Leaf micromorphological adaptations of resurrection ferns in Northern Pakistan

Flora 255 (2019) 1–10

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Leaf micromorphological adaptations of resurrection ferns in Northern Pakistan

T

Syed Nasar Shaha,b, , Mushtaq Ahmada, Muhammad Zafara, Fazal Ullaha,d,e, Wajid Zamana,c,d, Jaideep Mazumdarf, Izaz Khuramg, Shujahul Mulk Khana ⁎

a

Department of Plant Sciences, Quaid- i- Azam University Islamabad, 45320, Pakistan Science Laboratory, Government High School, Dherai Puran, Shangla, Pakistan c State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, China d CAS Key Laboratory of Mountain Ecological Restoration and Bioresources Utilization, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China e University of Chinese Academy of Sciences, Beijing, China f Department of Biological Sciences, Burdwan Town School, Burdwan, 713101, India g Department of Botany, University of Peshawar, 25120, Pakistan b

ARTICLE INFO

ABSTRACT

Edited by Alessio Papini

The resurrection plant species, termed desiccation-tolerant plants have evolved remarkable ability to withstand extreme dehydration and rapid rehydration of vegetative tissue without damage. Pteridophytes include almost 70 desiccation tolerant species, and there is limited information of vegetative desiccation tolerance in ferns. A field examination of the representatives of the ferns flora of the Northern Pakistan disclosed 5 ferns species belonging to 2 genera with foliage which can revive after dehydration. These species are Asplenium dalhousiae, Asplenium ceterach, Cheilanthes acrostica, Cheilanthes bicolor, and Cheilanthes nitidula. We undertook a comprehensive leaf micromorphological investigation in all the five resurrection fern species. The study were accomplished using light microscopy (LM) and scanning electron microscopy (SEM). The detailed investigation of adaxial and abaxial leaf surfaces revealed species specific variation in the size and number of epidermal cells, size of stomata, and stomatal pore, stomatal density, and stomatal index and other foliar micromorphological features. In all studied species, adaxial surface lack stomata, i.e., all species are hypostomatic, stomata is polocytic, and epidermal cells shape in all species on both surface is similar, and are irregular shaped. The quantified leaf micromorphological traits are discussed in order to detect their possible role in the desiccation tolerance of resurrection fern species.

Keywords: Resurrection ferns Micromorphology Ecological adaptations Desiccation tolerance

1. Introduction Ferns are estimated to contain 10,578 species worldwide (PPG 1, 2016). It is often viewed that ferns growth are related to humid and shady forests. Indeed, the highest diversity of ferns is found in tropical rain forest, where some 65% of extant ferns species are found (Page, 1979). For instance, in South Africa only 35 fern species in areas with less than 400 mm of rainfall, and many of these are restricted to humid microsites, where 60 species are recorded exclusively in areas with more than 1200 mm of rainfall (Jacobsen, 1983). In the humid tropics, the highest fern diversity is often found in cloud forest at mi D-E levation ranging from 1000 to 2500 m a. s. l., which is basically a result of water availability (Kessler, 2001). The restriction to moist environments and evolutionary substitution of ferns by seed plants as dominant land plants is likely due to morphological, anatomical and physiological ⁎

features that distinguish ferns from seed plants. Among the reasons refer to liking of moist environments by the ferns are lack of ability of leaves to acclimatize changing environmental condition, poor control of water loss, inefficient water transport system, and lower photosynthetic rates than in seed plants (Page, 2002). Nevertheless, a number of specialized xerophytic ferns suggest that their evolutionary background does not exclude them from xeric habitats. Ferns contain a fair number of species from xeric environment are desiccation tolerant. Most resurrection plants (roughly 90 percent) live in ecological niches on rock outcrops principally in tropical and subtropical areas of southern hemisphere, even though some species are also found in more temperate regions (Porembski and Barthlott, 2000). Nonetheless, ferns growing in temperate climates or tropical climates with a dry period shows seasonal leaf phenology (Sharpe and Mehltreter, 2010). Resurrection plant is any poikilohydric plant which have the ability

Corresponding author at: Department of Plant Sciences, Quaid- i- Azam University Islamabad, 45320, Pakistan. E-mail address: [email protected] (S.N. Shah).

https://doi.org/10.1016/j.flora.2019.03.018 Received 18 September 2018; Received in revised form 26 March 2019; Accepted 27 March 2019 Available online 02 April 2019 0367-2530/ © 2019 Elsevier GmbH. All rights reserved.

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to survive desiccation and fully recover from a loss of 90% of protoplasmic water in vegetative tissues (Rascio and Rocca, 2005). The vegetative tissues of most of the plants are sensitive to water deficit and cannot persist of low water availability. However, the resurrection plants tolerate desiccation to an extent where almost all protoplasmic water is lost, and upon rewetting, regain full physiological functionality in existing tissues (Oliver et al., 1998; Farrant et al., 2004; Oliver et al., 2004). Desiccation tolerance is relatively common among bryophytes and lichens, but is rare in pteridophytes and angiosperms, occurring in only 0.02% of vascular plants (Black and Pritchard, 2002). Resurrection plants are widespread but uncommon in different taxonomic groups, including bryophytes (158 species), (Wood, 2007), fern and ferns allies (60–70 species), (Bewley and Krochko, 1982), and angiosperms (350 species), (Proctor and Tuba, 2002). Among pteridophytes, poikilohydry has been observed particularly in some terrestrial or saxicolous species of the genus Actiniopteris, Asplenium, (incl. Ceterach sens. lat.), Chielanthes, Cosentinia, Notholaena, Pellaea, Pyrrosia, Pityrogramma, Schizaea, and Selaginella (Christ, 1910; Kornas, 1985; Walter, 1931; Porembski and Barthlott, 2000; Aldasoro et al., 2004).

Plants are able to cope with environments in which the availability of liquid water within their tissues is severely restricted. Three main strategies are commonly employed by plants to cope with this stress: escape, avoidance, and tolerance. In the first strategy, plants rapidly complete their life cycle, so the reproductive structures are developed before the plants dehydrates. The second one implies that plants delay water loss and prioritize the maintenance of cell turgor by imposing structural barriers to dehydration (cuticle, stomatal closure, enhanced boundary layer etc.). For the third strategy, tolerance, the plants equilibrates its water content with that of the air by an organized restructuration of cell that prevents irreparable damage and allows resumption of normal metabolic activity after rewatering (FernándezMarín et al., 2016). Resurrection plants can survive desiccation only when water loss occurs at a sufficiently slow rate. If dehydration is too fast, the behave like desiccation sensitive plants and die (Navari-Izzo and Rascio, 1999; Bartels and Salamini, 2001). It should be pointed out that, in contrast to bryophytes, more complex orders of plants possess morphological and physiological characteristics that serve to retard water loss, such as

Fig. 1. Resurrection ferns species collected from northern Pakistan A-B-C. Asplenium ceterach D-E-F. Asplenium dalhousiae G-H-I. Cheilanthes acrostica J-K-L. Cheilanthes bicolor M-N-O. Cheilanthes nitidula. C-F-I-L-O = shows images of sori of desiccants tolerant plant species. B- E-H -K-N = shows substantial changes in the appearance of resurrection ferns species that could occur period of drought. 2

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3

and and and and Shah Shah Shah Shah Shah S.N. S.N. S.N. S.N. S.N. SNS-3720 SNS-3923 SNS-4134 SNS-4222 SNS-4331 Asplenium ceterach L. Asplenium dalhousiae Hook. Cheilanthes acrostica (Balb.) Tod Cheilanthes bicolor (Roxb.) Fraser-Jenk. Cheilanthes nitidula Hook.

Habitat description

1. 2. 3. 4. 5.

Details about studied species, their altitude, localities, habitat, and vouchers are presented in (Table 1). All fern specimens were processed and prepared following the method of De Vogel (1987). Initially the identification of the species was carried out by comparing taxa with verified herbarium specimens housed in the National Herbarium of Pakistan (NARC), Islamabad, and Herbarium of Pakistan (ISL) QAU, Islamabad, and the species were confirmed on the basis of characters mentioned in the original descriptions of flora from neighboring China and those used by previous authors (Nakaike and Malik, 1992, 1993; Stewart, 1967; Stewart et al., 1972). Plant species names were confirmed from TPL (www.Theplantlist. org) and the IPNI (International Plant name index). For several taxa the nomenclature was updated. The voucher specimens were deposited for

Taxon name

2.2. Studied materials

S.No

Table 1 List of resurrection ferns species collected from northern Pakistan.

Elevational distribution (m)

All the five desiccation tolerant species of fern (Fig. 1) were collected during 2015–2017 in Malakand Division (35°29′59.99″ N 72°00′0.00″ E), a region in the Khyber Pakhtunkhwa province of Northern Pakistan. The area has high altitudinal variation ranging from 600 m a.s.l. to 8500 m a.s.l. at Tirich Mir the highest mountain peak of Hindukush, and one of the highest in the world (Awan et al., 2001). The division comprises of 7 districts which includes; Buner, Swat, Shangla, Chitral, Dir Lower, Upper Dir and District Malakand (Akhtar and Bergmeier, 2014; Ali and Qaiser, 2009). Malakand Division, due to geographical position have great importance and floristically it is placed in western Himalayan province (Ali and Qaiser, 1986). The annual rainfall varies from 600 mm in the lowland of District Malakand to 1500 mm in the upper part of the division (Ibrar and Hussain, 2009; Muhammad et al., 2016).The average annual temprature is approximatley 19.9 °C in Malakand Division, reaching up to 30 °C between April and October. The dry period begins in June and end in August (100 mm of total rainfall).

Shahpur, Malam Jabba (Shangla Swat) Kikore, Damorai, Shahpur (Shangla), Chitral, upper dir Malam Jabba, Chinar coat, Kikore (Swat, Dir, Shangla) Spin ghar (Shangla) Kikore (Shangla)

2.1. Study area and climate

1600-1800 1600- 2000 2300 3000 2000

2. Materials and methods

Terrestrial Terrestrial Exposed rocks Exposed rocks Exposed rocks

Localities

Voucher number

Collector Name

sclerenchymatous or strongly cutinized leaves (Dalla Vecchia et al., 1998). Thus, the endurable dehydration rate of a resurrection plant will depend on its structural and functional features (Farrant et al., 1999). Under desiccation, resurrection plants stop growing and their leaves often shrink in size and curl up until water becomes available (Scott, 2000; Mitra et al., 2013). Leaf epidermis typically consists of several cell types that carry out specific functions and can undergo adaptive changes in response to dehydration (Glover, 2000). In mature leaves, adaxial and abaxial cells could have different size and shape, depending on their biological function. They could also be affected by environmental factors. Stomatal distribution, frequency and size are considered defining characteristics of leaf growth rate and water use efficiency because of their contribution in determining leaf transpiration (Jones, 1977; Dillen et al., 2008). Drought tolerance is associated with smaller stomatal area and guard cells because of the reduced water loss (Jäger et al., 2014). In contrast to the few desiccation tolerant but much better studied angiosperms, a large proportion of desiccation tolerant ferns have probably gone unrecorded. There is limited information available about the adaptations which enable the ferns to survive in drought condition, and few studies have been carried out on ferns, at least compared with seed plants. Still very less information is available for general comparison of drought tolerant and drought sensitive ferns. The present work aims to document resurrection fern species recorded from various dry habitats in Northern Pakistan, in order to discuss leaf micromorphological features with respect to their possible contribution to the desiccation tolerance and ecological adaptation of resurrection fern species.

W. Zaman W. Zaman F. Ullah W. Zaman

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Table 2 Leaf morphological and anatomical traits of investigated resurrection fern species. Morphological traits

A. ceterach

A. dalhousiae

C. acrostica

C. bicolor

C. nitidula

Type of lamina Length (cm) Width (cm) Thickness (μm) Tissues thickness Adaxial cuticle (μm) Abaxial cuticle (μm) Mesophyll (μm) Adaxial epidermis(μm) Abaxial epidermis (μm) Vascular bundle Diameter of Vascular bundle (μm) Tissues density Adaxial epidermis (n mm2) Abaxial epidermis (n mm2) Mesophyll (n mm2) Epidermal features Adaxial epidermis length (μm) Adaxial epidermis width (μm) Abaxial epidermis length (μm) Abaxial epidermis width (μm)

Pinnatipartite 12.4 ± 3.2 3.07 ± 1.3 86.8 + 12.9

Pinnatipartite 20.8 ± 4.7 5.8 ± 1.8 106.7 ± 14.5

Tripinnate 6.09 ± 3.6 2.4 ± 0.9 56.8 ± 8.6

Tripinnatifid 16.5 ± 5.4 6.9 ± 1.9 96.3 ± 10.7

2-pinnate-pinnatifid 8.5 ± 2.3 3.4 ± 1.2 46.8 ± 7.7

6.23 ± 0.98 5.7 ± 1.1 131 ± 12 16.9 ± 3.45 13.9 ± 2.8

8.34 ± 1.03 6.3 ± 0.98 96 ± 10 13.67 ± 2.4 14 ± 2.8

4.85 ± 1.9 4.32 ± 1.5 116 ± 28 25 ± 4.7 22 ± 2.6

3.6 ± 0.3 4.7 ± 0.8 87 ± 16 34 ± 5.6 30 ± 4.8

5.06 ± 1.5 6.09 ± 1 73 ± 23 40 ± 7 37 ± 6.5

205 ± 20

230 ± 24

168 ± 14

190 ± 14

146 ± 10

270 ± 20 260 ± 17 500 ± 30.1

296 ± 23 305 ± 24 750 ± 47.5

196 ± 16 190 ± 20 450 ± 34

210 ± 18 184 ± 14 360 ± 27

156 ± 13 146 ± 10 600 ± 54

112 ± 13 24 ± 7 108 ± 12 20.09

96 ± 10 32 ± 9 110 ± 12 34 ± 10

88 28 98 24

117 ± 14 35 ± 13 100 ± 10 30 ± 8

78 19 90 23

± ± ± ±

12 10 13 9

± ± ± ±

9 9 10 6

one from the lower surface and one from the upper surface, were mounted with double adhesive tape on stubs, sputter coated with goldpalladium and then observed under SEM. The observations were made with a scanning electron microscope (JEOL JSM-5910), and the pictures were taken with Polaroid P/N 665 film housed at the Central Resource Laboratory, Department of Physics, University of Peshawar.

future reference in the Herbarium of Pakistan (ISL), Department of Plant Sciences, Quaid-i-Azam University, Islamabad. 2.3. Light microscopy For the leaf epidermal examination mature dried leaves of the fern specimens were analyzed under light microscope. The plant specimens were prepared according to the modified method of Clarke (1960). A small portion from the middle of a leaf was cut and placed in a test tube with a solution containing 3 parts nitric acid (75%) and one part lactic acid (25%), which was then heated to 100 °C for 5–10 min, so as to decolorize the plant material. The sample was then transferred to a petri dish and washed twice in water. Debris was removed by adding bleach to the surface of the plant material on the slide. A drop of lactic acid was used to soften plant tissue which facilitated peeling. A section of frond was prepared and then mounted on a permanent standard glass slide and covered with a cover slip. For all species, this uniform method was applied. About 6–8 slides were prepared for the lower and upper surfaces of each species. To check the consistency of epidermal characters, 4–5 leaf samples were taken from each species and a minimum of 5–7 slides, and in some cases up to 10 slides, were prepared from both surfaces of the leaf. For stomatal and epidermal cell densities we followed the method of Bondada et al. (1994). The leaf epidermal features, of each specimen were observed using a Nikon and Meiji (Tokyo, Japan) light microscope. Micrographs were taken using a LEICA- DM-1000 light microscope (Tokyo, Japan) fitted with a Meiji infinity DK- 5000 camera. For the observation of cuticle, mesophyll and other anatomical features of leaf, adult and totally expanded fronds were used. The transverse sections of leaves were made on hand microtome and photograph were captured with a camera Omax 18 Mp 3.0 fitted in Fluophot microscope. All the slides studied for foliar anatomy were deposited in the Plant Systematics and Biodiversity Laboratory of Quaid-i-Azam University.

2.5. Stomata and other epidermal cell measurement The recorded characters were analyzed using different quantitative measures i.e., stomatal length, no of epidermal cell, epidermal cell size (L × W), stomata size (L × W), stomatal pore size (L × W) and stomatal index. Statistical data analysis was carried out in SPSS software (ver 16). The quantitative characters are represented as: (mean ± standard error), for instance (23 ± 5). All the epidermal cell measurements were determined by bright field optics of 40 x magnification on Nikon and Meiji (Tokyo Japan) compound microscope with fitted ocular scale. Cells on both adaxial and abaxial surfaces were measured. The number of cell over the surface areas was determined. The length of stomatal pores (length between the junctions of the guard cells at each end of stomata were measured. The pore width was also measured. The stomatal and guard cell length and width were also measured. The stomatal index value was calculated as described by Salisbury (1928, 1932).

S. I =

S × 100 E+S

S.I = Stomatal index, S = No of stomata per unit area, E = No of epidermal cell per unit area The number of stomata per unit leaf area and the number of epidermal cell per unit area based on the observation of 5 samples and the area for counting the stomata and epidermal cell of each field was (0.0940 mm2). Stomatal density (D) is expressed as number of stomata per mm2 (st/mm2).

2.4. Scanning electron microscopy

3. Results

The scanning electron microscopic study of fern leaves was carried out on the upper side and underside surfaces of the pinnae lamina and expanded portion of leaflet; a section of the middle portion of the pinnae leaflet was used for analysis. Samples of the pinnae leaflet were carefully cut into square pieces of similar size. Two sections of the leaf,

The leaf micromorphological characters of resurrection fern species in Northern Pakistan are summarized in Tables 2 and 3. Selected light microscope (LM) and scanning electron microscope (SEM) micrograph are presented in Figs. 2–4. 4

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Table 3 Quantitative analysis of epidermal cells, stomata and other anatomical features in resurrection fern species. Taxon name

Epidermal cell number AD/AB

Epidermal cell size Length (μm) (Mean ± SE) AD/AB

Width (μm) (Mean ± SE) AD/AB

Stomatal size Length (μm) (Mean ± SE) AD/AB

Width (μm) (Mean ± SE) AD/AB

Stomatal pore size Length (μm) (Mean ± SE) AD/AB

Width (μm) (Mean ± SE) AD/AB

Stomatal index % AD/AB

A. ceterach

23 18 27 31 22 24 20 26 18 20

81 ± 4 116 ± 13 78 ± 12 81 ± 4 56 ± 2 62 ± 3 178 ± 16 72 ± 6.6 163 ± 23 178 ± 19

50 ± 3 40 ± 2 27.5 ± 1 32 ± 4 37 ± 3 32 ± 3 22 ± 3 30 ± 2 38 ± 4 22 ± 1

_ 39 _ 33 _ 25 _ 20 _ 24

_ 30 _ 22 _ 21 _ 16 _ 24

_ 37 _ 23 _ 22 _ 14 _ 29

_ 4 _ 4 _ 3 _ 4 _ 3

_ 21 _ 10 _ 14.2 _ 16 _ 20

A. dalhousiae C. acrostica C. bicolor C. nitidula

±2 ±2 ±1 ±1 ±1

3.1. Survey of leaf characters

±3 ± 0.7 ±1 ±1 ±2

±2 ±1 ±1 ±1 ±5

± 0.7 ± 0.7 ± 0.3 ± 0.7 ± 0.4

Stomatal density (st/ mm2)

20.3 ± 3 18.2 ± 2 30.1 ± 10 32.4 ± 12 30 ± 10

2-pinnate-pinnatifid lamina. The length of lamina ranged from 6.09 ± 3.6 cm in C. acrostica to 20.8 ± 4.7 cm in A. dalhousiae, and width of lamina varied from 2.4 ± 0.9 cm in C. acrostica to 6.9 ± 1.9 cm in C. bicolor. The lamina thickness ranged from 46.8 ± 7.7 μm in C. nitidula to 106.7 ± 14.5 μm in A. dalhousiae.

3.1.1. Leaf morphology The five resurrection fern species had different leaf morphology. In particular, A. ceterach and A. dalhousiae had pinnatipartite lamina, C. acrostica a tripinnate lamina, C. bicolor a tripinnatifid lamina, C. nitidula

Fig. 2. Light micrograph of foliar anatomical features of desiccation tolerant species of ferns A. Asplenium ceterach B–C Asplenium dalhousiae D–E Cheilanthes nitidula F. Cheilanthes bicolor. A-C-E = Abaxial surface contain stomata. B-D-E = Adaxial surface lack stomata. 5

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Fig. 3. SEM images of anatomical features of resurrection ferns A–B. Asplenium ceterach C–D. Asplenium dalhousiae E–F Cheilanthes acrostica G. Cheilanthes bicolor H. Cheilanthes nitidula A-C-E-H = Adaxial surface lack stomata. D-F-G = Abaxial surface contain stomata.

3.1.2. Morphology of epidermal cells When comparing the leaf epidermal cells in the investigated resurrection fern species, statistically significant differences in the size, and number on both adaxial and abaxial surface are detected. The length of epidermal cell on adaxial surface ranged from 56 ± 2 μm in C. acrostica to 178 ± 16 μm in C. bicolor, and width is varied from 22 ± 3 μm in C. bicolor to 50 ± 3 μm in A. ceterach. On abaxial surface mean epidermal cell length varied from 62 ± 3 μm in C. acrostica to 178 ± 19 μm in C. nitidula, and width ranged from 22 ± 1 μm in C. nitidula to 40 ± 2 μm in A. ceterach. All the species studied shared a common features of having similar shape of epidermal cells. The epidermal cells are irregular on both adaxial and abaxial surface in the examined species. The anticlinal walls are curved and wavy in majority of taxa. The highest cell count in per unit area on adaxial surface was noted 27 in A. dalhousiae, and lowest cell count on this surface is 18 in C. nitidula. While on abaxial surface the lowest cell count was 18 found in A. ceterach, and highest on this surface is 31 in A. dalhousiae.

3.1.3. Stomatal characters on epidermis All species have hypostomatic leaves. The stomata are polocytic in all examined species. Notable variation were detected in the stomatal index, stomatal size, stomatal pore size and stomatal density among the species. The shortest stomatal length were found in C. bicolor (20 ± 1 μm), while the longest was found in A. ceterach (39 ± 2 μm). The width of stomata varied from 16 ± 1 μm in C. bicolor to 30 ± 3 μm in A. ceterach. The mean length of stomatal pore ranged from 14 ± 1 μm in C. bicolor to 37 ± 2 μm in A. ceterach, while stomatal pore width varied from 3 ± 0.3 μm in C. acrostica to 4 ± 0.7 μm in A. ceterach and A. dalhousiae. The stomatal index percentage of species varied from 10% in A. dalhousiae as lowest to highest 21% in A. ceterach. C. bicolor had the highest stomatal density (32.4 ± 12 n st/ mm2), while A. ceterach had lowest stomatal density (20.3 ± 3 n st/ mm2).

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Fig. 4. Anatomical adaptation of resurrection ferns. A–D Transverse section of leaf showing epidermis, cuticle and mesophyll A–B. Asplenium ceterach C–D. Asplenium dalhousiae; E–H Transverse section of stipes showing complete sclerification of epidermis and sclerenchyma layer ensheathing vascular bundles E–F. Cheilanthes acrostica G. Cheilanthes bicolor H. Cheilanthes nitidula. Scale bar = 200 μm. AdC = Adaxial cuticle, AbC = Abaxial cuticle, Me = mesophyll cell, Vb = vascular bundles, Sc = sclerenchyma, Cu = cuticle.

3.2. Leaf transverse section

the epidermis is thicker on the adaxial surface than the abaxial surface in majority of taxa. On adaxial surface the size of epidermis (Length × Width) ranged from (78 ± 9 × 19 ± 9 μm) in C. nitidula to (117 ± 14 × 35 ± 13 μm) in C. bicolor. The epidermis length on abaxial surface is varied from 90 ± 10 μm in C. nitidula to 110 ± 12 μm in A. dalhousiae, while width of epidermis is ranged from 20.09 ± 3 μm in A. ceterach to 34 ± 10 μm in A. dalhousiae. The abaxial epidermal cells appear slightly longer than those on the adaxial surface in many species. Highest number of cells counted in unit area on adaxial surface is 296 ± 23 (n mm2) in A. dalhousiae, while lowest cell

3.2.1. Epidermis, cuticle and vascular bundles Abaxial and adaxial epidermis were characterized by one layer of cell covered by thick cuticle. The epidermis anticlinal wall are curved and wavy on both upper and lower surface in all cases. The epidermal cell had an irregular thickness on both surfaces. Cells thickness on adaxial surface ranged from 13.67 ± 2.4 μm in A. dalhousiae to 40 ± 7 μm in C. nitidula. On abaxial surface cell thickness varied from 13.9 ± 2.8 μm in A. ceterach to 37 ± 6.5 μm in C. nitidula. Relatively 7

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count on this surface is 156 ± 13 (n mm2) in C. nitidula. While on abaxial surface the same lowest and highest value of cell count were observed in the same above mentioned species. The adaxial cuticular layer ranged from 3.6 ± 0.3 μm in C. bicolor to 8.34 ± 1.03 μm in A. dalhousiae, while abaxial cuticular layer varied from 4.32 ± 1.5 μm in C. acrostica to 6.3 ± 0.98 μm in A. dalhousiae. The vascular bundle found in the examined species was of the collateral type, ranging in diameter from 146 ± 10 μm to 230 ± 24 μm for C. nitidula and A. dalhousiae, respectively (Table 3). A layer of sclerenchyma was found surrounding the vascular bundle in C. acrostica, C. bicolor and C. nitidula.

1972). Cheilanthes is by far the largest and most diverse genus of xericadapted ferns. The species of the genus is widely spread in Afghanistan, India, Iran, Turkey, Africa, North America, China, Taiwan, Madagascar, Nepal, North America (Nayar, 1963; Belling and Heusser, 1975). A number of resurrection ferns species in this genus is previously reported (Gaff and Latz, 1978). All of these plants must be able to endure periods of at least a few days desiccation. Previous study shows that some desiccation tolerant ferns (e.g Ceterach officinarum, Asplenium trichomanes, Polypodium vulgare) in humid temprate regions grows as epiphytes or on rock outcrops (Proctor and Tuba, 2002). The shallow soil and rock habitat are some of the most characteristics situations for desiccation tolerant plants are on thin soils over and around rock outcrops in such areas as Northern Pakistan (Fig. 1). In the present investigation we compared the leaf morphological and anatomical traits in order to assess the ecological adaptations of these resuurection ferns to survive desiccation in their environment. In order to survive in such a rigorous environment, a perennial plant has to be able to resist water loss and endure desiccation resulting from high temprature, low water supply and harsh edaphic situations. Nearly all of the strutural modifications suggetsed by Maksimov and Yapp (1929b), Maximov (1931); Eames and MacDaniels (1947); Shields (1950); Hevly (1963); Hietz (2010); Vasheka et al. (2017); López-Pozo et al. (2018); Vassileva et al. (2018) as important in the control of water loss in other xerophytes, may be shown to exist in the studied resuurection Asplenium and Cheilanthoid ferns by utilization of field observations, and survey of leaf charaters by light microscopy (LM), and scanning electron microscopy (SEM), and free hand or microtome sections of critical organs (epidermis, stomata, cuticle, mesophyll etc). The most common modification of xerophytic rock ferns is microphylly. The fronds of majority of the studied taxa here are small, the length being less than 20.8 ± 4.7 cm, and the width less than 6.9 ± 1.9 cm. The fronds are highly divided in the Cheilanthoid ferns here, and frequently 2–3 times pinnate with greatly reduced segments. Reduction in the leaf size or leaf surface has been thought to reduce water loss because of reduction in total exposed surface; however, it has been shown that xerophytes under optimal water conditions have hight rate of transpiration due perhaps to maximize ratio of internal to external exposed surface (Turrell, 1939, 1936, 1940). The lamina thickness was significantly different among the species (Table 2). The two species of Asplenium; can be placed into series of increasing the lamina thickness: 86.8 + 12.9 μm, 106.7 ± 14.5 μm for A. ceterach and A. dalhousiae, respectively. The lamina thickness of the three Cheilanthoid ferns varied from 46.8 ± 7.7 μm to 96.3 ± 10.7 μm. The increase in the lamina thickness due to increase of the mesophyll fraction. During drought periods in some xerophytic ferns, it is noted that the thickness of lamina is reduced (Pande, 1935). The desiccation tolerant ferns that may have a coriaceous lamina do not lose water easily. Not only are the fronds of the studied species of Asplenium, and Cheilanthoid ferns here are small, and equipped with various epidermal appendages, but the ultimate segments and even the entire fronds are able to curl up during period of water stress. The leaf curling is an adaptation seen in many desiccation tolerant ferns but are rare in angiosperms, which reduces surface area, thereby reducing water loss and light demage. The way that leaf curl is predetermined and species specific, in difrrent species the leaves may curl lengthwise or it may spirally curled (Tryon and Tryon, 1991). Various leaf micromorphological characters were observed within resuurection ferns here and appear to be associated with the enviornmental conditions of the habitat and depict the desiccation tolerance properties of these ferns. The epidermal cells characteristics were different in terms of size, and number and some similar features observed in all species were the shape of cells and patteren of anticlinal cells walls among the species. All the studied species exhibited curved and wavy anticlinal walls and shapes of the epidermal cells are irregular. The shape of anticlinal walls is considered as environmental adaptation, usually curved and straight anticlinal walls are characteristics of many

3.2.2. Mesophyll and sclerenchyma cells All the species have mesophyll of 3–4 layers composed of mainly rotund cells. Size and number of mesophyll cell differed among the species. The mesophyll cells of all the species not clearly differentiated into palisade and spongy parenchyma. Leaf mesophyll cells have greater intercellular spaces in A. dalhousiae and A. ceterach, while leaf mesophyll cell of the three Cheilanthes species are tightly packed as compared to above-mentioned Asplenium species. The mesophyll cell thickness varied from 73 ± 23 μm in C. nitidula to 131 ± 12 μm in A. ceterach. The highest number of mesophyll cells were 750 ± 47.5 (n mm2) found in A. dalhousiae, while the lowest cell number found were 360 ± 27 (n mm2) in C. bicolor. The stipe cross section in the three species of Cheilanthes depict the presence of complete and incomplete black layers surrounding the steles. These layers are sclerenchyma cells, and these layers are present in all Cheilanthes species studied. The sclerenchyma cells have different pattern among the studied species. The sclerenchyma cells layer completely ensheathing the vascular bundles in C. acrostica. There is incomplete ensheathing vascular bundle with sclerenchyma cells in C. nitidula and C. bicolor. 4. Discussion Resurrection plants have an amazing ability to withstand drought. These plants are widespread but uncommon in different taxonomic groups, there are about 60–70 species of resurrection ferns and ferns allies in the world. Here, we have collected five resurrection fern species belonging to two genera i.e., Cheilanthes and Asplenium L., from northern Pakistan. Resurrection ferns have been reported in the abovementioned two genera from different eco climatic zones, and desiccation tolerance has been tested and confirmed in many ferns belonging to different taxonomic groups including Asplenaiceae (Asplenium, Ceterach), and Pteridaceae (Cheilanthes) (Gaff and Latz, 1978; Diamond et al., 2012; Živković et al., 2010; López-Pozo et al., 2018). Resurrection plants are major pioneer species on shallow soil, particularly during the early stages of xeroseres on the rock surfaces. Most of the ferns species grow around the base of rocks or under rock shelf where they are shaded for at least part of the day. The two species of Asplenium L. studied here grows abundantly on the base of rocks and rocks ledges along mountains and village paths. The Asplenium ceterach L. is resurrection fern species which abundantly occurs in western and central Europe, Pakistan, Afghanistan, Brazil, Scotland, Tibet, Ireland, Africa, Siberia and Ireland, and the second species of Asplenium L. studied is Asplenium dalhousiae Hooker., and it is wide spread in Pakistan, Afghanistan, Yemen, North Africa, Ethiopia, Bhutan, Nepal, North America and India (Pinter et al., 2002; Živković et al., 2010). The data concerning desiccation tolerance included vegetative DT in the studied Asplenium L. species are previously available in the literature (Schwab et al., 1989; Schwab and Heber, 1984; Živković et al., 2010). The species of Cheilanthes studied here all are epilithic, normally exposed to full sunlight, and are xeric adapted. The ecological data regarding habitat of Pakistani ferns and lycophytes are previously given by (Nakaike and Malik, 1992, 1993; Stewart, 1967; Stewart et al., 8

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xerophytes, including ferns (Hevly, 1963; Stace, 1965; Esau, 1965; Gifford, 1989). Statistical quantitative analysis of the collected data showed specific differences in the adaxial and abaxial epidermis (Tables 2 and 3). As illustrated, the size and number of epidermal cells on adaxial and abaxial surface varying among the species. In previous study on resuurection plants it is noted that extreme dehydration did not induce the significant alterations in the size and number of leaf epidermal and mesophyll cells, compared to hydrated leaves (Vassileva et al., 2018). Here in the present study it is suggested that the applied stress did not considerably alter cell patterning mechanisms of these two tissues types. On adaxial surface the epidermal cells had an irregular thickness from 13.67 ± 2.4–40 ± 7 μm, while on abaxial its varied from 13.9 ± 2.8 μm to 37 ± 6.5 μm (Table 2). The irregularly thicknened epidermal cells may serve to roll the lamina into a cylindrical tube to survive desiccation (Helseth and Fischer, 2005). The pinnae of many ferns, particularly the glabrous species, produce a heavily cutinized epidermis. In all the studied species here abaxial and adaxial epidermis of leaves and stipe cross section were characterized by one layer of cell covered by thick cuticle. The cuticle is considered to be effective in reducing transpiration (Esau, 1965). The observed vascular bundles in this study were usually arranged in collateral shape. The sclerenchyma cells layers ensheathing the vascular bundles in the Chielanthoid ferns studied here. The sclerenchyma cells layers are commonly observed in many ferns including desiccation tolerant species (Ogura, 1972). All the species possesed hypostomatous leaves, and no stomata were found on the adaxial surface in either resurrection species. Stomata were present on abaxial surface only. The stomatal parameters i.e., length and width of stomata and stomatal pore, density of stomata and stomatal index were analyzed on the lower surface of leaf. The plant species with hypostomatous leaves are commonly found in drier areas (Syvertsen et al., 1995). Polocytic stomata were observed in all investigated species which corroborates with the observations of (Sen and De, 1992). The results shows specific differences in the size of stomata and stomatal pore were observed in the resuurection ferns (Table 3). Transpiration is reduced by reduction of stomata size and number as well as various modifications of accessory cells which reduce movement of air across the stoma (Hevly, 1963; Maksimov and Yapp, 1929a). Usually the epdermis of xerophytic plants has sunken stomata, but there is not such evidence in either resurrection species here. There is a strong correlation between stomatal density and size. In general, larger stomatal length is associated with lower stomatal density (Pyakurel and Wang, 2014; Tian et al., 2016). In agreement with these general expectations the resurrection ferns studied here shows similar behaviour. Acclimatization of stomatal frequency to stomatal size is believed to be of impoportant for the optimal balance between fixed carbon and water loss (Larcher et al., 2015). All the species studied here have 3–4 layers mesophytic organization mainly composed of rotund cells, and variation in the mesophyll thickness and in number were detected among the species (Table 2). The leaves of these species contract its mesophyll and decreased leaf area as the leaves curled during desiccation. The similar contraction of mesophyll in desiccation tolerant plants have been observed decreasing their specific leaf area during dehydration to minimize water loss (Tuba et al., 1996). The stipe cross section of the three species of Cheilanthes studied here shows the presence sclerenchyma cells surrounding the steles. These specialized cell layers probably play the same role in these ferns as in sclerophyllous plants where they are believed to prevent water loss through their impermeability to water (Eames and MacDaniels, 1947; Proctor and Tuba, 2002). Leaf micromorphological data with other morphological investigation also been used in taxonomic identification and morphogenetic studies of many plant families including ferns (Bahadur et al., 2018; Rashid et al., 2018a, 2018b; Saqib et al., 2018; Shah et al., 2018a, 2018b, 2018c; Ullah et al., 2018a, 2018b, 2018c, 2018d, 2018e; Zaman et al., 2018). This study was based on the field observations, and survey of leaf characters by microscopy

both LM and SEM, and free hand or microtome sections of critical organs (epidermis, stomata, cuticle, mesophyll etc.) in order to reveal desiccation tolerance in these species. The detailed, morphological, physiological and molecular studies for each species are recommended to confirm true poikilohydry in these plants. 5. Conclusion In comparison with lower vascular plants the desiccation tolerance in pterdiophytes is very low, and about 70 resurrection fern species have been disclosed so far. In the present study we have collected 5 resurrection fern species belonging to 2 taxonomically complex genera of ferns i.e., Cheilanthes and Asplenium from Northern Pakistan. There are limited information on the vegetative desiccation tolerance in ferns. The present investigation point out that some of the leaf micromorphological characters (cuticle, mesophyll, stomata, and other epidermal features) could serve as structural modifications to tolerate desiccation. Our results provided new insights into the response of resurrection species to desiccation and potential adaptation strategies at the micromorphological level. Acknowledgments The authors thank staff of the Central resource laboratory, University of Peshawar for access to their scanning electron microscopy. References Aldasoro, J., Cabezas, F., Aedo, C., 2004. Diversity and distribution of ferns in sub‐Saharan Africa, Madagascar and some islands of the South Atlantic. J. Biogeogr. 31 (10), 1579–1604. Ali, S.I., Qaiser, M., 1986. A phytogeographical analysis of the phanerogams of Pakistan and Kashmir. Proc. R. Soc. Edinburgh Sect. B Biol. Sci. 89, 89–101. Awan, M., Shah, M., Akbar, G., Ahmad, S., 2001. Traditional uses of economically important plants of Chitral District, Malakand Division, NWFP, Pakistan. Pak. J. Bot. 587–598. Bahadur, S., Ahmad, M., Zafar, M., Sultana, S., Begum, N., Ashfaq, S., Gul, S., Khan, M.S., Shah, S.N., Ullah, F., 2018. Palyno‐anatomical studies of monocot taxa and its taxonomic implications using light and scanning electron microscopy. Microsc. Res. Technol. 1–21. https://doi.org/10.1002/jemt.23179. Bartels, D., Salamini, F., 2001. Desiccation tolerance in the resurrection Plant Craterostigma plantagineum. A contribution to the study of drought tolerance at the molecular level. Plant Physiol. 127 (4), 1346–1353. Belling, A.J., Heusser, C.J., 1975. Spore morphology of the Polypodiaceae of Northeastern North America. II. Bull. Torrey Bot. Club 115–127. Bewley, J., Krochko, J., 1982. Desiccation-Tolerance, Physiological Plant Ecology II. Springer, pp. 325–378. Black, M., Pritchard, H.W., 2002. Desiccation and Survival in Plants: Drying without Dying. Cabi. Bondada, B.R., Oosterhuis, D.M., Wullschleger, S.D., Kim, K.S., Harris, W.M., 1994. Anatomical considerations related to photosynthesis in cotton (Gossypium hirsutum L.) leaves, bracts, and the capsule wall. J. Exp. Bot. 45, 111–118. Christ, H., 1910. In: Jena, G. (Ed.), Die Geographie der Farne, 358 Ss. Fischer Verlag. Clarke, J., 1960. Preparation of leaf epidermis for topographic study. Stain Technol. 35, 35–39. Dalla Vecchia, F., El Asmar, T., Calamassi, R., Rascio, N., Vazzana, C., 1998. Morphological and ultrastructural aspects of dehydration and rehydration in leaves of Sporobolus stapfianus. Plant Growth Regul. 24, 219–228. De Vogel, E., 1987. Manual of Herbarium Taxonomy. Unesco. Diamond, H.L., Jones, H.R., Swatzell, L.J., 2012. The role of aquaporins in water balance in Cheilanthes lanosa (Adiantaceae) gametophytes. Am. Fern J. 10 (2), 11–31. Dillen, S.Y., Marron, N., Koch, B., Ceulemans, R., 2008. Genetic variation of stomatal traits and carbon isotope discrimination in two hybrid poplar families (Populus deltoides ‘S9-2’× P. nigra ‘Ghoy’and P. deltoides ‘S9-2’× P. trichocarpa ‘V24’). Ann. Bot. 102 (3), 399–407. Eames, A.J., MacDaniels, L.H., 1947. An Introduction to Plant Anatomy. Mcgraw-Hill Book Company, Inc, London. Esau, K., 1965. Plant anatomy. Plant Anatomy, 2nd edition. . Farrant, J.M., Cooper, K., Kruger, L.A., Sherwin, H.W., 1999. The effect of drying rate on the survival of three desiccation-tolerant angiosperm species. Ann. Bot. 84 (3), 371–379. Farrant, J.M., Bailly, C., Leymarie, J., Hamman, B., Côme, D., Corbineau, F., 2004. Wheat seedlings as a model to understand desiccation tolerance and sensitivity. Physiol. Plantarium 120 (4), 563–574. Fernández-Marín, B., Holzinger, A., García-Plazaola, J.I., 2016. Photosynthetic strategies of desiccation-tolerant organisms. Handbook of Photosynthesis. pp. 719–737.

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