Introduction
Silene L. is the largest genus of the family Caryophyllaceae Juss., consisting of ca 870 spp. (Jafari et al. 2020) and often grows in dry habitats including rocky or gravelly places, sandy soils in steppes, chalky soil, and deserts, with little competition from other plants. According to a recent study (Jafari et al. 2020), Silene includes three subgenera and one section which is considered as incertae sedis, S. sect. Atocion Otth as circumscribed by Toprak et al. (2016).
Silene sect. Silene s.l., which is part of subg. Silene, consists of about 93 species. It is distributed in Europe, Asia, and Africa from the Mediterranean to Pakistan, some species extending to southern Africa (Jafari et al. 2020: appendix S6).
In the Flora of Iraq (Townsend et al. 2016), the annual species S. villosa Forssk. and S. arabica Boiss. are included in S. sect. Bipartitae (Boiss.) Melzh. The current infrageneric classification of Silene places the two species in S. sect. Silene s.l. (Jafari et al. 2020). The circumscription of section Silene was expanded to encompass species that previously were placed in S. sect. Atocion, S. sect. Dipterospermae (Rohrb.) Chowdhuri, S. sect. Fruticulosae (Willk.) Chowdhuri, S. sect. Nicaeenses (Rohrb.) Talavera, S. sect. Rubellae (Batt.) Oxelman & Greuter, S. sect. Scorpioides (Rohrb.) Chowdhuri, S. sect. Silene, and S. sect. Succulentae (Boiss.) Chowdhuri (Chowdhuri 1957; Talavera 1988; Greuter 1995; Jafari et al. 2020: appendix S6).
Silene villosa was described from Egypt (Forsskål and Niebuhr 1775); the type specimen is preserved in the Copenhagen herbarium. The species is distributed from SW Asia to North Africa, Egypt, and Algeria. The diagnostic characters in the protologue “petalis bifidis, oblongis, basi sub germine in tubum connatis” are not helpful here. Furthermore, in the description, S. villosa was identified by opposite branches, long calyx, violet corolla, and exserted petals. Boissier (1867) described this species with more details, as follows: stems with numerous fleshy leaves, the calyx shorter than peduncles (pedicels) in the reflexed fruit, and petals white. Rohrbach (1868) presented a description similar to Boissier. He recognized the species with stem ramose, calyx shorter than the pedicel, and petals whitish. Zohary (1966) described S. villosa in the Flora Palaestina as follows: stem much-branched, leaves somewhat fleshy, pedicels shorter than or as long as or longer than calyx, calyx 10–25 mm, and reflexed in fruit, petals white, rarely pink. Melzheimer (1988) identified the species with these characteristics in the Flora Iranica: calyx shorter than the pedicel, the pedicel straight, bent backward in fruit, calyx at anthesis subcylindrical, fruiting ± clavate, (16–)18–22(–23) mm long, yellowish at the base, pale green above, petals white to pink. Chamberlain (1996) indicated that two forms, ‘A’ and ‘B’, of this species occur in Arabia. The forms were separated based on calyx length ((10–)12–18 vs 17–23(–25) mm), the position of the calyx (pendulous vs erect), and plant length (10–45 vs 7–18 cm). Townsend et al. (2016) described S. villosa with these characteristics: pedicels shorter than calyx, reflexed in fruit, calyx subcylindrical at anthesis, ± clavate in fruit, petals white to reddish, exserted from the calyx.
Silene wendelboi Assadi was reported as a new species for the southwestern part of Iran (Assadi 1977). It was distinguished by a globose habit, long and narrow calyx, and the sculpture of the seed surface. Silene villosa was recognized as a closely related species to S. wendelboi (Assadi 1977). However, Melzheimer (1988) mentioned in his note that S. wendelboi could be part of S. arabica and he did not accept it as a separate species. Although S. wendelboi was also reported in Iraq (Lazkov 2006), it has not been treated as a separate species in a recent flora of Iraq (Townsend et al. 2016). The specimen “110 km SSW of Basra, Rechinger 8793; B [B10 1107154]” that was reviewed by Lazkov (2006) and identified as S. wendelboi was listed as S. villosa by Rechinger (1964). A specimen from the same locality (110 km SSW of Basra; Guest, Rawi & Rechinger, Natl. Herb. Iraq 17208; K [K000609857]) was listed under S. villosa by Townsend et al. (2016).
We recognize the second form of S. villosa noted in the Flora of the Arabian Peninsula and Socotra (Chamberlain 1996) with a longer and erect calyx as S. wendelboi. We examined many herbarium specimens of these two forms of S. villosa s.l. from the Persian Gulf region, including a few specimens at E that were annotated by Chamberlain, and by using molecular tools we investigated if they could be treated as two distinct species or if they are the same.
Material and methods
Taxonomic study
We examined material of the two species (S. villosa s.l. and S. wendelboi) in the following herbaria: IRAN, M, MIR, MSB, S, TUH, and TARI (abbreviations according to Thiers continuously updated). In addition, field trips were carried out during the period 2016–2019 in various regions of Iran. Plants were identified using the following references: Flora Aegyptiaco-Arabica (Forsskål and Niebuhr 1755), Flora Orientalis (Boissier 1867), Monographie der Gattung Silene (Rohrbach 1868), Flora Palaestina (Zohary 1966), Flora Iranica (Melzheimer 1988), Flora of the Arabian Peninsula and Socotra (Chamberlain 1996), and Flora of Iraq (Townsend et al. 2016). For studying the type specimen of S. villosa, we used online images at the virtual Copenhagen herbarium (http://www.daim.snm.ku.dk/search-in-types) and, for additional comparison of herbarium specimens, we used online images available from the Global Biodiversity Information Facility (GBIF) (https://www.gbif.org/), JSTOR (https://plants.jstor.org), and/or JACQ database (https://www.jacq.org/#database), as well as the herbarium websites of E, P, and US, and images provided by staff at B and K.
Phylogenetic study
We created two datasets containing 84 and 46 accessions of nrDNA ITS and cpDNA rps16 regions, respectively, which were extracted mainly from GenBank (Supplementary file 1). Four accession numbers are newly sequenced. The datasets mainly consist of species that belong to Silene sect. Silene s.l. sensu Jafari et al. (2020). We tried to include all accessions belonging to this section in the datasets, with only some repetitions removed. Genomic DNA was extracted from herbarium material using the Sinaclon Plant DNA extraction kit (Tehran, Iran) according to the manufacturer’s protocol.
Polymerase chain reaction (PCR) amplifications were performed in 25 μL reactions, containing 10 μl of deionized water, 12.5 μl of 2X Reddy® to use PCR Master Mix, 0.5 μl of each primer (10 pmol/μl), and 1 μl template DNA. Amplification of the ITS region was performed using the primers P17 and 26S-82R (Popp and Oxelman 2001). We used the primers rpsF and rpsR2R for amplification (Oxelman et al. 1997). Cycle sequencing was done using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, California, U.S.A.). DNA samples were sequenced with an ABI3130XL DNA Analyser 16-well capillary sequencer (Applied Biosystems) performed by Niagene Noor (Tehran, Iran).
Sequence alignment was performed in MAFFT v.7 (Kuraku et al. 2013; Katoh et al. 2019) at the web service (http://mafft.cbrc.jp/alignment/server/). The default setting was applied for all options. The preliminary alignments were then corrected manually. PAUP* 4.0a169 (Swofford 2003) was used to select the best-fitted model of nucleotide substitution based on the corrected Akaike information criteria (AICc), and the General Time Reversible model with Gamma shaped rate variation (GTR+G) model was selected for both regions. Maximum Likelihood (ML) analyses were conducted in RAxML HPC v.8.2.12 (Stamatakis 2014) using the GTRGAMMA model with 1000 pseudo-replicates to evaluate bootstrap support for each node. Bayesian gene tree inference was performed using MrBayes v.3.2.7a (Ronquist et al. 2012) with 10 and 5 million generations for the nrDNA ITS and cpDNA rps16 datasets. Four Metropolis-coupled chains were run with tree and parameter values saved every 1000th generation in two parallel runs. The first 25% of all trees were discarded as burn-in. Phylogenetic analyses were carried out on the CIPRES science gateway (Miller et al. 2010).
Discussion
Silene villosa and S. wendelboi are treated as two separate species in both the ITS and the rps16 phylogenetic tree (Figs 1, 2). Calyx texture, calyx length, the ratio of calyx length to pedicel, shape of the apex of the coronal scale, seed morphology, and molecular data are reliable characters that confirm the distinction of these species. Herbarium investigations show that S. wendelboi was erroneously identified mainly as S. villosa or sometimes as S. arabica among collections from the Persian Gulf and E Mediterranean regions. The erroneous identification causes the description of S. villosa in local floras of the Persian Gulf and E Mediterranean regions (i.e. Zohary 1966; Melzheimer 1988; Chamberlain 1996; Townsend et al. 2016) to be mixed with S. wendelboi. Shahid and Rao (2014) erroneously identified S. wendelboi as S. arabica for the flora of the United Arab Emirates. Although the calyx length shows a very small degree of overlap, the characters coronal scales and seeds are diagnostic. The apex of the coronal scale is dentate and acute in S. wendelboi and seeds are recessed near the hilum, while the apex of the coronal scale is entire and obtuse and seeds are concave in S. villosa. Pedicels often are shorter than the calyx in S. wendelboi, while long pedicels and a pendent or reflexed calyx in fruit are characters indicated for S. villosa in most local floras (Zohary 1966; Melzheimer 1988; Chamberlain 1996; Townsend et al. 2016). Lazkov (2006) also indicated seed shape and colour as diagnostic characters for separating S. wendelboi and S. villosa.
In the African Plant Database (version 4.0.0), four additional varieties of S. villosa are listed. We refrain from treating these varieties since further morphological investigation and molecular data are needed. It seems that the seed shape and micromorphology of S. villosa and S. wendelboi were intermixed in both the Flora Iranica and the protologue of S. wendelboi; the seed image of S. villosa in Flora Iranica belongs to S. wendelboi and the seed illustration of S. wendelboi in the protologue shows the micromorphological characters of S. villosa. The seeds are reniform in both taxa, and compressed around the hilum in S. wendelboi, while concave in S. villosa.
Although similarities of morphological characters in S. villosa and S. wendelboi cause these taxa to have been considered as the same taxon, the phylogenetic trees show that S. wendelboi is distinct from S. villosa and that it is a close relative of S. arabica rather than of S. villosa.
Low-copy nuclear markers have sometimes been used due to the fast-evolving intron of low-copy nuclear genes when the variation within nuclear and chloroplast sequences was not sufficient to segregate closely related species (Sang 2002). However, here, the nrDNA ITS and cpDNA rps16 are informative and support the distinction of S. villosa and S. wendelboi; therefore, low-copy nuclear genes were not applied here.