Taxonomical identity of Sarcocornia fruticosa and S . hispanica in the Iberian Peninsula

Guilló, A., Alonso, M.A., Lendínez, M.L., Salazar, C. & Juan, A. 2014. Taxonomical identity of Sarcocornia fruticosa and S. hispanica in the Iberian Peninsula. Anales Jard. Bot. Madrid 71(2): e011. The recent description of Sarcocornia hispanica, from SE Iberian Peninsula evidences the notable morphological complexity of this genus. This species is quite close to S. fruticosa, as they both show an erect habit and lack of rooting stems. Besides, S. fruticosa has been traditionally recorded for the same geographical area of S. hispanica. However, the original description of S. hispanica did not include any mention about these two taxa would live together and which ones are the differential morphological characters. Our study aims to clarify their main morphological differences and to address two questions. Firstly, if two close species would be coexisting or secondly, if both species would not live together and then only one erect shrubby species should be recognised for the southeastern peninsular. Therefore, a detailed morphological study of these two species was carried out using vegetative and reproductive characters together with SEM features. Based on the original description, the main morphological features of S. hispanica were compared, plus other features which would support their identification. Our data pointed out the existence of a marked overlapping for most of the studied characters independently of the geographical procedence of the samples. As a result, no significant morphological differences have been found between both taxa, and S. hispanica is proposed as a mere synonym of S. fruticosa.

Two species of Sarcocornia are widely accepted to be present in the Iberian Peninsula and other adjacent Mediterranean territories (Greuter & al., 1986).Both specie are characterised by shrubby habit, opposite leaves fused to form a segment and spicate inflorescences with each fertile segment composed by 3-flowered cymes.The first one Sarcocornia fruticosa (L.) A.J. Scott can be recognized by erect and no rooting stems, with a seminal exotesta covered by papillae or short conic hairs with a wide base (Castroviejo, 1990;Guilló & al., 2012).This species, described by Linnaeus (1753), was typified on material from the Camargue in France (Ball, 2007).The second species, Sarcocornia perennis (Mill.)A.J. Scott, is distinguished by its creeping habit, with marked rooted branches and by a seminal exotesta covered by long and usually hooked hairs (Castroviejo, 1990;Guilló & al., 2012).This species was described by Miller (1768) from Sheppey Island (United Kingdom), and typified on material from the same locality (cf.Guilló & al., 2011).Additionally, a taxon related to S. perennis, S. alpini (Lag.)Rivas Mart.was described from southwestern territories (Cádiz province) of the Iberian Peninsula, (cf.Lagasca, 1817), though it is mostly considered under an infraspecific taxonomical range as S. perennis subsp.alpini (Lag.)Castrov.(Castroviejo, 1990).The separation between both subspecies are based on differences their habit and width of the inflorescence (Castroviejo & Coello, 1980;Castroviejo, 1990).For the time being, this infraspecific taxon has been mentioned to occur in other Mediterranean territories as Morocco, Portugal and Italy (Valdés & al., 2002;Molero & Monserrat, 2006;Costa & al., 2009;Biondi & al., 2010).Recently, a new taxon named S. hispanica Fuente & al. with an explicit distribution in the Murcian-Almeriensian, Betic and Mediterranean Central Iberian and Balearic-Catalan-Provençal biogeographic provinces (Fuente & al., 2011).Fuente & al. (2011) described this species as a shrub with erect and ascending branches, not rooting stems, characterized by a fused or partially fused pericarp to the seed and by a verrucate seminal exotesta covered with papillae.According to their diagnosis and in the absence of a comparative analysis by Fuente & al. (2011), S. fruticosa would be considered as the closest morphological taxonomic relative, since both taxa share many morphological features (e.g.habit, seminal coat ornamentation).Moreover, since S. hispanica occurs in part of the recorded distribution area of S. fruticosa in southeastern Iberian territories, apparently S. fruticosa and S. hispanica overlapp in their geographical area of distribution, (Álvarez Rogel, 1997;Alonso & de la Torre, 2002;Salazar & al., 2002;Fuente & al., 2011;Lendínez & al., 2011).As a consequence, the taxonomical identity of the Sarcocornia taxa, occurring in south-eastern Iberian territories, should be carefully reviewed as two mutually ecluding scenarios may apear: (i) either S. hispanica and S. fruticosa are two sympatric species which can grow together; or (ii) only one species characterised by an erect habit should be recognised and the consideration of S. fruticosa as a separate species results from a taxonomical misinterpretation.The main goal of this study is centred on clarifying the taxonomical identity of the southeastern Iberian Sarcocornia populations based on morphological comparative analyses between the two closest taxa: S. hispanica and S. fruticosa.Moreover, a nomenclature review has been carried out regarding to S. fruticosa in order to clarify the different used taxonomical denominations.

Sampling
A total of 45 populations distributed in 29 localities (L) around the western Mediterranean basin and Atlantic surrounding areas were studied (Fig. 1).Localities include from one to five populations.From the 45 populations, 40 correspond to S. fruticosa populations, and five populations traditionally considered as S. fruticosa but here referred as S. hispanica according to Fuente & al. (2011).The specific details of these populations are provided in Appendix 1.All studied plant material is mostly kept at the Herbarium of the University of Alicante (ABH, acronym according to Thiers, 2012).The type material of S. hispanica was also analysed (kept in MAF herbarium), but in the case of S. fruticosa, only the photographical material has been observed since the original type material could not be directly studied due to its conservation status (Ball, com. pers.).Besides, specimens from other herbaria (G, MA, MAF, VAL and W) were studied (see appendix 2).Besides, fresh material was particularly studied from the type localities of S. hispanica (Villena, Spain) and S. fruticosa (Camargue, France), which is kept at ABH.

Morphological study
A comparative morphological study was performed based on characters used in the literature to identify the species of the genus Sarcocornia (Valdés & al., 1987b;Castroviejo, 1990;Shepherd & al., 2005;Alonso & Crespo, 2008;Guilló & al., 2012), with a focus on those remarked for the identification of S. hispanica (Fuente & al., 2011).Three types of moprhological characters were selected.First, morphological vegetative characters: The habit, height of the plant and the presence/absence of rooting branches have been directly observed in the field.The size of vegetative segments was measured on five segments per individual and five individuals per population with maximum and minimum values are provided.The shape of the segments and their apical apexes were also observed on these same individuals.These latter observations were done using an optical binocular (Olympus SZX12) with micrometer.
A second set of morphological reproductive characters was included, those were: The size of terminal and lateral inflorescences (length×width) was measured in five individuals per population.Besides, total number of lateral spikes was also counted.Maximum and minimum values are provided.The fruit description is provided according to Font Quer (1989).The morphological relationship between the pericarp and the perianth was based on the suggestions done by Shepherd & al. (2005).Features related to seed shape, colour and seminal orientation were observed on three seeds per sample and population.These observations were done using an optical binocular (Olympus SZX12) with micrometer.
And finally a thrid group of micromorphological characters was studied by light and SEM microscopy in several structures.The seminal coat, stomata and pollen were studied using dried material.Seminal coats were observed on three seeds per sample and population.The features related to seed size (length×width), type of indumentum and shape of the trichomes were analysed using scanning electronic microscope (SEM) images.No special treatment was required prior to SEM observations.Samples were directly glued on metallic stubs, and then coated with about 30 nm gold for 5-10 minutes.Minimum and maximum values are provided for seed measurements.Stomatal morphology was studied using the vegetative segments according to Guilló & al. (2012).Pollen was also studied for six individuals from five localities (L2, L12, L14, L15, L19) using light microscope (Olympus SX 212) and SEM.The morphological nomenclature for seeds and stomata follows to Guilló & al. (2012), whereas pollen nomenclature was based on Valdés & al. (1987a).

Chromosome number
Samples from the southeastern Iberian populations of Laguna Salada de La Mata (Torrevieja, Alicante) and Salinas del Braç del Port (Santa Pola, Alicante) were analysed.We have followed the protocol proposed by Day & al. (2012), though with certain modifications due to the small genome of the genus Sarcocornia (Koce & al., 2008).Chromosomes were observed using a microscope Axioplan 2 (Carl Zeiss, Göttingen, Germany).The pictures were taken with a camera SPOT RT (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) using the software SPOT ST v. 4.0.4(Diagnostic Instruments).

RESULTS
Morphological vegetative characters values are summarized in Table 1.Habit is always erect for all the studied samples, independently of their taxonomical denomination.The height of S. fruticosa ranges from 50 to 180 cm high.This maximum value was observed in many samples from the salty soils of the river Antas (Almería, Spain).Besides, the plant height is 50-100 cm in the type population of S. fruticosa.In the case of S. hispanica, the samples show a similar range (50-100 cm), which is totally coincident with the obtained measures from the type population of this taxon.
In all the studied populations, adult plants do not develop rooting branches, since branches are mostly erect.Nonetheless, certain young branches of punctual young and short individuals would appear lied directly on the soil, and consequently, adventitious roots could be sporadically originated.This rare growth pattern should not be considered as the typical rooting habit defined for other related species as S. perennis, which always show rooting branches independently of the age of the branch and the sample (pers.obs.).

Morphological reproductive characters (Table 2)
Inflorescences.They are always branched being characterized by a terminal and several non-pedunculate lateral spikes.The thickness of the spikes was 2.0-4.0 mm for both species, independently of their geographical origin.The length of the spikes was also rather similar between both species and among all the populations (including the type localities).The obtained length range for S. fruticosa was 10-45 mm, whereas it was 11-45 mm for S. hispanica.The number of lateral spikes was 6-40 for S. fruticosa, being this range smaller in the type locality of the Camargue population (10-26 lateral spikes).Similar data were obtained for the populations of S. hispanica (14-40 lateral spikes), and its type population (10-28 lateral spikes).
Fruit.The fruit corresponds to an utricule, since it is dry, monosperm and dehiscent.Besides, its pericarp always appears totally jointed to the perianth, which becomes membranous.Subsequently, the seed remains inside this perianth-pericarp structure and partly protrudes being possible to be observed.Seeds can be mostly separated without difficulty from the perianth-pericarp structure, though some pericarp residues would remain adhered on the seed surface, and they are rather difficult to separate.This relationship between perianth-pericarp and seed has been widely observed for both S. fruticosa and S. hispanica.
Seeds.Seeds are always brownish and their shape varies from ovate to orbiculate, without any tendency between the studied species.The seminal orientation was vertical in relation to the ovary position.The size for S. fruticosa and S. hispanica populations (including the type localities) was 0.63-1.50×0.52-1.02mm and 0.93-1.37×0.69-0.85mm, respectively.
Table 1.Morphological vegetative characters studied for S. hispanica and S. fruticosa.For S. hispanica, data from their original diagnosis (Fuente & al., 2011), from the natural type population (Las Virtudes, Spain) and natural populations recognised as S. hispanica according to Fuente & al. (2011)

Segment apex shape
Subacute to rounded Obtuse to rounded Obtuse to rounded Obtuse to rounded Obtuse to rounded Micromorphological characters (Table 3) Stomata morphology.Both S. fruticosa and S. hispanica, stomata are paracytic and they are well characterized by their deeply sunken guard cells compared to both epidermal and subsidiary cells.This stomatal morphology is named as sunken stoma (Fig. 2).
Pollen characteristics.Pollen is constantly pantoporate, apolar, with an equinulate surface characterised by simple pores (Fig. 3).This morphology was identical for S. fruticosa and S. hispanica.
Seeds.Concerning to seminal coat, the epidermic cells are flattened with a polygonal morphology.Two different kinds of trichomes were observed in both S. fruticosa and S. hispanica samples: (1) papillae and (2) short and conic hairs with a wide base (Fig. 4).These two morphologies can be easily observed in different individuals of the same population and also within the same seed.

DISCUSSION
Sarcocornia is a notable complex genus as has been reported by several authors (Shepherd & al., 2005, Alonso & Crespo, 2008;Steffen & al., 2010;Guilló & al., 2012), who also highlighted the importance of SEM characters such as seminal coat and stomata to identify among taxa.These morphological characters are rather stable as compared to other vegetative and reproductive features (e.g.habit, size of inflorescences), which might vary depending on the fluctuations of certain ecological conditions (e.g.water level, salinity, etc.).
The original description of Sarcocornia hispanica was mainly based on the life form (habit), shape of leaf apex, pericarp, seminal features (Fuente & al., 2011).Concerning to life form, many authors settled it as a taxonomic character to differentiate between S. perennis and S. fruticosa.The former is well characterised by rooting branches and a prostrate habit against an erect habit without rooting branches for S. fruticosa (Quézel & Santa, 1963;Pignatti, 1982;Bolòs & Vigo, 1990;Castroviejo, 1990;Coste, 2007;Jeanmonod & Gamisans, 2007;Sánchez-Gómez & Guerra, 2007;Mateo & Crespo, 2009).Conversely, S. fruticosa and S. hispanica Table 2. Reproductive characters studied for S. hispanica and S. fruticosa.For S. hispanica, data from their original diagnosis (Fuente & al., 2011), from the natural type population (Las Virtudes, Spain) and natural populations recognised as S. hispanica according to Fuente & al. (2011)   are both characterised by an erect life form and the lack of rooting branches as previous authors have pointed out (cf.Castroviejo, 1990;Fuente & al., 2011).Besides, the plant height reported for S. hispanica (up to 100-150 cm, Fuente & al., 2011) is rather overlapped to the obtained data for S. fruticosa (up to 180 cm) and also with the maximum value (up to 150 cm) stated by Castroviejo (1990).As a consequence, these vegetative features are not useful to differentiate between them.
The apex of the segments was commonly used in the descriptions of some Sarcocornia species (Maire & Quézel, 1962;Castroviejo, 1990;Alonso & Crespo, 2008;Steffen & al., 2009Steffen & al., , 2010)).Fuente & al. (2011) pointed out the existence of a wide morphological range from a subacute to rounded apex for S. hispanica and Castroviejo (1990) reported a subacute morphology for S. fruticosa.Our data have showed a general tendency from obtuse to rounded apexes, which are noticeably included within the ranges reported by Fuente & al. (2011).Fuente & al. (2011) used the presence of the pericarp partly fused to the seed as an own character of S. hispanica against the rest of the Iberian Sarcocornia species.On the basis of our data, all the studied populations also showed the pericarp relatively fused to seeds, and these are kept included in it though they are clearly visible because of the dehiscence of the utricule.Therefore, seeds are separated from the pericarp with difficulty, and pericarp residues are usually detectable in most of the seeds.These data are totally concordant with Shepherd & al. (2005).
Seed shape was also stated as one of the main characters to distinguish S. hispanica from other Sarcocornia taxa (Fuente & al., 2011).These authors reported the presence of rounded seeds, which falls within the variability here observed (from ovate to orbiculate).Additionally, seed size measured in the type locality of S. hispanica (Las Virtudes, Spain) was somehow lower than the original data done by Fuente & al. (2011), but these measures matched with  S. fruticosa data.As for previous morphological features, no tendency was newly observed between the studied species.Concerning to seed coat has been widely described for taxonomical purposes within the genus Sarcocornia (Shepherd & al., 2005;Alonso & Crespo, 2008;Steffen & al., 2009Steffen & al., , 2010)), and specially to distinguish between S. fruticosa   and S. perennis (Maire & Quézel, 1962;Meikle, 1977;Valdés & al., 1987b;Bolòs & Vigo, 1990;Castroviejo, 1990;Cabello 2011;Guilló & al., 2012).For S. fruticosa, the mentioned authors reported a seed ornamentation well characterised by the presence of short hairs, papillae or mamelons.Our data reveals the presence of this seminal ornamentation for all the studied seeds from the different Mediterranean populations, including those type localities from southeastern Iberian Peninsula and southern France populations.No tendency was observed among the studied populations.Fuente & al. (2011) reported a seminal morphology for S. hispanica, which is totally coincident with the morphology of the studied populations independently of the taxonomical designation and their geographical origin.
Additionally, other vegetative and reproductive features not mentioned by Fuente & al. (2011) have been analysed in detail to extend the morphological study between these two species.The segment size and shape were totally coincident among all the studied populations.The number of lateral spikes and their size were rather equivalent between the two type localities, and none difference was found among all the studied populations.Moreover, the wide spike reported by Fuente & al. (2011) is identical to our data from Camarga populations.Although some features related to the inflorescences have been previously used to identify among Sarcocornia taxa (e.g.Castroviejo, 1990), our results pointed out that these two features are not usefulness to discriminate between the two Sarcocornia species.Similarly, the pollen has shown the identical morphological features among all the studied populations, and hence it is similar among other Sarcoconia species and also among the genera of this family (Valdés & al., 1987a).As a result, pollen morphology should not be considered as a taxonomic character to support the identification among Sarcocornia species.Finally, fruit identification or any other related feature is not usually included in the Sarcocornia treatments (Maire & Quézel, 1962;Quézel & Santa, 1963;Valdés & al., 1987b;Bolòs & Vigo, 1990;Stace, 1991;Coste, 2007;Jeanmonod & Gamisans, 2007;Cabello, 2011), but certain contradictory descriptions have been given about its definition.Meikle (1977) reported the fruit of this genus as an utricule, whereas Castroviejo (1990) indicated it as an achene.Recently, Fuente & al. (2011) described the fruit of Sarcocornia hispanica as an achene, based on the pericarp appears partially fused to the seed.According to Font Quer (1989), the main differences between utricules and achenes are principally based on their dehiscence.This author defined an utricule as a dehiscent fruit against the permanent indehiscence of the achene.According to our observations, the fruit of Sarcocornia is always dehiscent, and consequently it should be considered as an utricule as Meikle (1977) initially reported.
Finally, Castroviejo & Coello (1980) and Castroviejo & Lago (1992) reported the hexaploid (2n=54), or even octoploid (2n=72) levels for S. fruticosa in many populations from the Iberian Peninsula, basically from southeastern territories (including the potential area of S. hispanica).Fuente & al. (2011) stated the presence of the hexaploid level for S. hispanica species, just based on the counts from the populations of Villena (type locality) and El Hondo, both in Alicante province.Likewise, our data have yielded the presence of the hexaploid level for Alicante populations, exactly from the populations Laguna Salada de La Mata (Torrevieja) and Salinas del Braç del Port (Santa Pola).Related to these two latter populations, Castroviejo & Coello (1980) also reported the highest level of ploidy (octoploid) for Sarcocornia samples of these areas, and they stated that no difference was found among individuals and localities characterised by different levels of ploidy.Moreover, the hexaploid level has been stated for S. fruticosa in other European territories as Portugal (Castro & Fontes, 1946) and France (Contandriopoulos, 1968;Labadie, 1976).Consequently, the level of ploidy within Sarcocornia would not provide any additional information to support the taxonomical differentiation between S. fruticosa and S. hispanica.

CONCLUSIONS
According to our data, no significant differences have been found between the widely distributed S. fruticosa and the recently described species S. hispanica on the basis of habit, branches, inflorescences, seeds and chromosome counts.The morphological comparison between individuals from the type populations of both S. hispanica (Las Virtudes, Spain) and S. fruticosa (Camargue, France) showed the lack of morphological differences between them.Besides, the morphological quantitative and qualitative charachters here obtained from the locality Las Virtudes (type locality of S. hispanica) and from other European populations fall within the variation reported by Fuente & al. (2011) for S. hispanica.Therefore, all this evidence together with the wide morphological plasticity observed in wild populations of the two taxa supports treating S. hispanica as a synonym of S. fruticosa, which was originally described by Linnaeus (1753).

Fig. 1 .
Fig. 1.Map of the studied populations.The squares correspond to Sarcocornia fruticosa populations and the circles to Sarcocornia hispanica populations.

Fig. 2 .
Fig. 2. Stomata.a, General aspect of the sunken stomata from the population of Saint Marie de la Mer, Camargue (France, ABH 52278); b, General aspect of the sunken stomata from the population of Las Virtudes, Villena (Spain, ABH 57792); c, Detailed aspect of a sunken stoma from the population of Saint Marie de la Mer, Camargue (France, ABH 52278); d, Detailed aspect of a sunken stoma from the population of Salinas de Braç del Port, Santa Pola (Spain, ABH 53139).