Arthrocaulon meridionalis ( Chenopodiaceae ) , a new species of Mediterranean flora

A new species of Arthrocaulon Piirainen & G. Kadereit, A. meridionalis is described. This diploid taxon is known from the islands of Sicily and Sardinia and from circum-Mediterranean territories (from North Africa to the Anatolian Peninsula in Turkey and as far as the Persian Gulf on the Asian continent). The distinctive macro-, micromorphological and chorological features of this taxon are given.


Introduction
Arthrocnemum was described as a new genus in the monograph on the Chenopodiaceae family published by Moquin-Tandon (1840:111).Among the basic morphological characters included by the author we highlight the following: 'hermaphrodite flowers, squamate, arranged at the branch nodes, swollen or bulging perigon, trigonous or tetragonous truncate apex, ending abruptly in a more or less straight line, finally spongy, without appendages.Stamen 1 or 2, inserted on the receptacle.Adnate style with 2 stigmas.Utricle flattened on the sides, swollen, fleshy and rolled inwards towards its upper adaxial surface; separate membranaceous pericarp.Vertical, lenticular seeds, with a persistent farinaceous perisperm.Semiannular embryo.
The same authors (Sukhorukov & Nilova, 2016: 237-239) indicate the need to typify the genus in a highly restrictive and conservative sense respecting the protologue and the original materials used by Moquin-Tandon in his description ['Type of the genus: not yet typified (the genus should be typified with a conserved name')].We absolutely agree with these authors on the need to make a thorough revision of the typification of the genus Arthrocnemum by publishing a justified proposal for a conserved generic name with a conserved type (in prep.).
This line is overlooked in the recent work by Piirainen et al. (2017), where they support the old proposal of Pfeiffer who, in his Nomenclator Botanicus (1872:279 -cf. Rye & Wilson 1999:794), mentions 'Salicornia fruticulosa L.' as a representative of the genus Arthrocnemum.The authors therefore publish a proposal for a new genus: Arthrocaulon Piirainen et al. which would include Arthrocaulon macrostachyum (Mediterranean Europe and N Africa, E Africa, SW Asia) and the one recently described from Cape Verde Islands, Arthrocaulon franzii (Sukhorukov & Nilova, 2016:239).They also propose the new genus Arthroceras (A. subterminale) for the west of North America.
Our review of a broad spectrum of circum-Mediterranean populations of Arthrocaulon macrostachyum points to the existence of contrasting genetic diversity.The tetraploid level is confirmed by Castro & Fontes (1946) and Queirós (1975) with Portuguese material, by Contandriopoulos (1968) with material from the south of France, by Castroviejo & Coello (1980) in Spain, and by Runemark (1996) with material from Greece.Ghaffari (2006:129) publishes the diploid level for this species on the coasts of the Persian Gulf ['Between Gavbandi and Kangan, Bidkhoon, 2.12.1987, Assadi & Akhani 64014 (TARI Herbarium), 2n=18'].
The phylogenetic trees presented by Piirainen et al. (2017) also reveal a clear separation between the Turkish and Iberian populations.Both genetic and phylogenetic data suggest a differentiation of the genus Arthrocaulon among the circum-Mediterranean populations.We therefore propose the new taxon Arthrocaulon meridionalis spec.nova to include the diploid populations distributed in southern Mediterranean halophytic habitats.

Plant material
Complete fresh specimens from various Mediterranean coastal and inland territories of Arthrocaulon populations were collected directly from the field in different stages: spring-summer (flowers) and summer-autumn (fruits and seeds).Plant material was preserved at -20°C for subsequent analysis.Voucher specimens of the collected and analysed material are preserved in the MAF Herbarium (Faculty of Pharmacy, Complutense University, Madrid, Spain).
The morphological study of seeds and fruits was done using voucher specimens from several herbaria: MA Herbarium (Real Jardín Botánico, CSIC, Madrid, Spain), MAF Herbarium, and PAL Herbarium (Herbarium Mediterraneum Panormitanum, Palermo Botanical Garden, Sicily, Italy) and our personal collections (V.de la Fuente, UAM, Spain).A complete list of the plant material collected and the voucher specimens for this study is shown in Figure 1 and the attached appendix (Appendix 1).

Morphological and micromorphological characters
Optical microscope.The Olympus SZX10 stereomicroscope was used to take photographs of the morphological characters of interest and to take measurements of leaf lengths (mm), leaf hyaline margin width (mm), inflorescence length-width (cm), bract hyaline margin width (mm), central and lateral perianth length (mm), anther length (mm), style length-width (mm), stigma length (mm) and seed length-width (mm) (Appendix 2).The measurements were made with the program tool associated with the stereomicroscope (Olympus Stream Image Analysis Software) and the photographs with the Olympus SC30 camera, also attached with the equipment.
Scanning electronic microscopy analysis (SEM).Cross-sections of flower, fruits and seeds were cut with a sharp blade.Dry samples were fixed in situ with formyl acetic alcohol (FAA).After washing with a 0.1M phosphate buffer (pH 7.4), they were dehydrated through a graded ethanol series.Dry samples were mounted flat on the surface of conductive graphite stubs and sputtered and gold coated in a Bio-Rad SC 502 apparatus for electrical conductivity and to prevent charging under the electron beam.Samples were examined with a Hitachi S-3000N (Japan) SEM using an acceleration voltage of 20 kV and a working distance of 15 mm.The samples were analysed at room temperature.
Description.Perennial woody shrub up to 100 cm tall, almost always erect and less frequently prostrate.Secondary branches erect and ascending.Regular decussate ramification.Opposing amplexicaule scale-like leaves 2-3 mm long, fused at their base and to the stem forming a cyathiform structure of fleshy segments, with a scarious hyaline margin 0.3-0.5 mm wide with an acuminate apex.
Inflorescence (2.5) 3.8 (5.5) cm long and (0.2) 0.35 (0.5) cm, spicate, terminal and lateral, segmented; each fertile segmented composed of two 3-flowered cymes, decussate, immersed in the pair of opposite scale like bracts with a hyaline margin 0.4-0.65 mm wide.Cymes formed of three sessile flowers in a row, fused at the base, with the central flower arranged slightly higher than the lateral ones.Central perianth slightly longer than the two lateral sections of the perianth (1.2) 1.4 (1.9) / (1) 1.3 (1.5) mm respectively.Fleshy perianth formed of four tepals fused to the tip, which has a floral opening formed by 2 lateral orbicular flaps and two more scarious frontals.Stamens 2 with yellow anthers (0.6) 0.9 (1.2) mm long, apiculate at the base; superior ovary with style from 0.1 to 0.7 mm in length and 0.05 to 0.1 mm in width.2(3) stigmas.
Distribution.This species occurs on the islands of Sicily and Sardinia and in circum-Mediterranean territories from North Africa to the Anatolian Peninsula in Turkey.Other populations not included on the map: Iran: between Gavbandi and Kangan, Bidkhoon, 2.12.1987,Assadi & Akhani 64014 (TARI Herbarium), 2n=18, Figure 1.Numbers refer to localities and populations listed in Appendix 1.
Ecology and Phytosociology.This species grows in the most inland and xeric zones of saline habitats and can withstand high concentrations of salt.The communities undergo dry periods in summer (Brullo & Furnari, 1976).
According to our field observations, it grows alongside Sarcocornia fruticosa (L.) A.J. Bolòs 1950 (Rufo et al., 2016).In contrast, Arthrocaulon macrostachyum is described as a perennial woody shrub growing up to 150 cm tall, from prostrate to erect, often forming rounded mats.Secondary branches erect and ascending.Regular decussate ramification.Opposing amplexicaule scalelike leaves 2-3 mm long, fused at their base and to the stem forming a cyathiform structure of green, glaucous or reddish fleshy segments, with a scarious hyaline margin 0.3-0.4mm wide with a slightly to very acuminate apex.Inflorescence (2.5) 2.9 (4) cm long and (0.3) 0.4 (0.5) cm wide, cylindrical, spicate, terminal and lateral, segmented; each fertile segment composed of two 3-flowered cymes, decussate, immersed in the pair of opposing scale like bracts with a hyaline margin 0.3-0.5 mm wide.Cymes formed of three sessile flowers in a row, fused at the base, and with the central flower arranged slightly higher than the lateral ones.Central perianth slightly longer than the two lateral sections of the perianth (1.9) 2.1 (2.4) / (1.8) 1.9 (2.3) mm respectively.Fleshy perianth formed of four tepals fused to the tip, which has a floral opening formed by two lateral orbicular flaps and two more scarious frontals.Stamens 2 with yellow anthers (0.9) 1.1 (1.5) mm long, apiculate at the base; superior ovary with style from 0.1 to 0.6 mm in length and 0.1 to 0.2 mm in width.2(3) stigmas.Fruit an utricle-like.Membranaceous pericarp partially fused to the seed.Vertical, round or ellipsoidal, reddish-black seeds (0.9  et al., 2004, 2014) (Figure 1).Other populations not included in Figure 1 are from the Canary Islands (GenBank number KU975176).Arthrocaulon macrostachyum occurs in salt marshes on the Atlantic and Mediterranean coasts in mid-and high-intertidal areas, occupying territories that are only occasionally flooded by tides, and in saline depressions where it is not usually reached by seawater.It can also be found far from the coast in endorreic lakes derived from Tertiary materials, and even in canals in salt works.It usually grows on saline sandy to clayey gypsiferous soils where it may commonly be accompanied by Sarcocornia and Salicornia species (Salicornietea fruticosae phytosociological class) (Rufo et al., 2016).

Discussion
Arthrocaulon macrostachyum (2n: 36) differs from Arthrocaulon meridionalis (2n: 18) in both, its ploidy level and the different morphological characteristics of its stems, leaves, flowers and seeds, as shown in Figure 2, 3 and Appendix 2. The main differences can be summarised in the following morphological characters: Inflorescence size.In general terms, the size of the inflorescence was found to be shorter and wider in the Iberian populations (2.5) 2.9 (4) / (0.3) 0.4 (0.5) mm long/width than in the Sicilian and Sardinian populations, which are characterized by slightly longer and thinner inflorescences (2.5) 3.8 (5.5) / (0.2) 0.35 (0.5) mm long/width.Bract hyaline margin.The scarious margin of the bracts is slightly thinner in the Iberian populations of A. macrostachyum compared to those from Sicily and Sardinia: 0.3-0.5 mm wide and 0.4-0.65 mm wide respectively.
Perianth.The central flower is slightly larger than the lateral ones in both species; however, the perianth in the Iberian populations ((1.9) 2.1 (2.4) mm central perianth and (1.8) 1.9 (2.3) in both lateral perianth sections) has been observed to be longer than in the Italian populations ((1.2) 1.4 (1.9) mm central perianth and (1) 1.3 (1.5) in both lateral perianth sections).
Anthers.The anthers of A. macrostachyum (0.9) 1.1 (1.5) mm are generally longer than those observed in the Italian populations ((0.6) 0.9 (1.2) mm long).Style.Although the styles in the individuals observed in both species have a very similar length, differences have been found in the width; the style of the Iberian populations is wider (0.1-0.2) mm than in the Sicilian and Sardinian ones (0.05-0.1) mm.
Seeds.The seeds of both species are morphologically similar and generally slightly larger and wider in the Iberian populations ((0.9) 1.1 (1.25) mm long and (0.7) 0.8 (0.95) mm wide) compared to the Italian ones ((0.8) 0.9 (1.1) mm long and (0.6) 0.65 (0.8) wide).Amount and size of papillae in testa cells is variable in both species.Combined use of macro-micromorphological and biogeographical features are essential to correctly delimit plant species.This type of data has already been used by several authors for other genera of Chenopodiaceae close to Arthrocaulon (Alonso & Crespo, 2008;Biondi et al., 2013;Fuente et al., 2013Fuente et al., , 2015;;Rufo et al., 2016).In regard to phylogenetic relationships, the molecular phylogeny was studied in depth based on the combined analysis of ETS, ITS, atpB-rbcL and matK-trnK (Piirainen et al., 2017, Supplementary Material, Figure S3), in greater detail for the genus Arthrocaulon than the phylogeny chosen for the main document, and revealed a separation with a moderately to well-supported bootstrap (BS 83) for the Turkish populations (ETS: KU975228*, ITS: KU975175; ETS: EF433587, ITS: AY489240, matK-trnK: KU975311) compared to the Spanish and Portuguese populations, which are grouped in politomy (ETS: KU975227, ITS: KU975174, atpB-rbcL: KU975274, matK-trnK: KU975310; ITS: AY489239, atpB-rbcL: EU484419 = DQ340101; ETS: KU975229, ITS: KU975176, atpB-rbcL: KU975275, matK-trnK: KU975312).Our data for the ITS marker corroborate this separation at the molecular level and incorporate all the sequences taken from various populations in Sicily and Sardinia in the eastern block (data not showed).It is highly likely that these separations are related with the genetic and morphological differences implied by the two types of chromosome levels present in the genus.
On the other hand, individuals sequenced in the Canary Islands in the same study (Piirainen et al., 2017), in which no exact location is specified within these islands, is added to the clade of A. macrostachyum.The sequences of Piirainen et al. 2017 for the populations of Senegal (GenBank numbers: ETS: KU975265, ITS: KU975211, atpB-rbcL: KU975300 and matK-trnK: KU975357), present a clear separation from the rest of the sequences grouped in A. macrostachyum.In a later work (Ball et al., 2017), the names of these sequences were transferred from A. macrostachyum to A. franzii.Thus, the sequencing of new samples in Cape Verde, Senegal and other African areas could clarify the ascription of these populations within the genus Arthrocaulon.In future studies it is necessary to advance in the characterization of this genus, especially focused on the phylogeny of the populations present in the African.

Figure 1 .
Figure 1.Map showing the distribution of Arthrocaulon macrostachyum (points) and A. meridionalis (stars).Numbers refer to localities and populations listed in Appendix 1.

Figure 2 .
Figure 2. Arthrocaulon meridionalis (A-F) and A. macrostachyum (G-K).A, Sterile branch showing the oppossite amplexicaule leaves with a scarious hyaline margin with an acuminate apex.Scale bar: 2 mm; B,Inflorescence.Scale 2 mm; C, Three sessile flowers of a cyme, the central one slightly bigger than the lateral ones.Scale bar: 500 µm; D, Detail of the perianth of a flower.Scale bar: 500 µm; E, Detail of a three flowered cyme.Scale bar: 1 mm; F, Detail of the opening of the perianth with the four flaps.Scale bar: 500 µm; G, Sterile branch showing the oppossite amplexicaule leaves with a scarious hyaline margin with an acuminate apex.Scale bar: 2 mm; H, Inflorescence.Scale bar: 2 mm; I, Perianth of a flower showing two exerted stamens.Scale bar: 2 mm; J, Detail of an inflorescence with flowers with exerted styles.Scale bar: 1 mm; K, Detail of a perianth of a flower.Scale bar: 1 mm.

Figure 3 .
Figure 3. Representative SEM images.A) Cross section of a three flowered cyme of A. macrostachyum, s: seed.Scale bar: 500 µm; B) Three sessile flowers of a cyme of A. meridionalis.Note the central flower slightly bigger than the lateral ones.Scale bar: 1 mm; C) A fleshy perianth of a flower of A. macrostachyum.Scale bar: 1 mm; D) Opening of the perianth of a flower showing the four flaps of A. meridionalis, sg: stigma.Scale bar: 500 µm; E) Style and two stigmas with the two stamens of a flower of A. meridionalis, sg: stigma, st: style, sm: stamen.Scale bar: 500 µm; F) Seed of A. meridionalis.Scale bar: 300 µm; G) Seed of A. macrostachyum.Scale bar: 500 µm.