The morphological and anatomical reinvestigation of the Psilotum nudum, in Hyrcanian forests, N Iran

Introduction

Psilotaceae, as a family of Psilotales, comprises two genera (PsilotumSw., TmesipterisBernh.) and about 15 species of fern-like plants (Kenrick, 2000). The genus Psilotum consists of two species, P. nudum (L.) P. Beauv.(= P. triquetrumSw) and P. flaccidumHook. & Grev.(= P. complanatum Sw.). Most commonly, it grows erectly on the ground or in crevices among rocks, but it may also grow as an epiphyte on tree-ferns or among other epiphytes on tree branches (Sporne, 1962). It is sometimes found in botanical gardens and in greenhouses (Nazarian et al., 2010). Psilotum nudum is fairly common in tropical and subtropical parts of both hemispheres (Singh et al., 2010).

This genus is often grouped with the extinct Psilophytes, the Rhyniales and Zosterophyllales dating from the Devonian some 400 million years ago (Roux, 2003). Among the vascular plants, they considered as one of the oldest and the simplest. The word Psilotum is derived from the Greek word Psilos which means naked or bare which is in regard to its lack of true leaves on the stem (Amanda, 2010). Psilotum nudum is more commonly known as the Whisk Fern; this name comes from the likeness of the branched stems to whisks and alludes to its use in the past as a small broom.

Recently, study of its morphology and anatomy suggests that the ancestral features present in living Psilophytes represent a reduction from a more typical modern fern plant. Likewise, molecular evidence strongly confirms that Psilotum is a fern (Smith et al., 2006).

The genus Psilotum is characterized by dichotomously branched stems, the absence of roots, a relatively simple vascular structure, small reduced leaves and thick-walled, homosporous synangia (Roux, 2003). These small plants consist of a brown underground rhizome which is likewise much branched. Psilotum gametophytes apparently have a lipid coating (Bierhorst, 1971), and they are also subterranean, mycorrhizal, and not ephemeral. Likewise, it has two cytological races, a diploid with a chromosome number 2n = 52; and a tetraploid with 4n = 104 (Khullar, 2008).

So far, only a few anatomical and morphological studies have been published on the genus Psilotum (Ford, 1904; Stiles, 1910; Sporne, 1962; Schulte et al., 1987; Khoshravesh et al., 2009; Nazarian et al., 2010; Singh et al., 2010). However, the current study can relate these data to other available morphological and anatomical data of this genus and will certainly enrich the botanical information on this species.

In the present study, firstly we report a new locality with a new habitat in the Hyrcanian area. Then, we systematically examined the morphology and anatomy of aerial stem and rhizome in Psilotum species growing in two lowland forests (Ata-Kuh and Ramsar), in order to determine the presence of Psilotum nudum in forest ecosystem of Guilan.

Materials and Methods

Plant samples were collected from natural habitats and the voucher specimens were deposited in the Herbarium of Guilan University. For each sample, replicates were collected from two populations. Morphological data evaluated based on 10-15 plant specimens. Anatomical studies carried out on the samples conserved in a solution of alcohol-water-glycerine. The cross sections were stained with methylene blue and congo red and mounted with glycerine jelly to make permanent slides (Vardar, 1987). Well-stained sections of the aerial stem, apex aerial stem and rhizome were studied under the light microscope (using ocular and stage micrometer) and photographed by camera. The aerial stem was placed in a tube filled with 70 % lactic acid for 4-5 days. The sections were then prepared by a sharp scalpel or by hand.

Results

Habitat preferences and morphological evidences

Psilotum nudum is a perennial plant which grows as an epiphyte on the trunk of Parrotia persica C.A.May. in the Ramsar riparian forest or river bank (586 m a.s.l.) and as a lithophyte in crevices among rocks near streams in the Ata-Kuh forest (207 m a.s.l.) (Figure 1A, B). Aerial stems are 26 cm (Ramsar population) to 31 cm (Ata-Kuh population) long, and 2 mm in diameter, repeatedly dichotomously branched above with 20-36 branches, often pentagonal towards the first dichotomy and in the most distal portions triangular, erect, glabrous and chlorophyllous, with longitudinal parallel lines and dichotomous bract. Sporangia are 2.5 mm in diameter, large, with walls two cells thick, two or three sporangia fused to form a synangium, and orange-brown when mature. Leaves are 1.5-2 mm long, with spiral phyllotaxy on stem, lanceolate to ovate in outline, scale-like, pointed, with no stomata, lacking a midrib and without ligules (Figure 1C, D). Rhizomes are 3-4 cm (Ata-Kuh) and 2-4 cm (Ramsar) long, to 1.5 mm in diameter, cylindrical, lack chlorophyll (non-photosynthetic), subterranean, rootless, with short and long rhizoids and mycorrhizal. This is the first report of the species in the Hyrcanian forests of Guilan (Figure 2).

Figure 1. Psilotum nudum (L.) P. Beauv. A and B. Habit and Habitat (Ramsar on Parrotia persica C.A.Mey.); C. Synangia (b. Bracts; Sc. Scaly leaves; Sy. Synangium); D. Close up of synangium. Scale Bar D = 0.1 mm

Figure 2. Map showing the distribution of the sampled population

Anatomical studies

Aerial stem

Cross sections of the aerial stem were irregular in outline (pentagonal in Ramsar samples and star-like in Ata-Kuh samples) due to the presence of grooves and ridges. Epidermis was composed of oblong cells that were heavily cutinized and interrupted by large numbers of stomata. Stomata were of the anomocytic type (Figure 3). Each stoma had two dumbbell shaped guard cells that are somewhat sunk below the level of the other epidermal cells and had heavily cutinized walls like those of epidermal cells (Figure 4D, J). The cortex was differentiated into three zones (I) outer cortex consisting of 2-4 layers of chlorophyllous cells (since the leaves did not perform their photosynthetic function, the green stem capture this activity), (II) middle cortex comprising 3-6 layers of sclerenchymatous cells, and (III) inner cortex, including thin-walled parenchymatous cells (Figure 4B, H). The stele was a protostele (actinostele) with xylem rays (star-like and pentarch to octarch) and surrounded by distinct layers of endodermis with characteristic casparian bands on the radial walls of its cells. The center of the stele was occupied by sclerenchymatous pith (Figure 4C, I). The characteristics of theaerial stem, apex aerial stem and rhizome anatomy of Psilotum species of 2 localities were given in Table 1.

Apex aerial stem

In cross section, apex aerial stem was triangular. Epidermal cells are quadrangular. Cuticle was present on the epidermis. Stomata were of the anomocytic type. The cortex was undifferentiated and the stele was a protostele (actinostele) that was surrounded by a clear layer of endodermis. In the apical part there were only 2-3 radiating rays (diarch or triarch) (Figure 4E, F, K and L).

Rhizome

Cross sections of the rhizome are elliptic-circle in outline. Rhizome epidermis is composed of quadrangular cells that are cutinized. Beneath this layer is a cortex that is differentiated into three zones. The outer and middle cortex includes parenchymatous cells. Inner cortex is dark brown in colour because of the presence of phlobaphene. Endodermis is distinguishable. It is followed by single layered pericycles which encircle the stele; the stellar type is a protostele (haplostele) (Figure 5).

Figure 3. Stomatal type in the aerial stem in two studied localities (A. Ata-Kuh and B. Ramsar samples). Scale Bar = 0.1 mm.

Figure 4. Transverse sections of the aerial stem (A-D. Ata-Kuh; G-J. Ramsar samples): A and D. General aspect; B and H. A part of aerial stem; C and I. Stele region. D and J. Stoma cell with cutinized walls. Transverse sections of the apex aerial stem (E-F. Ata-Kuh; K-I. Ramsar samples): E and K. General aspect; F and I. Stele region. Abbreviations: E. Epidermis; C. Cuticle; St. Stomata; Ph. Phloem; Xy. Xylem; P. Parenchyma; Sc. Sclerenchyma; En. Endodermis. Scale bar = 0.1 mm

Table 1. Anatomical characters of Psilotum nudum in Ata-Kuh and Ramsar forests

Discussion

Habitat preferences

Psilotum nudum was discovered in a lowland Hyrcanian forest of Ramsar in 2003 (Rezaei, 2003). It was mainly growing on the trunks slots and roughness of Parrotia persica trees in a very shady habitat. During last ten years after the first discovery of the species, this species was also reported elsewhere as epiphyte on some other species e.g. Alnus glutinosa (L.) Gaertn. in Chalus valley (Nazarian et al., 2010), Nowshahr (Dr. Habib Zare, personal communication) and in Ramak valley in Ramsar (Naqinezhad and Zarrei, in preparation). In all mentioned habitats, P. nudum was found as epiphyte on lowland trees. Moreover, Khullar (2008) stated that it grew on the ground or in rock crevices, and Morgan (1962) reported that P. triquetrum had been found in rock fissure through which water is flowing. In the current study, a new different habitat, rocks, was found for this species in Ata-Kuh forest and also, as an epiphyte on Parrotia persica, in the Ramsar forest, confirming previous findings.

These habitats are under the threat of damage and destruction by road building and human activites. Therefore, they should be regarded as threatened species according to IUCN categories and criteria.

Anatomical and morphological reexamination

We decided to investigate Psilotum nudum morphologically and anatomically in detail. Our morphological findings of P. nudum were consistent with the morphological description of the taxon, of Ford (1904), Stiles (1910), Sporne (1962), Khoshravesh et al. (2009), Nazarian et al. (2010), and Singh et al. (2010).

According to Singh et al. (2010) P. flaccidum was 90 cm long and the leaves were in opposite position on the edges of plane branches, while P. nudum was 25-35 cm long and the leaves were placed in alternate position. Another comparable feature with respect to P. nudum and P. flaccidum in stem morphology was in the form of the aerial stem. In P. flaccidum the lower part was triangular and the upper part flattened in one plane, while in P. triquetrum this is multiangular in the lower parts and triangular in the ultimate branches (Stiles, 1910). Our results showed that the cross section of P. nudum stem supports this idea. Moreover, in another report by Sporne (1962), P. nudum had upright habit and P. flaccidum had pendulous habit.

Our results, like those of Ford (1904), Sporne (1962), Khullar (2008), Khoshravesh et al. (2009), Nazarian et al. (2010), and Singh et al. (2010), showed that the cortex in the stem was differentiated into three zones (outer photosynthetic, middle sclerenchymatous and inner parenchymatous) and the sclerenchymatous layers were responsible for mechanical protection of the plant. The result of this study showed that stele in the aerial stem was protostele (actinostele). The stele has been interpreted as a protostele (actinostele) by Schulte et al. (1987), Khoshravesh et al. (2009), and Singh et al. (2010) and as a siphonostele by Nazarian et al. (2010) and Gifford and Foster (1988). Khullar (2008) also mentioned that if the pith was considered as composed of sclerenchymatous cells, then the stele was a siphonostele, but if these sclerenchymatous cells belonged to the xylem, then it was a protostele. Also, our results showed that the cortex in the rhizome was differentiated into three zones (outer, middle and inner); this finding was in agreement with results obtained by Sporne (1962) and Ford (1904). Also, we found that stele in the rhizome was a protostele (haplostele), since non-differentiated tracheids to protoxylem and metaxylem in the pith are surrounded by phloem. Nazarian et al. (2010) have reported the stele is protostel (actinostele) in the rhizome. However, Singh et al. (2010) have reported that the stele was a protostele (haplostele), which supported our observations in this research.

The size and shape of the stomata are taxonomically important characters (Tahir and Rajput, 2009). In our study, stomata type was anomocytic; this is in agreement with observations reported by Roux (2003) and Mickle (2012).

Conclusion

In the current investigation, the morphological and anatomical studies on P. nudum indicate similarity between the samples collected from the two habitats, and a comparison of our results with other researcher’s studies on Psilotum species indicates apparent differences with the P. flaccidum species and confirms the existence of P. nudum in forest ecosystem of Guilan.

Acknowledgements

This research was supported by the project of University of Guilan and Mazandaran University. We would like to thank H. Vahdati for help in the field study.