Onobrychis

Khan, Amjad, Sultan, Amir, Shah, Sayed Afzal, Khan, Raees, Khan, Nazar, Mumtaz, Abdul Samad, Kaleem, Muhammad, Abdullah, Muhammad, Al- Qahtani, Wahidah H. & Khan, Tahir, 2024, Morpho-anatomical and palynotaxonomic study of the genus Onobrychis Miller (Hedysareae-Fabaceae) in Pakistan, and its systematic significance, Phytotaxa 650 (2), pp. 121-147 : 132-142

Identification key to Onobrychis species based on morphological characters

1+ Plant spiny, corolla reddish purple, vexillum dorsally glabrous ........................................................................................ O. cornuta

- Plant not spiny, corolla not reddish purple, vexillum dorsally more or less pilose ............................................................................2

2+ Stipule 6–12 mm long ....................................................................................................................................................... O. dealbata

- Stipule less than 6–12 mm long..........................................................................................................................................................3

3+ Bracts upto 2 mm long, calyx upto 3.5 mm long, teeth ovate........................................................................................... O. stewartii

- Bracts more than 2 mm long, calyx more than 3.5 mm long, teeth subulate .....................................................................................4

4+ Leaflets linear, acute, glabrous above, sparsely pubescent below.................................................................................. O. micrantha

- Leaflets not linear and acute, sparsely or densely pubescent on both surfaces ................................................................................ 5

5+ Fruit 8.5–12× 4–4.5 mm diam, semiorbicular, unilocular disc-shaped, disc areolate, covered with densely hairs cottony outgrowth ........................................................................................................................................................................................... O. laxiflora

- Fruit 10–11.5× 9–11 mm diam, orbicular, bilocular, deeply pitted, cottony-pubescent becoming glabrescent; margin 2–3 mm broad......................................................................................................................................................................... O. tavernierifolia

Anatomy

Anatomy includes the cross-sectional examination of the stem, petiole, peduncle and leaflet.

Comparative stem anatomy

Anatomical study of stem revealed that the uppermost epidermal layer is comprised of squarish shaped cells in Onobrychis cornuta , rectangular in O. dealbata , rectangular to oval in O. laxiflora and O. micrantha , squarish to rectangular O. stewartii , and circular to slightly rectangular in O. tavernierifolia . On the epidermal layers, eglandular trichomes are observed in the stem of Onobrychis dealbata , O. laxiflora , O. micrantha and O. stewartii , while Onobrychis cornuta and O. tavernierifolia has a smooth surface. Few layers of collenchyma lying beneath the epidermis are observed in all taxa except Onobrychis tavernierifolia . Some variations are found in vascular tissues such as: many, continuous, cylindrical indistinguishable in O. dealbata , O. laxiflora , O. stewartii and O. tavernierifolia , whereas vascular tissues few, discontinuous and polygonal or in various shapes are observed in O. cornuta and O. micrantha . The vascular tissue has an outer layer with phloem sclerenchyma distinguishable in some extent in all taxa. The thick lignified walled phloem sclerenchyma is observed lying above the cambium layer in the Onobrychis cornuta . Xylem vessels radially arrangement along with parallel to medullary rays are present in all studied taxa, but in Onobrychis laxiflora and O. micrantha has a solitary and radially chains pores. There is also xylem parenchyma forming oval to oblong cluster lying in between the vessels and pith regions of all examined taxa, except in Onobrychis cornuta . The sclerenchymatous tissues are scattered in the periphery region of pith in Onobrychis cornuta . Pith shape in Onobrychis cornuta , O. dealbata and O. tavernierifolia are spheroidal, while Onobrychis micrantha and O. stewartii have an oval to oblong pith shape. Pith cavity is observed in Onobrychis laxiflora ( Fig. 10 ).

Comparative petiolar anatomy

On the cross section of petiolar outline is oval in Onobrychis cornuta , ellipsoidic in O. dealbata , subcordate in O. laxiflora , cordate in O. micrantha , slightly ellipsoidic in O. stewartii and slightly obovate is observed in Onobrychis tavernierifolia . The outer epidermal layer is comprising of rectangular to semi-circular cells. In addition, epidermis of Onobrychis dealbata , O. laxiflora , O. micrantha and O. tavernierifolia are covered with eglandular trichomes ( Fig. 9 ). There were continuous collenchymatous 3–5 celled layers with rectangular to polygonal shape, followed by discontinuous thick-walled collenchymatous cortical region lying tangentially to the epidermis. Petiole of each studied species of Onobrychis varied in shape, size and numbers of vascular bundles. All they have a single large prominent vascular bundle with distinct phloem parenchymatous fibres and radially parallel xylem vessels, and many small subsidiary bundles arranged in circular cylindrical ring. The large centralized vascular bundle lied in between phloem sclerenchyma and xylem sclerenchyma around the pericyclic fibres within the cortical region. Onobrychis cornuta has phloem fibres above the phloem consisting of thick-walled sclerenchyma that gives woody texture to the petiole. Pith shape also varied in shape, that were more or less polygonal with lobes in all examined species except in Onobrychis laxiflora and O. micrantha , that have a more or less circular and composed of oval to polygonal thick-walled parenchymatous and isodiametric cells.

Comparative peduncle anatomy

Peduncle outline at the cross section is elliptic lobed in Onobrychis cornuta , more or less circular in O. dealbata , O. laxiflora and O. micrantha , oblong in O. stewartii and hexagonal in O. tavernierifolia . The epidermal layer is composed of semi-circular to rectangular cells in all the studied species. The out layer is covered with sparsely eglandular trichomes in Onobrychis dealbata , O. laxiflora and O. tavernierifolia . There are a few collenchymatous layers lies beneath the epidermis consisting of tangentially oval to oblong cells followed by inner cortex with continuous 3–5 layers of tangential collenchymatous and some thick-walled parenchyma making a cortical region in the peduncles. There is a continuous single vascular cylinder comprises with a few numbers of discrete collateral primary bundles. Vascular bundles are distinctly arranged in a circular ring to make pericycle region in the peduncle. The pericycle region is composed of distinguishable fibres upto 4–7 celled layer lie under the inner cortex in all studied species. In peduncle, vascular bundles are varied in number: 7–9 bundles in Onobrychis cornuta , 13–16 in O. dealbata , 12–14 in O. laxiflora , 6–8 in O. micrantha , 12–14 in O. stewartii and 7–8 bundles are observed in O. tavernierifolia ( Fig. 12 ). Each bundle consisted of thick-walled parenchymatous phloem fibers above and many radially elongated xylem vessels and xylem rays lied toward pith. Pith shape is also varied among the examined taxa: Onobrychis cornuta , O. stewartii and O. tavernierifolia having a semi-circular to ellipsoidic, while Onobrychis dealbata , O. laxiflora and O. micrantha having a more or less circular pith consisting of large isodiametric thick-walled rectangular to polygonal cells.

Comparative leaflets anatomy

Leaflets at the cross section are displayed variation in outline: Onobrychis dealbata and O. micrantha have a concave shaped lamina, while O. laxiflora , O. stewartii and O. tavernierifolia have a more or less straight lamina ( Fig. 13 ). In addition, some variations are observed in the outline of midrib, i.e., Onobrychis stewartii and O. tavernierifolia having a more or less flat shape, while Onobrychis cornuta , O. dealbata , O. laxiflora and O. micrantha having a prominent curved shape. In leaflets, both lower and upper epidermises are composed of rectangular to polygonal cells in all species. The outer most layers leaflets are covered with eglandular trichomes are observed in Onobrychis cornuta , O. dealbata , O. laxiflora , O. micrantha and O. stewartii , while O. tavernierifolia is lacking trichomes. Palisade parenchyma layers are comprised of tangentially elongated cells up to 6–7 layers in Onobrychis cornuta and 4–7 layers in O. dealbata . The palisade parenchymatous tissues are found indistinguishable in Onobrychis laxiflora , O. micrantha , O. stewartii and O. tavernierifolia . Similarly, spongy parenchyma layers are also composed of compactly arranged with small oval to rectangular cells; up to 3–5 layers in Onobrychis cornuta , 3–6 layers in O. dealbata , 4–7 layers in O. micrantha , while indistinguishable in O. laxiflora , O. stewartii and O. tavernierifolia . There is a distinct vascular bundle surrounded by collenchymatous region in the midrib varying among the investigated species.

Identification key to Onobrychis species based on anatomical characters

1+ Petiolar outline oval, in stem sclerenchymatous tissues scattered in the periphery of pith region .................................... O. cornuta

- Petiolar outline not oval, in stem sclerenchymatous tissues not scattered in the periphery of pith region ........................................2

2+ Pith cavity present ............................................................................................................................................................. O. laxiflora

- Pith cavity absent................................................................................................................................................................................3

3+ Peduncle outline more or less circular............................................................................................................................... O. dealbata

- Peduncle outline oblong or hexagonal ...............................................................................................................................................4

4+ In peduncle numbers of vascular bundles upto 6–8 .......................................................................................................... O. stewartii

- In peduncle numbers of vascular bundles more than 6–8 ..................................................................................................................5

5+ In leaflets both surfaces covered with eglandular trichomes.......................................................................................... O. micrantha

- In leaflets both surfaces lack eglandular trichomes.................................................................................................. O. tavernierifolia

Comparative pollen Morphology

Pollens grains of studied Onobrychis taxa are tricolpate, isopolar and radially symmetrical ( Fig. 14 ). By employing Erdmann’s classification, it was found that pollen grains are medium size, ranging 20–49 μm, prolate in shape and obtuse convex in general view. Surface ornamentations are micro-reticulate in ( O. cornuta and O. laxiflora ), supra-reticulate in ( O. dealbata ), micro-reticulate perforate in ( O. micrantha ), micro-reticulate granulate in ( O. stewartii ) and reticulate in ( O. tavernierifolia ) ( Fig. 15 and Table 4). The mean size of pollen at polar view is ranged from 28.77±0.09 μm in O. laxiflora to 34.16±1.05 μm in O. tavernierifolia , whereas mean size of pollen at equatorial view is ranged from 17.91±0.76 μm in O. dealbata to 23.33±1.05 μm in O. stewartii . Polar to equatorial ratio (P/E ratio) is ranged from 1.35 in O. laxiflora to 1.65 in O. dealbata . Mean of colpi length is ranged from 22.97± 1.19 in laxiflora to 27.50± 0.91 in O. stewartii , while width of colpi is ranged from 1.95± 0.11 in O. dealbata to 2.12 in O. cornuta and O. tavernierifolia . The mean of apocolpium is ranged from 3.54± 0.30 in O. cornuta to 4.00± 0.12 in O. laxiflora . Similarly, mean of mesocolpium is ranged from 12.91 in O. dealbata to 15.00± 0.91 in O. cornuta . Mean of exine thickness is ranged from 1.16± 0.08 in O. cornuta to 2.00± 0.04 in O. laxiflora , whereas mean of intine thickness is ranged from 0.37± 0.05 in O. dealbata and O. stewartii to 0.45±0.07 to 0.62± 1.07 O . laxiflora ( Table 3).

Identification key to Onobrychis species based on palynological characters

1+ Polar to equatorial ratio upto 1.35 ..................................................................................................................................... O. laxiflora

- Polar to equatorial ratio more than 1.35 .............................................................................................................................................2

2+ Exine ornamentation micro-reticulate ................................................................................................................................ O. cornuta

- Exine ornamentation not micro-reticulate ..........................................................................................................................................3

3+ Polar axis 34.16 μm.................................................................................................................................................. O. tavernierifolia

- Polar axis less than 34.16 μm .............................................................................................................................................................4

4+ Equatorial axis is 23.33 μm ............................................................................................................................................... O. stewartii

- Equatorial axis is less than 23.33 μm .................................................................................................................................................5

5+ Length of colpi is 25.83 μm, width of colpi is 2.04 μm ................................................................................................. O. micrantha

- Length of colpi is less than 25.41 μm, width of colpi is 1.95 μm ..................................................................................... O. dealbata

Ordination (PCA) and clustering (UPGMA) analysis

Statistical method ordination (PCA) and clustering (UPGMA) analyses are performed based on pollen quantitative characters. We observed the examined species at two components of principal component (PCA) analysis and revealed that some differences are found among them ( Fig. 16 ). The first component is explained 61% differences with positive correlation. The positive correlated pollen characters are noted with polar, equatorial axis, length and width of colpi, apocolpium and mesocolpium.The species are distinctly apart from each other due to high variation in their quantitative values. The second component showed 39% differences with negative correlation. This is revealed a negative correlated feature with P/E ratio, exine and entine thickness are found among the quantative characters.

Clustering analysis is used for the estimation level of variation among the studied Onobrychis species and also determined relationship among its close allies. The UPGMA main cladistic tree is divided into two clades (Clade-I and Clade-II). The Clade-I is comprises with O. stewartii and O. tavernierifolia , while the Clade-II sub-divided into primary clade and secondary clade ( Fig 17 ). The primary clade is comprised with only O. laxiflora , while the secondary clade is composed of further two sub-clades. The first sub-clade comprised two species, namely O. dealbata and O. micrantha , while the second sub-clade has a single species ( O. cornuta ). The grouping of species with ordination and clustering analyses did not provide authentic correlation like molecular study, due to pollen characters has a high homogeneity among the studied species. However, the current results are supported ordination and clustering analyses due to some differences that are noticed in the pollen morphology of studied taxa.

Figures

FIGURE 9. Comparative illustrations of seeds in Onobrychis species: (A) O. dealbata, (B–C) O. laxiflora, (D) O. micrantha, (E) O. stewartii, (F) O. tavernierifolia (scale bar according to Table 2).

FIGURE 10. Comparative transverse sections of stem in Onobrychis species: (A) O. cornuta, (B) O. dealbata, (C) O. laxiflora, (D) O. micrantha, (E) O. stewartii, (F) O. tavernierifolia. Acronyms: ep—epidermis, tr—trichomes, cl—collenchyma, co—cortex parenchyma, sc—sclerenchymatous tissue, ca—cambium, ph—phloem, phs—phleom sclerenchyma, xy—xylem cells, xyp—xylem parenchyma, xys—xylem sclerenchyma, pc—pith cavity, pi—pith.

FIGURE 12. Comparative transverse sections of peduncle in Onobrychis species: (A) O. cornuta, (B) O. dealbata, (C) O. laxiflora, (D) O. micrantha, (E) O. stewartii, (F) O. tavernierifolia. Acronyms: ep—epidermis, tr—trichomes, cl—collenchyma, sb—subsidiary bundles, co—cortex, in—inner cortex, pc—pericycle, ph—phloem parenchyma, xy—xylem parenchyma, xys—xylem sclerenchyma, pi—pith.

FIGURE 13. Comparative transverse sections of leaflet in Onobrychis species: (A) O. cornuta, (B) O. dealbata, (C) O. laxiflora, (D) O. micrantha, (E) O. stewartii, (F) O. tavernierifolia. Acronyms: ue—upper epidermis, le—lower epidermis, tr—trichomes, pp—palisade parenchyma, sp—spongy parenchyma, vs—vascular bundle, cl—collenchyma.

FIGURE 14. Comparative pollen morphology in Onobrychis species: (A–R) General view at light microscopy; (A–C) O. cornuta, (D–F) O. dealbata, (G–I) O. laxiflora, (J–L) O. micrantha, (M–O) O. stewartii, (P–R) O. tavernierifolia (scale bar 10 µm).

FIGURE 15. Comparative pollen morphology in Onobrychis species at scanning electron microscopy; O. cornuta in equatorial view (A), in exine sculpturing view (B), in polar view (C); O. dealbata in equatorial view (D), in exine sculpturing view (E), in polar view (F); O. laxiflora in equatorial view (G), in exine sculpturing view (H), in polar view (I); O. micrantha in equatorial view (J), in exine sculpturing view (K), in polar view (L); O. stewartii in equatorial view (M), in exine sculpturing view (N), in polar view (O); O. tavernierifolia in equatorial view (P), in exine sculpturing view (Q), in polar view (R).

FIGURE 16. Principal component analysis (PCA) plot for examined Onobrychis taxa based on pollen quantative characters (according to Table 3).

FIGURE 17. UPGMA cladistic tree of Onobrychis taxa based on pollen quantative characters (according to Table 3).