K–L) A. bassii var. maculata (Sicily); M–N) A. bassii var. reticulata (Sicily); O–P) A. vulgare from Sicily. Details on populations in Table 1.

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The number of the vertical ribs across 2 µm is 3.89 ranging from 3.33 in Sic-Mac and Sic-Ret to 4.58 in Corsica (Table 2). The highest thickness of the ribs is found in Sic-Mac and Sic-Ret (0.32 and 0.37 respectively), and their distance is the lowest in Corsica and Tunisia populations (0.27 and 0.25 respectively), the highest in Sic-Ret (0.43) (Table 2). The pollen of A. vulgare is also inaperturate (Fig. 1O), smaller in length (42.42 ± 2.35 SD × 14.57 ± 2.28 SD) and with a L/W ratio of 2.91 ± 0.78 SD. Exine is striate in “equatorial” zones with striae showing small foramina: “polar” zones are smooth and covered by very numerous foramina. Compared to Ambrosina, these “polar” foramina are smaller in size and the ribswork is absent.

Figure 2. Scatter plot of the first two principal components (PCA) based on seven pollen traits (Table 2) from Ambrosina bassii samples (mean data for the studied populations). Names of the populations as in Table 1.

Statistical analysis by ANOVA showed significant (p < 0.01) difference in all the examined pollen traits. The principal component analysis (PCA) for the complete set of variables (but without L/W ratio due to autocorrelation with L and W variables) resolved 3 principal components with eigenvalues higher than 1, which altogether described 99% of the observed variation. The factor one (PC1) explained 42.9% of variance, it was positively correlated with “width” and negatively correlated with “n striae”. In the case of factor two (PC2), accounting for 36.1% of total variation, a clear positive correlation was with “length”. The scatter plots of standardised scores for the first two components (Fig. 2) highlighted a clear separation of different populations, forming well delimited groups with moderate overlaps: Tunisia population is clearly isolated in the positive part of PC1, as well as Sic-Mac population in the negative part of the same axis. The other populations fall more or less close, in the negative part of PC2, with Sardinia and Corsica showing the lowest values on the axis and Sic-Ret in an intermediate position between this group and Sic-Mac. The pattern observed from cluster analysis (Fig. 3) partially agreed with results of PCA. Tunisia population is again clearly isolated, Sic-Ret and Sic-Mac populations are close, Corsica and Sardinia populations are close but embedded between two groups of Sicilian populations. Reproductive structures of Ambrosina (Araceae)

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Figure 3. Dendrogram obtained from agglomerative cluster analysis using Ward’s algorithm of Euclidean distance, based on seven pollen traits (Table 2) from Ambrosina bassii samples (mean data for the studied populations). Names of the populations as in Table 1.

Fruit and seed morphology The fruit of Ambrosina is dry, and can be defined as a capsule (Fig. 4A): it is globose or subglobose, greenish to brown when mature, not dehiscent, with style and stigma persistent, and many-seeded (10−40 seeds). Also in Arisarum, the dry fruit can be defined as a capsule, few-seeded (