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Re-establishment of Silene neglecta Ten. (Caryophyllaceae) with taxonomic notes on some related taxa

Silene neglecta has been misunderstood and confused with S. nocturna , although several morphological characters (petal shape, calyx indumentum, hairiness of stamen filaments, seed size, seed-coat surface and shape) allow separation of these species. Moreover, S. mutabilis (which has been considered conspecific with S. neglecta ) and S. martinolii (an alleged endemic species to south-western Sardinia) are considered here as taxonomic synonyms of S. nocturna and S. neglecta , respectively. These taxonomic conclusions are strongly supported by multivariate morphometric analyses of 21 characters.


Introduction
Silene L. is one of the large genera in Caryophyllaceae, comprising around 850 taxonomic species (Jafari et al. 2020). The genus is widely distributed in temperate regions mainly in the Northern Hemisphere, with the centre of its diversity is in western Asia and the Mediterranean Basin.
Silene sect. Silene is one of the largest sections of the genus, as classified by Jafari et al. (2020). It comprises about 93 species mainly distributed in the Mediterranean. It is characterised by monochasial (sometimes dichasial) inflorescence, usually nonauriculate petal claws and often excavate or flat seeds with long and narrow testa cells (Jafari et al. 2020). Silene nocturna L., S. neglecta Ten. and S. mutabilis L. are annual species grouped in this section (Jafari et al. 2020;Mesbah et al., in prep.). Tenore (1826) described Silene neglecta from southern Italy, whereas twelve years later (Tenore 1838), illustrations of the species which revealed its key characteristics were published. The taxon has been recognised at different levels, either as a separate species (Maire 1963;Pignatti 1982Pignatti , 2017Talavera 1990) or at subspecies level within S. nocturna (Arcangeli 1882;Fiori and Paoletti 1896;Chater et al. 1993). In a taxonomic study of the S. nocturna complex in Italy, Peruzzi and Carta (2013) proposed the species rank for S. neglecta, by providing morphological and karyological data and typifying the name. Later, in a study of original materials of some Linnaean names currently included within Silene, Peruzzi et al. (2014) concluded that the first available name at species level for the plants called S. neglecta was S. mutabilis. However, the application of the name S. mutabilis remains uncertain at present, since some authors have continued to use the name S. neglecta (Pignatti 2017;Bosch et al. 2019). Furthermore, S. martinolii Bocchieri & B.Mulas, an alleged endemic species to islets of south-western Sardinia, is morphologically very close to S. neglecta (Bocchieri and Mulas 1988), so it is advisable to clarify its taxonomic position.
This study aims to provide distinction between these taxa (Silene neglecta, S. nocturna, S. martinolii and S. mutabilis), based on macromorphological features and Scanning Electron Microscopy (SEM) observations of hairs and seeds.

Plant material
The present morphological and comparative study is based on the examination of specimens in the field and in herbarium/laboratory and on the analysis of the protologues. The names of the specimens were applied a priori following Bocchieri and Mulas (1988) and Pignatti (2017) for Silene martinolii; Linnaeus (1753) for S. mutabilis; Talavera (1990) and Pignatti (2017) for S. neglecta and Talavera (1990), Peruzzi and Carta (2013) and Pignatti (2017) or in the herbarium specimens (BC, BCN,  CAG, GB, ENSA, HJBS, JACA, LINN, MA, NAP, PI and WAG; acronyms according to Thiers (2022 [continuously updated]). Digital images from online databases for the Herbaria MPU, K, P and US were also examined. Morphological observations of materials were carried out under a binocular stereoscopic microscope Zeiss Stemi DV4 with eyepiece micrometer. Micromorphology was observed on calyces and mature seeds which were glued directly to aluminium stubs, coated with 40-50 nm gold and examined with a scanning electron microscopy (Hitachi 2300-SEM) at 20 kV. Given that detailed information on the seeds of S. nocturna has been provided in recent works (Peruzzi and Carta 2013;Peruzzi et al. 2014), seed data, based on SEM, provided for this species refers only to specimens that were misidentified as S. neglecta or S. mutabilis.

Data analysis
A total of 21 characters were selected and scored in 71 specimens. From the total 21 morphological characters, 15 were quantitative, three were calculated ratios and three were qualitative (Table 1). A non-metric multidimensional scaling (NMDS; Kruskal 1964), which represents the relationships amongst individuals in a reduced dimension scatterplot and Cluster Analysis (CA) using the average linkage method (UPGMA; Michener and Sokal 1957), which allows the classification of individuals by similarity, were performed with PAST 4.08 (Hammer et al. 2001). The similarity matrix was calculated using the Gower coefficient, suitable for mixed data (Gower 1971

Results and discussion
The variation, based on morphometric analysis (NMDS and CA) and the morphological characters of Silene neglecta, S. nocturna, S. martinolii and S. mutabilis, are described and the taxonomic value of the characters is discussed.

Morphometric analysis
The NMDS, performed with three dimensions, yielded a value of stress of 0.10 corresponding to a good ordination result (Clarke 1993). The scatterplot showed two clearly defined groups, where Silene martinolii is intermingled with S. neglecta and both are separated from S. nocturna (Fig. 1). The UPGMA dendogram ( Fig. 2) yielded two well-defined clusters, one formed by individuals of S. neglecta and S. martinolii and a second one formed exclusively by individuals of S. nocturna. The cophenetic correlation coefficient was 0.98, indicating a good fit between the cophenetic value matrix and the similarity matrix.

Habit and hairiness
All the studied species are annuals, except some specimens of Silene neglecta from maritime sands in north-western Tunisia (Tabarka)

Leaves
Leaf outline in Silene neglecta, S. martinolii and S. nocturna varies from spatulate to linear, usually tapering more or less progressively from the base towards the inflorescence. Silene neglecta has usually wider leaves than S. nocturna, but there is a notable variability with respect to this character so that it cannot be used for identification purposes.
Silene nocturna sometimes has narrowly linear leaves in the middle and upper part of the stem (e.g. the lectotype of S. mutabilis), whereas in S. neglecta, they are spatulate to lanceolate or linear-lanceolate.

Pedicels
The pedicels are accrescent and their length is somewhat variable within a single taxon.
Our study reveals that this character presents much more variability than has been documented so far. The presence of long pedicels has been attributed to Silene neglecta (Peruzzi and Carta 2013) and, based on this character, S. neglecta and S. mutabilis were later considered as synonyms . Indeed, S. neglecta can have remarkably long lowermost pedicels, up to twice the length of the calyx (Italy,MPU300592). However, there are specimens of S. neglecta with lowermost pedicels equal to or shorter than the calyx [Algeria: Kabylie de Collo (P), Bône (P); Spain: Roca del Barret (LS7707), Tunisia: Tabarka  martinolii are usually shorter than to equal to the calyx length (Bocchieri and Mulas 1988), although in some specimens (Sa Corona su Crabi, CAG), the pedicel is longer than the calyx, even without being the lowermost flower. Therefore, this character cannot be used for taxonomic purposes. The inclination of the lowest flower's pedicel (in fruiting period) has been used by Peruzzi and Carta (2013) as a character to separate Silene neglecta (up to 90°) from S. nocturna (up to 40°). Although this relatively strong inclination is certainly observed in some specimens of S. neglecta (including the lectotype), there are also several specimens of this species with the lowest flower's pedicel suberect or erect-patent (Spain: LS7707, Fig. 3; Tunisia: Tabarka). Bocchieri and Mulas (1988) attributed erect-patent pedicels to S. martinolii. Although this is true in most cases, in two specimens (S'Aquasa Canna and Sa Corona su Crabi, CAG), several clearly patent lowest flower's pedicels (ca. 90°) are observed.

Gonophore
Both species have puberulent gonophores, being longer in Silene neglecta and S. martinolii than in S. nocturna (Table 2). Our study has revealed the existence of longer gonophores (up to 2.6 mm long) in S. neglecta than previously documented (Peruzzi and Carta 2013).

Calyx
As noted by Peruzzi and Carta (2013), Silene neglecta is distinct from S. nocturna by its larger calyx and larger calyx teeth. Our study reveals that there is a wide overlap in the calyx length (Table 2), so this character does not allow an unequivocal separation of both taxa. Bocchieri and Mulas (1988) attributed a higher maximum value of calyx length to S. martinolii (9-13 mm). However, we have observed specimens of S. neglecta from Spain that also reach 13 mm in length (this measurement refers to calyces that do not correspond to the lowermost flower). The length of the calyces and calyx teeth of the lectotype of S. mutabilis is 8.8-11.2 mm and 1.4-2.0 mm, respectively.
The hairiness type of the calyx has taxonomic value. Two types of hairs were identified: eglandular and glandular hairs. The eglandular hairs (unicellular or pluricellular, the latter with up to nine cells) are progressively tapering towards the apex. These hairs show striated or verruculate walls. The eglandular hairs are usually short and antrorse in Silene nocturna and in the lectotype of S. mutabilis. These hairs are somewhat longer on the veins of the calyx (Table 2), whereas between the veins, they rarely exceed 0.2-0.3 mm in length (Table 2,  . The hairs of the calyx of the lectotype of S. mutabilis are eglandular, short (up to 0.2 mm long) and antrorse (Fig. 5). This morphology matches the typical calyx hairiness of S. nocturna. In S. neglecta and S. martinolii, the eglandular hairs are longer, patent or antrorse. These hairs are somewhat longer on the veins of the calyx if compared to those located between the veins ( Table 2).
The glandular hairs are formed by a gland and stalk consisting of 1-8 cells. These glandular hairs show striated walls. The glandular hairs are found in Silene neglecta and S. martinolii, while they are usually absent in S. nocturna and the type material of

Corolla
Flower opening in Silene nocturna is mainly nocturnal, while in S. neglecta and S. martinolii, it is diurnal. The petals of S. nocturna and S. mutabilis are bifid (see also Linnaeus, 1753;1756). In cleistogamous variants of S. nocturna, the petal limbs are very short (usually included) or even absent. On the contrary, the limb of the petals of S. neglecta and S. martinolii is subentire to emarginate (Fig. 3); the sinus of the limb can reach almost a third of its length. The lobes of S. neglecta and S. martinolii, when present, are much wider than those of S. nocturna, which are narrowly oblong to sublinear. After the description of S. neglecta, Tenore (1838) provided detailed illustrations of the species, showing the presence of pink petals with broad, not bifid limbs. The study of the lectotype of S. neglecta reveals that the limb of the petals is emarginate. The colour of the corolla of Silene neglecta and S. nocturna varies from pale pink (rarely white) to pinkish-purple. The petal colouration is variable within S. nocturna, even in the same population. The petals can be white (sometimes greenish in the abaxial surface), pale rose and even tinged with pink-purple (mainly on the abaxial side). This is remarkable, since this colouration was invoked by Linnaeus (1756) to describe his S. mutabilis ("petalis post florescentiam extus purpurescentibus"). We have observed specimens in populations from Spain (Barcelona Province) with this colouration attributed by Linnaeus to S. mutabilis and that perfectly fits the current concept of S. nocturna (Fig. 4).

Stamens
The hairiness of the stamen filaments is of taxonomic significance and, based on our observations, it is always related to the calyx hairiness (see above). The filaments are glabrous in Silene nocturna, whereas in S. neglecta and S. martinolii, alternate filaments are hairy at base (sometimes all are hairy at base). In those specimens in which all the stamen filaments are hairy (plants from Tabarka, Corona su Crabi, Torre gaveta, Campania and Volcano), the hairy portion of the filament is noticeably shorter in those stamens adjacent to the petals. Possibly, the difficult observation of this character can explain why it has gone unnoticed so far.

Seeds
The seeds are reniform with excavate lateral faces and a dorsal furrow. The seeds of Silene nocturna are somewhat smaller than those of S. neglecta and S. martinolii. There are differences in colouration (grey or greyish-brown in S. nocturna, blackish to dark- brown in S. neglecta and S. martinolii). Peruzzi et al. (2014) stated that the lectotype of S. mutabilis has polygonal (star-shaped) dorsal cells. However, based on our observations, S. neglecta has elongate (more or less polygonal), not star-shaped dorsal cells  (Fig. 6). The surface of the dorsal cells of S. neglecta has a more or less prominent central tubercle (up to 30 µm long), while in S. nocturna, these tubercles, when present, are not very prominent (less than 15 µm long). The pattern of morphological variability of S. nocturna seeds is complex and requires further study.

Taxonomic treatment
After a critical macro-and micromorphological analysis and detailed studies of the protologues, we conclude that Silene mutabilis is not conspecific with S. neglecta. S. mutabilis shows characters which fall within the range of morphological variation of the currently recognised S. nocturna. Further research is needed to identify the existence of taxonomic units within S. nocturna, which could be a polyphyletic species. Our study shows that S. neglecta and S. nocturna are distinct, based on macro-features (leaf shape and petal limb shape) and micromorphological characters (calyx and stamens indumentum and SEM analysis of seeds). An amended description is here also provided for S. neglecta, based on herbarium and live specimens collected from North Africa, Italy, Sardinia, Sicily and Spain. Based on this macro-and micromorphological evidence, in addition to morphometric analysis, we can conclude that S. martinolii falls within the variation of S. neglecta. Our preliminary phylogenetic results (Mesbah et al., in prep.) suggest that S. neglecta is not related to S. nocturna, but more closely related to S. gallica L., the type species of the genus Silene. Description. Annual herb (rarely biennial) green or greyish-green, hairy. Stems (10-)15-50(-70) cm, more or less erect (rarely prostrate-ascending), unbranched to much-branched, densely hairy, usually viscid above. Lower leaves spatulate to obovate; cauline leaves obovate to linear-lanceolate. Flowers 2-9(-10) in raceme-like monochasial cymes; lowermost pedicels shorter or longer than calyx (0.45-2.00 times as long as calyx in fruit), erect to patent, pubescent-glandular. Calyx 8-13 mm long, subcylindrical to cylindrical-ovoid in flower, attenuate, becoming subovoid to ovoid in fruit, usually densely hairy, covered by long eglandular hairs (up to 1.9 mm long) and glandular hairs (up to 2 mm long); calyx teeth triangular to linear-lanceolate or linear, acute; veins anastomosing. Petals pale pink to pinkish-purple, rarely white; limb 4.5-6.0 mm, subentire or emarginate; coronal scales 1.2-2.2 mm, whitish to pink. Stamens with alternate filaments hairy at base, sometimes all hairy at base; anthers 1.0-1.5 mm long, lilac to purple, exserted from corolla mouth. Gonophore (1.8-)2.0-2.4(-2.6) mm long, puberulent. Capsule 6.0-8.5 mm, subcylindrical, enclosed within the calyx. Seeds (0.90-)0.91-1.02(-1.05) × (0.76-)0.80-0.89(-0.91) blackish to dark brown; faces deeply concave, tuberculate; back wide, slightly canaliculate. Habitat. Rocky places, maritime sands and grassland, usually on siliceous substrata, 0-1700 m a.s.l.

Silene neglecta
Distribution. Central and southern Italy, south-western Sardinia, Sicily, northern Algeria and Tunisia and north-eastern Spain.