Comparative anatomy of some selected species of the poppy family (Papaveraceae) in Iran

Introduction

Poppy family (Papaveraceae) comprises of approximately 26 to 42 genera and 690 to 800 species in the world (Judd et al., 1999). The members of Papaveraceae are shrub, herbaceous perennials and annuals distributed in the temperate and the subtropical regions of the world. Among five genera of family Papaveraceae in Iran, Glaucium, Hypecoum, Chelidonium and Roemeria consist of 10, 1, 1 and 2 species, respectively (Rechinger and Cullen, 1966). Morover, Mobayen (1985) introduced two subspecies G. fimbrilligerum Boiss. subsp. annuum and G. fimbrilligerum subsp. Ophyocarpum.

Anatomical structure of leaves (Dickson,1935; Esau, 1977; Fahn, 1990; Batanouny, 1992) and laticifers (Solereder, 1908) have been presented for some species. Solereder (1908) claimed that the type of laticifers, composition and location of them in different organs are among traits that are taxonomically valuable. Metcalfe and Chalk (1950) reported several anatomical traits of poppy family that exclusively can be taxonomically useful in the identification and delimitation of the species.

Furthermore, anatomical characteristics of Glaucium flavum Cr. have been investigated (Dickson,1935; Esau, 1977; Fahn, 1990; Batanouny, 1992). The most important studies,in respect of anatomy,are restricted to few economical species such as Papaver somniferum (Dickinson and Fairbairn 1975) and Glaucium flavum (Nessler,1992; Bercu et al.,2006) which mainly concerned to ultra-structure of alkaloid sac and laticifers,respectively. Azizian and Alishahi Norani (1997) studied anatomical characteristics of fruit and blade with emphasis on latex tubes in species of Glaucium. Furthermore, Carlquist and Hoekman (1985) studied anatomical structure of wood in Romneya and Dendromecon. Carlquist and Zona (1988) continued his studies in cooperation with Zona on structure of wood in Papaveraceae. Some anatomical features of midrib and fruit of Glaucium are of diagnostic value (Solereder, 1908; Metcalfe and Chalk, 1950). Anatomical characteristics of the fruit,stem and petiole in four studied genera confirm the results of the previous studies. Because of the high variation of morphological characters of Glaucium,this research was aimed to 1) provide some anatomical characters of above- named genera,specifically Glacium;
2) assess these characters’ value in sorting out of the species.

Materials and methods

In this survey,all voucher specimens are deposited at TUH (acronyms according to Holmgren et al.,1990) listed in Table 1. Because of the high variation of morphological diagnostic features of these species and difficulty of their identification,only representative specimens of any species were used in the study. For anatomical studies,dried basal leaves,fruits and stems were fixed in FAA 70 (Formalin, Glacial acetic acid and 70% Ethanol, 5: 5: 90, respectively), cross sections were made at the middle of blade, fruit, stem and were stained white methyl green and bismarck brown colors and then photographed by Leitz light microscope model Wetzlar, Nikon camrera (Coolpix S10). For measuring required characters, Mesurepro software model HASP 2.17 was used.

Table 1. Glaucium species,their localities and voucher specimens

Results and Discussion

Glaucium

Midrib and Stem: In transverse section of the midrib, upper surface was flat and the lower surface was raised (Figure 1, a-t). A thick cuticle was observed on the outer surface of the epidermis (Figure 1: a, d, g, j, m, o and r). Several laminar layers of collenchyma were recognized under the epidermis (Figure 1: a, d, g, j, m, o and r). Under the collenchyma, parenchyma and in the central part, vascular bundles could be seen (Figure 1: a-t). Which were surrounded by sclerenchymatous sheaths (Figure 1: a-t). The continuity of the epidermal cells was interrupted by the presence of hairs. Some of the hairs were short, others were long (Figure 1: c, f, i, l, q and t). There were multilayer hairs (covering hairs) in some species such as G. pulchrum (Figure 1: l). These hairs were shorter (3 to 4 cells) and thicker than single row ones and had more cells (Figure 1: a, c, f, i, l, q and t).

Figure 1. ...

Figure 1. Cross section of midrib in species: G. corniculatum (a,b,c), G. grandiflorum (d,e,f), G. haussknechtii (g,h,i), G. pulchrum (j,k,l), G. contortuplicatum (m,n). G. elegans (o,p, q), G. oxylobum (r,s, t), Roemeria hybrida (u,v), R. refracta (w,x, y), Hypecoum pendulum (z,aa), Chelidonium majus (bb,cc,dd).

Scale bars in the photos are as: a=200μm, b=30μm, c=30μm, d=200μm, e=30μm, f=30μm, g=200μm, h=30μm, i=30μm, m=200μm, n=30μm, o=200μm, p=30μm, q=30μm, r=200μm, s=30μm, t=30μm, u=400μm, v=30μm, w=400μm, x=30μm, y=30μm, z=400μm, aa=30μm, bb=200μm, cc=30μm, dd=30μm.

Minimum thickness (5 µm) of cuticle belonged to G. grandiflorum (Figure 1: d) and maximum (20 µm) was observed in G. contortuplicatum, G. pulchrum and G. haussknechtii (Figure 1: g, j and m). Upper epidermis cells were usually larger than the lowers (Figure 1: a, d, g, j, m, o and r). Diameter of epidermal cells varied from 15 µm in G. corniculatum and G. haussknechtii to 40 µm in G. oxylobum and G. contortuplicatum (Figure 1: o and r). Thickness of upper collenchyma varied from 25 µm in G. corniculatum to 55 µm in
G. oxylobum (Figure 1: a, d, g, j, m, o and r). There was no upper collenchyma in
G. contortuplicatum. Thickness of lower collenchyma ranged from 40 µm in
G. grandiflorum and G. corniculatum to 280 µm in G. oxylobum; lower parenchyma thickness varied from 40 µm in G. grandiflorum and G. contortuplicatum to 280 µm in
G. oxylobum. Upper parenchyma thickness varied from 100 µm in G. haussknechtii to 430 µm in G. oxylobum. The range of vascular bundles varied from 110 µm in G. pulchrum to 230 µm in G oxylobum (Figure 1: k and s). The range of fiber thickness at the upper surface of vascular bundles varied from 25 µm in G. corniculatum to 50 µm in G. oxylobum. Also, in most studied species, phloem tissue was bicolateral except for G. contortuplicatum (Figure 1: n). The range of xylem thickness varied from 80 µm in G. elegans to 50 µm in
G. grandiflorum, G. corniculatum. Midrib cross section in G. oxylobum cleared a central cavity (Figure 1; r). In some species such as G. contortuplicatum, G. haussknechtii and
G. elegans no hairs were observed on the surface of blade (Figure 1: i and q). Transverse section of the stem included cuticle layer, epidermal cells, parenchyma and vascular bundles. Cortex composed of parenchyma cells and one layer of sclerenchymatous cells. At the central part of stem, the medulla could be seen which was composed of many separated spherical cells, the center was occupied by a large cavity (Figure 3: a-l). Cuticular thickness in G. fimbrilligerum Boiss. subsp. annuum was 5 µm and in other species was 10 µm (Figure 3: g). Mesaured characteristics were shown in the Tables 2, 3 and 4 (Figure 3: a-l).

Fruit: A cross section of fruit showed the placenta in the internal surface and two large cavities. There was a layer of small regular epidermis cells (Figure 2: b, d, f, h, j, l, n and p). Several layers of parenchyma cells were recognized below the epidermis (Figure 2: a-p). The external area included 3-4 layers of chlorenchyma cells while 10 to 12 layers of the sclerenchymatous cells were observed in the internal region (Figure 2: a-p). At the center, vascular bundles were arranged in one row (Figure 2: a-p). The sclerenchyma tissue of pericarp was considerably occupied by laticifer tubes (Figure 2: a-p). Cuticle thickness varied from 5 µm in G. fimbriligerum Boiss. subsp. annuum to 20 µm in G. pulchrum (Figure 2: i and m). Epidermal cell thickness varied from 15 to 50 µm in G. oxylobum and G. fimbriiligerum subsp. ophycarpum (Figure 2: g and o). Inner parenchyma layer thickness varied from 20 µm in G. corniculatum to 90 µm in G. fimbriiligerum subsp. ophycarpum. Number of internal layers of parenchyma ranged from three in the G. corniculatum to seven in G. grandiflorum and G. haussknechtii (Figure 2: a, c and e). Thickness of internal parenchyma varied from 60 µm in G. pulchrum to 220 µm in G. haussknechtii. Thickness of sclerenchyma tissue of fruit wall varied from 70 µm in G. pulchrum to 250 µm in
G. corniculatum (Figure 2: a and m). The thickness of the tissue showed evident differences in G. corniculatum with respect to other species (Figure 2: a-p). Laticifer tubes could be seen in the sclerenchymatous sheath of the vascular bundles, the fruit wall, placenta and internal parenchyma layers (Figure 2: a-p). Length of vascular bundles in fruit wall varied from 20 µm in G. fimbrilligerum Boiss. subsp. annuum to 90 µm in G. corniculatum (Figure 2: g and b). Carpellary angle varied from 95º in G. pulchrum to 170º in
G. oxylobum (Figure 2: m and o). Carpellary angle form was crescent in G. corniculatum, G. haussknechtii, G. elegans (Figure 2: a, e and k), linear in G. oxylobum and
G. fimbriligerum subsp. ophycarpum (Figure 2: g and o), V-shaped in G. grandiflorum,
G. fimbrilligerum Boiss. subsp. annuum (Figure 2: i and k) and semi-circular in
G. pulchrum (Figure 2: m). Thickness of ovary wall varied from 300 µm in G. oxylobum, G. grandiflorum and G. fimbrilligerum subsp. ophyocarpum to 450 µm in G. corniculatum. The connected wall of placenta to the ovary varied from 100 µm in G. haussknechtii to 1500 µm in G. fimbrilligerum Boiss. subsp. annuum (Figure 2: f-i). Thickness of placenta varied from 530 µm in G. elegans to 1100 µm in G. fimbriiligerum subsp. ophycarpum (Figure 2: g-k). Among studied species triangular-shaped placenta could be seen in
G. haussknechtii and G. corniculatum (Figure 2: b and f); dentiform in G. elegans (Figure 2: l), heart-shaped in G. oxylobum and G. pulchrum (Figure 2: n and p) and the bean-shaped could be found in G. grandiflorum, G. fimbrilligerum Boiss. subsp. annuum and
G. fimbrilligerum subsp. ophyocarpum (Figure 2: d, h and j). Placental thickness varied from 530 µm in G. elegans to 1100 µm in G. fimbrilligerum subsp. ophyocarpum (Figure 2: a, s, e, g, i, k, m and o). Total numbers of placental vascular bundles were two in
G. oxylobum, four in G. corniculaum, G. haussknechtii, five in G. fimbrilligerum subsp. ophyocarpum, six in G. elegans and G. pulchrum, seven in G. fimbrilligerum Boiss. subsp. annuum and eight in G. grandiflorum (Figure 2: a, s, e, g, i, k, m and o). Multicellular appressed trichomes in some species such as G. corniculatum, G. grandiflorum and
G. haussknechtii were recognized, while glandular hairs could be found in G. oxylobum and G. fimbrilligerum subsp. ophyocarpum (Figure 2: a, s, e, g, i, k, m and o).

Figure 2. Cross section of fruit in species: G. corniculatum (a,b), G. grandiflorum (c,d),G. haussknechtii (e,f), G. fimbrilligerum subsp. ophyocarpum (g,h), G. fimbrilligerum subsp. annuum (i,j), G. elegans (k,n), G. pulchrum (m,n), G. oxylobum (o,p), Roemeria hybrida (q,r), Roemeria refracta (s,t), Hypecoum pendulum (u,v), Chelidonium majus (w,x).

Scale bars in the photos are as: a=400μm, b=200μm, c=400μm, d=200μm, e=400μm, f=200μm, g=400μm, h=200μm, i=400μm, j=200μm, k=400μm, l=200μm, m=400μm, n=200μm, o=400μm, p=200μ, q=400μm, r=30μm, s=400μm, t=200μm, u=400μm, v=200μm, w=400μm, x=30μm.

Figure 3. Cross section of stem in species: G. grandiflorum (a,b), G. haussknechtii (c,d), fimbrilligerum subsp. annuum (e,f), G. fimbrilligerum subsp. ophyocarpum (g,h), G. pulchrum (i,j), G. oxylobum (k,l), Roemeria hybrida (m,n), Roemeria refracta (o,p), Hypecoum pendulum (q,r), Chelidonium majus (s,t).

Scale bars in the photos are as: a=400μm, b=200μm, c=400μm ,d=200μm, e=400μm, f=200μm, g=400μm, h=200μm, i=400μm, j=200μm, k=400μm, l=200μm, m=400μm, n=200μm, o=400μm, p=200μm, q=400μm, r=200μm, s=400μm, t=200μm.

Roemeria, Hypecoum and Chelidonium

Midrib: Upper cuticle thickness did not show considerable differences in R. refracta,
R. heybrida, C. majus and H. pendulum but less thickness was measured in most species of Glaucium (Figure 1: u-z and aa, bb, cc, dd). Lower cuticle thickness in R. refracta was more than R. heybrida, C. majus and H. pendulum but in comparison with most species of Glaucium did not show considerable differences. Upper and lower epidermises in C. majus in comparison with H. pendulum, R. refracta and R. heybrida had less thickness (Figure 1: u-z and aa, bb, cc, dd).

Thicknesses of the upper parenchyma in C. majus and R. refracta in comparison with
R. hybrida, H. pendulum and species of Glaucium had considerable differences (Figure 1: u-z and aa, bb, cc, dd). Thicknesses of the lower parenchyma in C. majus in comparision with other genera had considerable differences. Against R. hybrida, C. majus and most species of Glaucium,upper collenchyma did not exist in Roemeria refracta and
H. pendulum and R. hybrida phloem was collateral (Figure 1: u-z and aa, bb, cc, dd). Vascular bundles of midrib in R. refracta were one, in R. hybrida three and five in C. majus and H. pendulum. The minimum number of midrib vascular bundles among all four genera belonged to R. refracta (Figure 1: u-z and aa, bb, cc, dd). There were some laticifer tubes around vascular bundles, parenchyma, especially sclerenchymatous sheath, cone-shaped, multicellular multi-row hairs were observed in R. refracta, while long multicellular and single row hairs were recognized in R. hybrida and C. majus. No hair was seen in
H. pendulum (Figure 1: u-z and aa, bb, cc, dd).

Stem: Cuticle and epidermal thickness in R. hybrida, H. pendulum, C. majus were similar, but more thickness was measured in R. refracta; maximum thickness of cuticle among four genera belonged to R. refracta (Figure 3: m-t). Parenchyma thickness did not show significant differences in the four mentioned species (Figure 3: m-t). scleranchyma cortex thickness,width of external and internal phloem in R. refracta and H. pendulum in comparison with R. hybrida, C. majus had considerable differences. Laticifer tubes in Roemeria species could be seen around vascular bundle and in C. majus and H. pendulum clearly in the cortex and sclerenchymatous sheath (Figure 3: m-t).

Fruit: Cuticular thickness in H. pendulum was more than three other species (Figure 2: q-x). Inner parenchyma layers were superseded by sclerenchymatous in H. pendulum (Figure 2: v). Placenta was planar and had a vascular bundle in Roemeria species, while in C. majus was dentiform with a vascular bundle (Figure 2: q-x). Semi-circular carpellary angle varies from 90º to 130º in R. refracta, R. hybrida and H. pendulum, respectively (Figure 2: q, s, u and w). Linear carpellary angle (170º) was seen in Chleidonium majus. Laticifer tubes could be found in placental sclerenchyma and in wall parenchyma in
R. hybrid. Multicellular spear-like hairs were observed in Roemeria, but the others lacked such a character (Figure 2: q and r).

Table 2. Anatomical characters of basal leaf in studied species.

Abbreviations: A:Papile presence, B:Papile layers' number, C:Papile's shape, D:upper cuticle's thickness,
E:lower cuticle's thickness, F:upper epidermis thickness, G:lower epidermis thickness, H: upper collenchyma thickness, I:lower collenchyma thicknes, J: upper parenchyma layer thickness, K:lower parenchyma layer thickness, L:upper phloem width, M:lower phloem width, N:xylem width, O:upper tissue strength thickness, P:lower tissue strength thickness, Q:Bundle vessle number, R: Length leaflet, S:Bundle vessel distance between. l:long, Mu:multiseriate, Mc:multicellural, o:oblong, On:Onion shape, g:glandular, u:uniseriate. The unit of all quantity is micrometer.

Table 3. Anatomical characters of the capsule in studied species

Abbreviations: A:Papile presence, B:Papile layers' number, C:Papile's shape, D:cuticle's thickness, E:epidermis thickness, F:external parenchyma layer number, G:upper parenchyma layer thickness, H:lower parenchyma layer number, I:lower parenchyma layer thickness, J:laticiferous tubes location, K:bundle vessel width, L:upper scleranchyma length, M:placenta's shape, N:Bundle vessle number of placenta, O:shape's Leaflet Angle between, P:Leaflet Angle between, Q: Wall thickness, R:placenta's thickness, S:Attache wall, an:angular, em:embowed, fi: Five form, g:globular, li:linear, l:long, Mo:Monoseriate, Mu:Multiseriate, o:oblong, re:renal, p:Paranchym, pd:Paranchym down, pu:paranchya up, s:short, sc:semicircle, su:sunken, sp:spear, to:tooth, tr:triangle. The unit of all quantity is micrometer. The unit of all quantity is micrometer.

Table 4. Anatomical characters of the stem in studied species

Abbreviations: A: cuticle's thickness, B:epidermis thickness, C: parenchyma cortex thickness, D:scleranchyma cortex thickness, E:external phloem width, F: internal phloem width, G:xylem width. The unit of all quantity is micrometer.

Results of present study showed, anatomical characters of midrib in Glaucium species led to finding some traits which could be used for identification for different species. In vascular structure of midrib in most Glaucium species, bicolateral phloem and scleranchymatous sheath of vascular bundles could be seen (Figure 1: n, v). Among species of this genus, G. contortuplicatum possessed colateral phloem and lacked external collenchyma which was distinguished from other species. This species was easily recognized from other species by its helical fruit. A central cavity can be observed midrib in G. oxylobum which in turn, could be used as a differentiating feature for this species. Multicellular single row hairs in G. grandiflorum and G. corniculatum could be seen (Figure 1: c, f). Although, Metcalfe and Chalk (1950) studied anatomical traits of Papaveraceae but they didn’t report on glandular hairs. In this study, glandular hairs were observed in G. oxylobum and G. pulchrum.

According to present study, some anatomical features of fruit in Glaucium can be used in grouping of the genus. The most important traits were the apparent shape of ovarian placenta (Figure 2: b, d, f, h, j, l, n and p). Placenta types were observed as following:

1-        Triangular: G. haussknechtii, G. corniculatum (Figure 2: a and e)

2-        Dentiformis: G. elegans (Figure 2: k)

3-        Heart-shaped: G. oxylobum, G. pulchrum (Figure 2: o , m)

4-        Bean-shaped: G. grandiflorum, G. fimbrilligerum Boiss. subsp. annuum, G. fimbrilligerum subsp. ophyocarpum (Figure 2: c, g and i)

Another distinguishing character was carpellary angle. Carpellary angle varied from 95º in G. pulchrum to 170º in G. oxylobum (Figure 2: m and o) which was classified as following:

Group I: Lunar-shaped: G. haussknechtii, G. corniculatum, G. elegans and G. pulchrum (Figure 2: a, e and k).

Group II: Linear-formed: G. oxylobum and G. fimbrilligerum subsp. ophyocarpum (Figure 2: g and o).

Group III: V-shaped: G. fimbrilligerum Boiss. subsp. annuum and G. grandiflorum (Figure 2: i).

In addition to the above mentioned traits, the differences in the number of external and internal layers of parenchyma in the ovary wall, the number of vascular bundles of placenta and type of hairs might be suitable in identification of unknown species. Considering the anatomical traits of fruit in Glaucium, identification of two species, G. haussknechtii and
G. grandiflorum, from each other and other species of Glaucium was easily possible. Anatomical characteristics of the stem in species of this genus did not show very important differences. According to anatomical study of Roemeria and the transverse incision of blade of two species there were some significant differences as following:

- Lack of external collenchyma (such as G. contortuplicatum) and external parenchyma in the main vein in Roemeria hybrida.

- Lack of strengthening tissue surrounding the vascular bundles in Roemeria refracta (like H. pendulum)

- Peresence of 1 and 3 vascular bundle in blade cross section provided from R. refracta and presence R. hybrida respectively.

Anatomical characteristics of the fruit in Roemeria which differed between these two species as follows: lack of hair in cross cutting the fruit of Roemeria refracta and its presence in R. hybrida and the difference in the overall shape of fruit in the transect so the shape of fruit in R. refracta was almost quadragonal (four corners) and the shape in
R. hybrida was triangular. Anatomical study of the stems of species in this genus did not show very important difference. Blades transect study in two other genera such as Hypecoum and Chelidonium showed structurally no significant difference with respect to Glaucium and Roemeria. Cross section of blade in C. majus showed more similarity with G. oxylobum. Transect of fruit in H. pendulum was different from other genera. The general form of the transect was rectangular and lacking any placental bulge so identification of the placental place was difficult. External sclerenchyma could be seen only on the vascular bundles. As like as Glaucium, fruit in C. majus posed prominent placenta and the placenta shape in latter species was dentiformis. Identification of some species based only on morphological characteristics was difficult and even sometimes impossible. Previous studies and this study on anatomical characters of Glaucium indicated that these traits could identify and separate these species more accurately in some cases.

Anatomical study of blade in these genera indicated,in spite of some differences,the fundamental structure was similar,so cross section of blade solely could not be a good scale for separating species of genus Glaucium and identification of four genera. Fruit anatomical characters not only were useful in separating species of genus Glaucium,but also,for identification and determination of the four other genera. Stem anatomical study indicated that these four genera were very similar in this character,so stem anatomy characters was neither a suitable scale for separating species of Glaucium,nor for other genera. However, anatomical characteristics of the fruit, stem and blade in the four genera could confirm previous studies and had a good agreement with previous studies. (Solereder, 1908; Metcalfe and Chalk, 1950).