Spore wall cytochemistry and structure in five species of the liverwort Riccia (Ricciaceae)

Ayub, Daniel Martins; Costa, Denise Pinheiro da; Santos, Rinaldo Pires dos

doi:10.1590/2175-7860202475009

Daniel Martins Ayub ^* * daniel.ayub@bento.ifrs.edu.br

Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Sul, Juventude da Enologia, Bento Gonçalves, RS, Brazil.

http://orcid.org/0009-0002-0704-3372

Denise Pinheiro da Costa

Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Jardim Botânico, Rio de Janeiro, RJ, Brazil.

http://orcid.org/0000-0001-9495-3029

Rinaldo Pires dos Santos

Universidade Federal do Rio Grande do Sul, Inst. Biociências, Depto. Botânica, Porto Alegre, RS, Brazil.

http://orcid.org/0000-0003-0723-1146

* daniel.ayub@bento.ifrs.edu.br

Area Editor:

Dr. João Paulo Basso-Alves

Figures (7)
Tables (3)

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Figure 1
a-k. Riccia plano-biconvexa - a. proximal face of the spore; b. distal face of the spore; c. spore under section, showing exine 1, with the typical orange natural color, a cavity (*) between exine 1 and exine 2, and exine 3, transparent; d. detail of the sporoderm after staining with Toluidine Blue O, showing exine 1 and exine 2 partially stained in greenish-blue, exine 3 stained blue, and the intine stained purple and filling the exine 3 discontinuities; e. detail of the sporoderm, stained with Coomassie Blue, showing exine 1 and exine 3; f. details of the sporoderm show that exine 3 and the intine are PAS-positive; g. detail of the sporoderm after staining with Basic Fuchsin, showing the difference in staining between exine 1, exine 2, and exine 3 (stained); h. detail of the sporoderm, with a positive reaction to Auramine O in exine 3; weak reaction in exine 1; i. detail of sporoderm, showing the intine with a positive reaction for Calcofluor White; j. detail of the sporoderm showing exine 3 stained with Sudan Black B; k. detail of the sporoderm, with intine stained by Alcian Blue. (E1 = exine 1; E2 = exine 2; E3 = exine 3; IN = intine). Bars = 10 μm.

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Figure 2
a-k. Riccia fruchartii - a. proximal face of the spore; b. distal face of the spore; c. spore under section, showing exine 1 with the typical orange color, a cavity (*), between exine 1 and exine 2, and exine 3, transparent; d. detail of the sporoderm after staining with Toluidine Blue O, showing exine 1 and exine 2, irregularly stained with greenish blue, exine 3 stained blue, and the intine stained purple, filling in the exine 3 discontinuities; e. detail of the sporoderm, stained with Coomassie Blue, showing weak exine 3 staining; f. detail of sporoderm showing exine 3 strongly stained by the PAS reaction. It is not possible to distinguish the limits between exine 3 and intine, both stained; g. detail of sporoderm, stained with Basic Fuchsin, showing differential staining between exine 1, exine 2 and exine 3; h. detail of sporoderm stained with Auramine O, showing exine 3 and a weak color on the surface of exine 1; i. detail of sporoderm, showing intine, after staining with Calcofluor White; j. detail of sporoderm, showing exine 3, stained with Sudan Black B and partial staining of exine 1; k. detail of sporoderm, with intine stained by Alcian Blue. (E1 = exine 1; E2 = exine 2; E3 = exine 3; IN = intine). Bars = 10 μm.

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Figure 3
a-k. Riccia cavernosa - a. proximal face of the spore; b. distal face of the spore; c. spore under section, showing exine 1 and exine 2, with typical orange color and exine 3, transparent; d. detail of sporoderm, stained with Toluidine Blue O, showing exine 1 and exine 2, greenish blue, exine 3 stained blue, and the intine, colorless and filling the discontinuities present in exine 3; e. detail of the sporoderm, stained with Coomassie Blue, showing exine 3; f. detail of the sporoderm of immature spore, showing all exine sublayers PAS-positive and the lamellation of exine 1; g. detail of the sporoderm of immature spore, showing differential staining of exine sublayers (1, 2 and 3) with Basic Fuchsin; h. detail of sporoderm, stained with Auramine O, showing exine 3; i. detail of sporoderm, showing intine after staining with Calcofluor White; j. detail of sporoderm, showing exine 2 and exine 3, stained with Sudan Black B and weak staining of exine 1 lamellae; k. detail of sporoderm with intine stained with Alcian Blue. (E1 = exine 1; E2 = exine 2; E3 = exine 3; IN = intine). Bars = 10 μm.

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Figure 4
a-k. Riccia stenophylla - a. proximal face of spore, showing the trilete scar; b. distal face of the spore; c. section of spore, showing exine 1 and exine 2, with typical yellow color, a cavity (*) between exine 1 and 2, and exine 3, transparent; d. detail of sporoderm, stained with Toluidine Blue O, showing exine 3, stained blue and the intine, colorless and filling the discontinuities present in exine 3; e. detail of sporoderm, stained with Coomassie Blue, showing exine 3, weakly stained; f. detail of sporoderm, showing the PAS-positive exine 3 and a weak reaction in exine 1 and the intine; g. detail of sporoderm, showing differential staining of exine sublayers (1, 2 and 3) with Basic Fuchsin; h. detail of the sporoderm, stained with Auramine O, showing exine 3 and a weak reaction on the surface of exine 1; i. detail of sporoderm, showing the intine, after staining with Calcofluor White; j. detail of sporoderm showing exine 3, stained with Sudan Black B and weak color of exine 1 lamellae; k. detail of sporoderm, with intine stained by Alcian Blue. (E1 = exine 1; E2 = exine 2; E3 = exine 3; IN = intine). Bars = 10 μm.

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Figure 5
a-k. Riccia curtisii - a-b. tetrad of spores; c. section of spores in the tetrad, showing the outer layers of exine 1, orange colored, and exine 3, transparent; d. detail of sporoderm, stained with Toluidine Blue O, showing exine 1 and 2, partially stained greenish-blue, exine 3, stained blue, and the intine, colorless; e. detail of sporoderm, stained with Coomassie Blue, showing exine 3, weakly stained; f. detail of sporoderm, showing the PAS-positive exine 3 and a weak reaction in the intine. Note a thin PAS-positive layer between the spores (*); g. detail of the sporoderm, showing differential staining of the exine sublayers (1, 2 and 3) with Basic Fuchsin. Note a thin layer stained with Basic Fuchsin between the spores (*); h. detail of sporoderm stained with Auramine O, showing exine 3 and a weak reaction on the surface of exine 1. A positive reaction occurred in the exine between the spores; i. detail of sporoderm, showing intine, after staining with Calcofluor White; j. detail of sporoderm, showing exine 3, stained with Sudan Black B, reaction in exine 1 and 2 and cavity (*) between exine 1 and 2; k. detail of sporoderm, with intine stained with Alcian Blue. (E1 = exine 1; E2 = exine 2; E3 = exine 3; IN = intine). Bars = 10 μm.

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Figure 6
a-d. Spores of Riccia plano-biconvexa submitted to acetolysis - a. detail of sporoderm of two iferente spores, showing a differential action of acetolysis. In the lower spore, greater extraction of iferente of exine 1 occurred, and there was a clear reduction of the natural color of this sublayer; b. the same section as the previous figure (a), stained with Auramine O, showing a more intense reaction in exine 1, after extraction due to acetolysis; c. spore stained with Toluidine Blue O, with a greater exine 1 and 2 staining after acetolysis (compared to d); d. detail of sporoderm, after acetolysis and stained with Toluidine Blue O, showing differential lamellae staining in exine 1. (E1 = exine 1; E2 = exine 2; E3 = exine 3). Bars = 10 μm.

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Figure 7
a-e. Schematic drawings of the stratification of the spore wall in R. plano-biconvexa (a) R fruchartii (b), R. cavernosa (c), R. stenophylla (d), and R. curtisii (e), showing the four layers of the sporoderm: exine 1 (black), exine 2 (dark grey), exine 3 (gray) and intine (light grey). The cavities between exine 1 and 2 are marked with an asterisk. (E1 = exine 1; E2 = exine 2; E3 = exine 3).

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Table 1
Terminology used to describe the sublayers of sporoderm in previous studies on the genus Riccia. The columns represent different authors’ nomenclature for the corresponding layers, while the rows represent the different sublayers of sporoderm identified in those studies.

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Table 2
Data from the Riccia species studied. The coordinates are given in degrees, minutes, and seconds format for latitude and longitude.

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Table 3
Sporoderm sublayers of Riccia species under different stains and cytochemistry tests. (+ = weak reaction; ++ = medium reaction; +++ = strong reaction; - = not reacted; 1. After the acetolysis treatment).

Beer (1906Beer R (1906) On the development of spores of Riccia glauca. Annals of Botany 20: 275-291. )	Black (1913Black CA (1913) The morphology of Riccia frostii Aust. Annals of Botany 27: 511-532. )	*Jovet-Ast (1966* Jovet-Ast S (1966) Riccia crystallina L. emend. Raddi et Riccia cavernosa Hoffm. emend. Raddi - II. Revue 361 Bryologique et Lichénologique 34: 82-90. )	Steinkamp & Doyle (1979Steinkamp MP & Doyle WT (1979) Spore wall ultrastructure in four species of the liverwort Riccia. American Journal of Botany 66: 546-556. )	Jovet-Ast (1975Jovet-Ast S (1975) Précisions sur les charactères de deux Riccia du sous-genre Thallocarpus. Revue Bryologique et Lichénologique 41: 449-456. ) and Thaithong (1982Thaithong O (1982) Fine structure of spore wall in fourteen species of Riccia. Journal of the Hattori Botanical Laboratory 53: 133-146. )	Thaithong (1982Thaithong O (1982) Fine structure of spore wall in fourteen species of Riccia. Journal of the Hattori Botanical Laboratory 53: 133-146. )
Primary spore wall	Primary spore wall	Couche externe a	Exine region III	Sexine	Sexine
Secondary spore wall	Secondary spore wall	Couche interne b1	Exine region II	Nexine
		Couche interne b2	Exine region I
Endospore	Endospore	-	Intine	Intine	Intine

Herbarium voucher number	Specie	Sample locality (city, state, country)	Coordinates
ICN 175632	R. plano-biconvexa	Caçapava do Sul, Rio Grande do Sul, Brazil	30^o45’2.57’’S, 53^o31’42.08”W
ICN 192153	R. fruchartii	Caçapava do Sul, Rio Grande do Sul, Brazil	30^o45’2.57’’S, 53^o31’42.08”W
ICN 175634	R. cavernosa	Barra do Ribeiro, Horto Florestal Barba Negra, Rio Grande do Sul, Brazil	30^o21’40.02’’S, 51^o16’18.93”W
ICN 175635	R. stenophylla	Barra do Ribeiro, Horto Florestal Barba Negra, Rio Grande do Sul, Brasil	30^o21’40.02’’S, 51^o16’18.93”W
ICN 175633	R. curtisii	Caçapava do Sul, Rio Grande do Sul, Brazil	30^o45’2.57’’S, 53^o31’42.08”W

Species	Stain / cytochemical test	Sporoderm layers
		Exine			Intine
		Exine 1	Exine 2	Exine 3
R. plano-biconvexa	Toluidine Blue O	+ (green), ++, (green)¹	+ (green), + (green)¹	++ (blue), + (blue)¹	+ (purple), -¹
	Comassie Blue	-	-	+	-
	PAS	-	-	+++	++
	Basic Fuchsin	-	-	+++	++
	Auramin O	-, +¹	-, -¹	+++, +++¹	-, -¹
	Calcofluor White	-	-	-	++
	Sudan Black B	-	-	+++	-
	Alcian blue 8GX	-	-	-	+
R. fruchartii	Toluidine Blue O	+ (green)	+ (green)	++ (blue)	+ (purple)
	Comassie Blue	-	-	+	-
	PAS	-	-	+++	+++
	Basic Fuchsin	-	-	++	-
	Auramin O	+	-	+++	-
	Calcofluor White	-	-	-	++
	Sudan Black B	+	-	+++	-
	Alcian blue 8GX	-	-	-	+
R. cavernosa	Toluidine Blue O	+ (green)	+ (green)	+ (blue)	-
	Comassie Blue	-	-	+	-
	PAS	+	+++	++	-
	Basic Fuchsin	++	+++	++	-
	Auramin O	-	-	++	-
	Calcofluor White	-	-	-	++
	Sudan Black B	+	++	+	-
	Alcian blue 8GX	-	-	-	+
R. stenophylla	Toluidine Blue O	-	-	+ (blue)	-
	Comassie Blue	-	-	+	-
	PAS	+	-	+++	+
	Basic Fuchsin	-	-	+++
	Auramin O	-	-	++	-
	Calcofluor White	-	-	-	++
	Sudan Black B	-	-	+	-
	Alcian blue 8GX	-	-	-	+
R. curtisii	Toluidine Blue O	+ (green)	+ (green)	++ (blue)	-
	Comassie Blue	-	-	+	-
	PAS	-	-	++	+
	Basic Fuchsin	-	-	++	-
	Auramin O	+	-	++	-
	Calcofluor White	-	-	-	++
	Sudan Black B	+	+	++	-
	Alcian blue 8GX	-	-	-	+

[1] * daniel.ayub@bento.ifrs.edu.br

Brasil

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Spore wall cytochemistry and structure in five species of the liverwort Riccia (Ricciaceae)

Abstract