Pollen morphology of Austrocylindropuntia Backeb, Maihueniopsis Speg., Opuntia Mill. and Tephrocactus Lem. (Cactaceae, Opuntioideae) of Argentina

Review of Palaeobotany and Palynology 146 (2007) 1 – 17 www.elsevier.com/locate/revpalbo

Pollen morphology of Austrocylindropuntia Backeb, Maihueniopsis Speg., Opuntia Mill. and Tephrocactus Lem. (Cactaceae, Opuntioideae) of Argentina Silvina Garralla ⁎, Graciela Ana Cuadrado CECOAL-CONICET Ruta 5, km. 2,5 cc: 291, 3400 Corrientes, Argentina Departamento de Biología, Universidad Nacional del Noreste UNNE, Av. Libertad 5500, 3400 Corrientes, Argentina Received 26 April 2006; received in revised form 17 January 2007; accepted 24 January 2007 Available online 31 January 2007

Abstract The pollen morphology of the Cactaceae (Opuntioideae) of Argentina was studied under optical and scanning electron microscopy. The following species were considered: Austrocylindropuntia shaferi (Britton and Rose) Backeberg, Austrocylindropuntia verschaffeltii (Cels ex F. A. C Weber) Backeberg., Austrocylindropuntia vestita (Salm-Dyck) Backeberg, Maihueniopsis boliviana (Salm-Dyck) R. Kiesling, Maihueniopsis darwinii (Henslow) F. Ritter, Maihueniopsis glomerata (Haworth) R. Kiesling, Maihueniopsis ovata (Pfeiffer) F. Ritter, Maihueniopsis pentlandii (Salm-Dyck) R. Kiesling, Opuntia anacantha Spegazzini, Opuntia arechavaletae Spegazzini, Opuntia aurantiaca Lindley, Opuntia chakensis Spegazzini, Opuntia colubrina A. Castellanos, Opuntia ficus-indica (L.) Miller, Opuntia monacantha Haworth, Opuntia salagria Castellanos, Opuntia quimilo K. Schumann, Opuntia schickendantzii F. A. C. Weber, Opuntia schulzii A. Castellanos and Lelong, Opuntia soehrensii Britton and Rose, Opuntia sulphurea Gillies ex Salm-Dyck, Tunilla corrugata (Salm-Dyck) D. R. Hunt and Iliff, Tephrocactus alexanderi (Britton and Rose) Backeberg, Tephrocactus aoracanthus (Lemaire) Lemaire, Tephrocactus articulatus (Pfeiffer) Backeberg, Tephrocactus geometricus (A. Castellanos) Backeberg, Tephrocactus halophilus (Spegazzini) Backeberg, Tephrocactus weberi (Spegazzini) Backeberg. The species of the studied genera are: spheroidal, apolar, radiosymetric. Amb circular. Pantoaperturate. Tectate perforate, tectate imperforate, spinulose, nanospinulose or reticulate. Morphological characteristics of the pollen grain that allows differentiation of the taxa include the type of exine (tectate/semitectate), shape and number of pores and supratectal elements. Differences among the species are discussed and a species key is presented. © 2007 Elsevier B.V. All rights reserved. Keywords: Pollen morphology; Cactaceae; Opuntioideae; Argentina

1. Introduction In Argentina, the family Cactaceae is represented by 36 genera and 225 species, according to Kiesling (1999). One more genus has since been described: Tunilla, D. R. ⁎ Corresponding author. Tel./fax: +54 3783 454417. E-mail addresses: [email protected], [email protected] (S. Garralla). 0034-6667/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.revpalbo.2007.01.002

Hunt and Iliff, was segregated from Opuntia Miller (Anderson, 2001); in this paper included as Opuntia longispina. In this first report, the palynological characteristics of species of the genera Austrocylindropuntia Backeberg, Maihueniopsis Spegazzini, Opuntia and Tephrocactus Lemaire., observed under optical and scanning electron microscope, are given, and interspecific and intergeneric differences in the pollen of this family are established.

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One of the motivations of this palynological study is that fossil pollen of Cactaceae were recently found for the first time in Tertiary sediments (Middle Miocene) from the northwestern part of Argentina (Mautino, 2002). However, there is very little information available on the pollen of this group, so it was not possible to assign the new specimens to a current genus or species. Studies of Quaternary sediments, as well as analyses of modern pollen dispersal have also found Cactaceae pollen, but it has been impossible to identify the grains to the generic level. Cactaceae are native to the New World, mainly distributed in arid regions, but also found in rainforests, and grow under a wide variety of conditions ranging from cold to tropical. They are cultivated extensively

as an ornamental plant in the Old World (Nowicke and Skvarla, 1979). In Argentina, plants of the Cactaceae are concentrated in the northwest, although some species are found in all types of environments (Kiesling, 1999). Previous palynological studies on Argentine Cactaceae species include Kiesling (1984), who was working on a revision of Mahiueniopsis, as proposed by Ritter (1980). He analyzed the pollen morphology, among other characteristics, from two species of Mahiueniopsis, four species of Tephrocactus, two species of Puna and two species of Opuntia in a very general way, including the pollen characteristics within the systematic descriptions of this genus. Later, Ferguson and Kiesling (1997) analyzed pollen of Punna bonnieae D. K. Ferguson and R. Kiesling. Leuenberger (1976a) studied some 600 species of this

Plate I. In E and G, the scale bar equals 41 μm; N scale bar equals 38 μm; A, D and M scale bar equals 33 μm; K scale bar equals 30 μm; H scale bar equals 27 μm; F scale bar equals 14 μM; B and I scale bar equals 8 μm; C and J scale bar equals 6 μm. (see on page 3) (A–D). (B). (C). (D). (E–G). (E). (F). (G). (H–K). (H). (I). (J). (K). (L–N). (L). (M). (N).

Austrocylindropuntia shaferi (Britton and Rose) Backeberg A. SEM general view. SEM detail of ornamentation. SEM exine structure, tectum with perforations and spines. LM general view, proximal focus. Austrocylindropuntia verschaffeltii (Cels ex F. A. C Weber) Backeberg. SEM general view. SEM detail of ornamentation and aperture. LM general view. Austrocylindropuntia vestita (Salm-Dyck) Backeberg. SEM general view. SEM detail of ornamentation and aperture. SEM exine structure. LM general view, proximal focus. Maihueniopsis boliviana (Salm-Dyck) (R) Kiesling. SEM detail of ornamentation and aperture. SEM general view. LM general view.

Plate II. In C, the scale bar equals 35 μm; G, F and I scale bar equals 33 μm; A scale bar equals 31 μm; D scale bar equals 28 μm; L scale bar equals 26 μm, J scale bar equals 24 μm; B scale bar equals 20 μm; K scale bar equals 17 μm; H scale bar equals 13 μm; E scale bar equals 5.6 μm. (see on page 4) (A–C). (A). (B). (C). (D–F). (D). (E). (F). (G–I). (G). (H). (I). (J–L). (J). (K). (L).

Maihueniopsis darwinii (Henslow) F. Ritter. SEM general view. SEM detail of ornamentation. LM general view, proximal focus. Maihueniopsis glomerata (Haworth) R. Kiesling. SEM general view. SEM detail of ornamentation. LM general view, proximal focus. Maihueniopsis ovata (Pfeiffer) F. Ritter. SEM general view. SEM exine structure, tectum with perforations and spines. LM cross-section. Maihueniopsis pentlandii (Salm-Dyck) R. Kiesling. SEM general view. SEM detail of ornamentation and aperture. LM general view.

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Plate I (caption on page 2 ).

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Plate II (caption on page 2 ).

family, distributed throughout the world, of which only a few are present in Argentina. Among those from Argentina, Opuntia aurantiaca Lindley and Opuntia schickendantzii F. A. C. Weber were included in this study. Brasiliopuntia (K. Schum.) Berger, Austrocylindropuntia, Quiabentia (Vaupel) Borg and Pterocactus K. Schumann are other

genera of the Opuntioideae taxa that are present in Argentina. Studies including pollen descriptions of Cactaceae species that are not found in Argentina include Kurtz (1948, 1963), Tsukada (1964), Heusser (1971), Kozar (1974), Nowicke (1975), Leuenberger (1976a,b), among others.

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Plate III (caption on page 7 ).

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Plate IV (caption on page 7 ).

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2. Materials and methods This study of Cactaceae pollen was carried out using material from the Darwinion Institute Herbarium (SI) and the Corrientes Herbarium (CTES). Details of the particular specimens examined are given below. The pollen was processed according to Erdtman's method (1960) and mounted in glycerin gelatin. Pollen samples have been deposited in the pollen herbarium of the National University of the Northeast, UNNE (PAL-CTES). Observations and counts were carried out using an Olympus BX40 light microscope. Photographs were obtained using a Canon Power Shot S50 digital camera. The following parameters were measured on each sample, for a minimum of 20 pollen grains: diameter

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of the grain, size of apertures (diameter or length and width, depending on the nature of the aperture), thickness of exine: sexine and nexine, and the shape and size of the sculptural elements. The observation and interpretation of the sculpture and apertures were supplemented by observations and photographs taken with a Jeol JSM-580 OLV scanning electron microscope (SEM) at the Secretary of Science and Technology of the National University of the Northeast, Argentina. For the SEM study, the pollen grains, previously acetolyzed, were mounted on a metallic slide and coated in palladium gold. Terminology used in this study is taken from Erdtman (1960, 1966), Saenz de Rivas (1978), Nilsson

Plate III. In G, the scale bar equals 48 μm; J and K scale bar equals 45 μm; F scale bar equals 40 μm; A and C scale bar equals 34 μm; D scale bar equals 31.5 μm; M scale bar equals 28 μm; H scale bar equals 24 μm; I scale bar equals 20 μm; B scale bar equals 17 μm; E scale bar equals 16 μm. (see on page 5) (A–C). (A). (B). (C). (D–F). (D). (E). (F). (G–I). (G). (H). (I). (J–L). (J). (K). (I). (M). (M).

Opuntia schulzii A. Castellanos and Lelong. SEM general view. SEM detail of ornamentation. LM general view, proximal focus. Tunilla corrugata (Salm-Dyck) D. R. Hunt and Iliff. SEM general view. SEM detail of ornamentation and aperture. LM general view, proximal focus. Opuntia schickendantzii F. A. C. Weber. SEM general view. SEM detail of ornamentation. LM general view. Opuntia quimilo K. Schumann. SEM general view. SEM detail of ornamentation. LM detail of aperture in proximal focus. Opuntia soehrensii Britton and Rose. LM general view.

Plate IV. In C, the scale bar equals 50 μm; G scale bar equals 42 μm; A, D, F, I and L scale bar equals 40 μm; J scale bar equals 36 μm; H and K scale bar equals 15 μm; B scale bar equals 13 μm; E scale bar equals 11 μm. (see on page 6) (A–C). (A). (B). (F). (D–F). (D). (E). (F). (G–I). (G). (H). (I). (J–L). (J). (K). (L).

Opuntia anacantha Spegazzini. SEM general view. SEM detail of ornamentation and aperture. LM general view, proximal focus. Opuntia arechavaletae. Spegazzini. SEM general view. SEM detail of ornamentation and aperture. LM general view, proximal focus. Opuntia chakensis Spegazzini. SEM general view. SEM detail of ornamentation and aperture. LM general view. Opuntia colubrina A. Castellanos. SEM detail of ornamentation and aperture. SEM general view. LM general view, proximal focus.

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Plate V (caption on page 10 ).

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Plate VI (caption on page 10 ).

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and Praglowski (1992), and Punt et al. (1994). The number of apertures shown in the Tables corresponds to that found in 90% of grains. 3. Specimens examined Austrocylindropuntia shaferi (Britton and Rose) Backeberg. Bolivia, Chuquisaca Department, Kiesling and Metzing 8390 (SI), PAL-CTES 6972; Potosí Department, Mariño 218 (SI), PAL-CTES 6973. Austrocylindropuntia verschaffeltii (Cels ex F. A. C Weber) Backeberg. Argentina, Salta Province, Rosario

de Lerma Department, Kiesling 1650 (SI), PAL-CTES 6970. Bolivia, Chuquisaca Department, Kiesling and Metzing 8391 (SI), PAL-CTES 6971. Austrocylindropuntia vestita (Salm-Dyck) Backeberg. Argentina, Salta Province, Guachipas Department, Kiesling and Tombesi, 10004 (SI), PAL-CTES 6974. Maihueniopsis boliviana (Salm-Dyck) R. Kiesling. Argentina, Catamarca Province, Tinogasta Department, Biurrum, Kiesling and Molina 4945 (SI), PAL-CTES 6894. San Juan Province, Iglesia Department, Kiesling 2991(SI), PAL-CTES 6965.

Plate V. In A, the scale bar equals 86 μm; C scale bar equals 74 μm; F and I scale bar equals 40 μm; M scale bar equals 46 μm; L scale bar equals 32 μm; G scale bar equals 31 μm; D scale bar equals 29 μm; B and N scale bar equals 15 μm; E scale bar equals 13 μm; H scale bar equals 12 μm. (see on page 8) (A–C). (A). (B). (C). (D–F). (D). (E). (F). (G–I). (G). (H). (I). (J–K). (J). (K). (L–N). (L). (M). (N).

Opuntia ficus-indica (L.) Miller. SEM general view. SEM detail of ornamentation and aperture. LM general view, proximal focus. Opuntia sulphurea Gillies ex Salm-Dyck. SEM general view. SEM detail of ornamentation and aperture. LM general view, proximal focus. Opuntia monacantha Haworth. SEM general view. SEM detail of ornamentation and aperture. LM general view, proximal focus. Opuntia aurantiaca Lindley. SEM general view. LM general view, proximal focus. Opuntia salagria A. Castellanos. SEM general view. LM general view. SEM detail of ornamentation and aperture.

Plate VI. In H, K and M the scale bar equals 46 μm; B scale bar equals 36,5 μm; E, G and J scale bar equals 35 μm; D scale bar equals 33 μm; A scale bar equals 32 μm; L and N scale bar equals 31 μm; C scale bar equals 16 μm; F scale bar equals 10 μm; I scale bar equals 4 μm. (see on page 9) (A–C). (A). (B). (C). (D–F). (D). (E). (F). (G). (H). (I). (J–K). (J). (K). (L–M). (L). (M). (N).

Tephrocactus alexanderi (Britton and Rose) Backeberg. SEM general view. LM general view, proximal focus. SEM detail of ornamentation and aperture. Tephrocactus aoracanthus (Lemaire) Lemaire. SEM general view. LM general view, distal focus. SEM detail of ornamentation and structure. (G–I) Tephrocactus articulatus (Pfeiffer) Backeberg. SEM general view. LM general view, proximal focus. SEM detail of exine structure. Tephrocactus geometricus (A. Castellanos) Backeberg. SEM general view. LM general view, distal focus. Tephrocactus halophilus (Spegazzini) Backeberg. SEM general view. LM general view, distal focus. Tephrocactus weberi (Spegazzini) Backeberg. (N) SEM general view.

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Maihueniopsis darwinii (Henslow) F. Ritter. Argentina, Chubut Province, Futaleufú Department, Kiesling 9975 (SI), PAL-CTES 6895. Río Negro Province, Kiesling 3372 (SI), PAL-CTES 6966. Maihueniopsis glomerata (Haworth) R. Kiesling. Argentina, San Juan Province, Iglesia Department, Cabrera et al. 31156 (SI), PAL-CTES 6896. Catamarca Province, Belén Department, Cabrera et al. 32531 (SI), PAL-CTES 6967. Maihueniopsis ovata (Pfeiffer) F. Ritter. Argentina, San Juan Province, Sarmiento Department, Kiesling and Saenz 4148 (SI), PAL-CTES 6897. Maihueniopsis pentlandii (Salm-Dyck) R. Kiesling. Bolivia, Potosí Department, Kiesling 220 (SI), PALCTES 6968; Tarija Department, Kiesling 22115 (SI), PAL-CTES 6969. Opuntia anacantha Spegazzini. Paraguay, Nueva Asunción Department, Nicora 9733 (SI), PAL-CTES 6993. Argentina, Entre Ríos Province, La Paz Department, Burkart and Bacigalupo 21294 (SI), PAL-CTES 6994. Opuntia arechavaletae Spegazzini. Argentina. Burkart 17911 (SI), PALCTES 6975. Opuntia aurantiaca Lindley. Argentina, Buenos Aires Province, Burkart, 23905 (SI), PAL-CTES 6978. Opuntia chakensis Spegazzini. Argentina, Corrientes Province, San Luis del Palmar Department, Schinini and Vanni 21644 (SI), PAL-CTES 6983. Buenos Aires Province, Villamil, 5603 (SI), PAL-CTES 6984. Tucumán Province, Capital Department, Venturi 3554 (SI), PALCTES 6985. Opuntia colubrina A. Castellanos. Paraguay, Boquerón Department, Nicora, Kiesling and Pin 9722 (SI), PALCTES 6979. Opuntia ficus-indica (L.) Miller. Argentina, La Rioja Province, Chamical Department, Biurrum, Paredes and Torres 2494 (SI), PAL-CTES 6987. Opuntia longispina, name to be changed for Tunilla corrugata (Salm-Dyck) D. R. Hunt and Iliff. Argentina, San Juan Province, Iglesia Department, Leuemberger, 4436 (SI), PAL-CTES 6980. Opuntia monacantha Haworth. Argentina, Cultivated Buenos Aires Province, San Isidro Department, Kiesling, 8713 (SI), PAL-CTES 6990. Opuntia quimilo K. Schumann. Argentina, Salta Province, Metán Department, Pedersen 15861 (SI), PAL-CTES 6991. Catamarca Province, Chumbicha Department, Biurrun and Agüero 1191 (SI), PALCTES 6992. Opuntia salagria Castellanos. Argentina, San Luis Province, Kiesling and Meregali 9320 (SI), PAL-CTES 6989.

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Opuntia schickendantzii F. A. C. Weber. Argentina, Jujuy Province, Santa Barbara Department, Kiesling et al. 8315 (SI), PAL-CTES 6995. Opuntia schulzii A. Castellanos and Lelong. Argentina, Corrientes Province, Capital Department, Meza Torres 100 (CTES), PAL-CTES 7003. Opuntia soehrensii Britton and Rose. Argentina, Tucumán Province, Tafí del Valle Department, Leuemberger, 4894 (SI), PAL-CTES 7002. Opuntia sulphurea Gillies ex Salm-Dyck. Argentina, Salta Province, Cafayate Department, Leuemberger 4852 (SI), PAL-CTES 7001. Tephrocactus alexanderi (Britton and Rose) Backeberg. Argentina, San Juan Province, Valle Fértil Department, Kiesling and Marengo 9329 (SI), PAL-CTES 6899. La Rioja Province, Arauco Department, Kiesling et al. 4338 (SI), PAL-CTES 6998. Tephrocactus aoracanthus (Lemaire) Lemaire. Argentina, San Juan Province, Valle Fértil Department, Biurrun, Márquez and Molina 5003 (SI), PAL-CTES 6900. Tephrocactus articulatus (Pfeiffer) Backeberg. Argentina, La Rioja Province, Capital Department, Biurrun, Molina and Luna 41 (SI), PAL-CTES 6901; Independencia Department, Biurrun, Molina and Luna 4461 (SI), PALCTES 6999. Tephrocactus geometricus (A. Castellanos) Backeberg. Argentina s.n. (SI), PAL-CTES 6904. Tephrocactus halophilus (Spegazzini) Backeberg. Argentina, San Juan Province, Albardón Department, Kiesling et al. 9234 (SI), PAL-CTES 6902. Tephrocactus weberi (Spegazzini) Backeberg. Argentina, San Juan, Province, Caucete Department, Leuemberger et al. 4470 (SI), PAL-CTES 6903. 4. Results 4.1. Pollen descriptions 4.1.1. Austrocylindropuntia Backeberg Pollen grains are spheroidal, apolar, radiosymetric. Amb circular. Pantoaperturate, 12–14 circular or elongated pores. Exine tectate 2–3 μm thick, with positive elements b 1 μm, more or less densely arranged in LM (light microscope). Tectum is spinulose or nanospinulose, perforate; each perforation surrounded by thin ring. Aperture membranes with granules or spinules in SEM (scanning electron microscope) (Plate I, A–K). The diameter of the grain, size of apertures, thickness of exine: sexine and nexine of each species of Austrocylindropuntia are shown in Table 1.

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Table 1 Comparative measurements of the diameter in μm, size of apertures (μm), number of apertures, thickness of exine: sexine and nexine of pollen grains from examined species of the genus Austrocylindropuntia Species

Diameter in μm

Size of apertures (μm)

Number of apertures

Exine (μm)

Sex/Nex (μm)

A. shaferi A. verschaffeltii A. vestita

63(72)89 84(93.7)101 56(61)68

12 24.5 × 20.5 10

16 12 14

2.5 3 2

2/0.5 2.5/1 1.5/0.5

4.1.2. Austrocylindropuntia shaferi (Britton and Rose) Backeberg SEM: Tectum spinulose and perforate. Circular or lightly elliptical perforations from 0.5 to 2.5 μm in diameter, spinule 1 to 1.5 μm high. Circular pores, covered by a granulate membrane, granules 1 μm high (Plate I, A–D). 4.1.3. Austrocylindropuntia verschaffeltii (Cels ex F. A. C. Weber) Backeberg SEM: Tectum spinulose and perforate. Circular perforations 0.2–0.5 μm in diameter, spinule 0.6 μm high. Elongated pores, covered by a spinulose membrane, spinules of the same size as the other spinules. Exine thickness decreases towards the apertures (Plate I, E–G). 4.1.4. Austrocylindropuntia vestita (Salm-Dyck) Backeberg SEM: Tectum nanospinulose and perforate. Circular perforations 0.1–0.4 μm in diameter, nanospinules ca. 0.4 μm high. Circular pores, covered by a spinulose membrane, spinules 1 μm high (Plate I, H–K). 4.1.5. Maihueniopsis Spegazzini Pollen grains are spheroidal, apolar, radiosymetric. Amb circular. Pantoaperturate, 14–20 circular or elongated pores. Exine tectate 2.5–3 μm thick, with positive elements b 1 μm, more or less densely arranged in LM. Tectum is perforate or imperforate, spinulose or nanospinulose. Aperture membranes with granules or spinules in SEM (Plate I, L–N; Plate II, A–L).

The diameter of the grain, size of apertures, thickness of exine: sexine and nexine of each species of Maihueniopsis are shown in Table 2. 4.1.6. Maihueniopsis boliviana (Salm-Dyck) R. Kiesling SEM: Tectum nanospinulose and perforate. Circular perforations 0.3–0.5 μm in diameter, nanospinules 0.3–0.4 μm high. Exine thickness decreases towards apertures. Circular pores covered by a spinulose membrane, spinules ca. 1 μm high (Plate I, L–N). 4.1.7. Maihueniopsis darwinii (Henslow) F. Ritter SEM: Tectum spinulose and perforate. Circular perforations 0.2–1 μm in diameter, spinules ca. 0.5 μm high. Elongated pores, covered by a spinulose membrane, spinules ca. 0.8 μm high (Plate II, A–C). 4.1.8. Maihueniopsis glomerata (Haworth) R. Kiesling SEM: Tectum nanospinulose and perforate. Circular perforations 0.1–0.3 μm in diameter, nanospinules ca. 0.3 μm high. Elongated pores covered by a granulate membrane, granules ca. 0.6 high (Plate II, D–F). 4.1.9. Maihueniopsis ovata (Pfeiffer) F. Ritter (Plate II, G–I) SEM: Tectum spinulose and perforate. Circular perforation surrounded by a thin ring, ca. 0.6 μm in diameter, spinules ca. 0.6 μm high. Elongated pores covered by spinulose membrane, spinules ca. 1 μm high.

Table 2 Comparative measurements of the diameter in μm, size of apertures (μm), number of apertures, thickness of exine: sexine and nexine of pollen grains from examined species of the genus Maihueniopsis Species

Diameter in μm

Size of apertures (μm)

Number of apertures (μm)

Exine (μm)

Sex/Nex (μm)

M. boliviana M. darwinii M. glomerata M. ovata M. pentlandii

63(77)84.7 70(77)84 50(64)70.5 67(74)84 40(60)80

17.6 15 × 7 14 × 7 19 × 11 14

14 20 12 14 14

3 2,5 3 3 3

1.5/1.5 2/0.5 2.5/0.5 2/1 1.5/1.5

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Table 3 Comparative measurements of the diameter in μm, size of apertures (μm), number of apertures, thickness of exine: sexine and nexine of pollen grains of Tunilla corrugata, Opuntia schickendantzii, Opuntia soehrensii and Opuntia schulzii Species

Diameter in μm

Size of apertures (μm)

Number of apertures

Exine (μm)

Sex/Nex (μm)

T. corrugata O. schickendantzii O. soehrensii O. schulzii

62.5(71.6)78 98(107.8)115 53.9(56.7)61 77(78.3)87

15 × 9 18 10 × 6 17 × 8.5

12 24 12 12

4 7 4 4

3/1 6/1 3/1 3/1

4.1.10. Maihueniopsis pentlandii (Salm-Dyck) R. Kiesling SEM: Tectum spinulose and imperforate. Spinules ca. 0.7 μm high. Exine thickness decreases towards apertures. Circular pores covered by granulate membrane, granules ca. 1 μm high (Plate II, J–L).

4.1.13. Opuntia schulzii A. Castellanos and Lelong SEM: Tectum with nanospinules ca. 0.1 μm high. Circular or slightly elongated pores, with nanospinulose membrane; these nanospinules are the same size as the other nanospinules (Plate III, A–C).

4.1.11. Opuntia Miller Pollen grains are spheroidal, apolar, radiosymetric. Amb circular except Opuntia sulphurea Haworth that is hexagonal. Pantoaperturate, circular or elongated pores with aperture membrane. Exine tectate or semi-tectate with different thickness in each species (Plates III, IV, and V). In the genus Opuntia we can recognise two main pollen types: (1) Pollen grains tectate with positive elements b 1 μm in LM. In SEM, tectum is spinulose and perforate; each perforation is circular and surrounded by a ring, from 0.1 μm to 0.3 μm in diameter (Plate III, A–I) in: Opuntia schickendantzii F. A. C. Weber, Opuntia schulzii A. Castellanos and Lelong, Opuntia soehrensii Britton and Rose and Tunilla corrugata (Salm-Dyck) D. R. Hunt and Iliff. The diameter of the grain, size of apertures, thickness of exine: sexine and nexine of these species are shown in Table 3.

4.1.14. Opuntia soehrensii Britton and Rose SEM: Tectum with spinules ca. 0.7 μm high. Elongated pores with spinulose membrane, these spinules are the same size as the other spinules.

4.1.12. Opuntia schickendantzii F. A. C. Weber SEM: Tectum with nanospinules ca. 0.3 μm high. Circular pores with spinulose membrane, spinule ca. 1.5 μm high (Plate III, G–I).

4.1.15. Tunilla corrugata (Salm-Dyck) D. R. Hunt and Iliff SEM: Tectum with spinules ca. 1 μm high. Exine thickness decreases towards apertures. Elongated pores with spinulose membrane, these spinules are the same size as the other spinules (Plate III, D–F). (2) Pollen grains are semitectate, reticulate, heterobrochate, muri simplibaculate with irregularly thick in LM. In SEM, the pollen grain includes some lumina of the reticulum with free columellae (Plates IV and V) in: Opuntia anacantha Spegazzini, Opuntia arechavaletae Spegazzini, Opuntia aurantiaca Lindley, Opuntia chakensis Spegazzini, Opuntia colubrina A. Castellanos, Opuntia ficus-indica (L.) Miller, Opuntia monacantha Haworth, Opuntia quimilo K. Schumann, Opuntia salagria Castellanos, Opuntia sulphurea Gillies ex Salm-Dyck. The diameter of the grain, size of

Table 4 Comparative measurements of the diameter in μm, size of apertures (μm), number of apertures, thickness of exine: sexine and nexine of pollen grains of Opuntia anacantha, Opuntia arechavaletae, Opuntia aurantiaca, Opuntia chakensis, Opuntia colubrina, Opuntia ficus-indica, Opuntia monacantha, Opuntia salagria, Opuntia quimilo and Opuntia sulphurea Species

Diameter in μm

Size of apertures (μm)

Number of apertures

Exine (μm)

Sex/Nex (μm)

O. anacantha O. arechavaletae O. aurantiaca O. chakensis O. colubrina O. ficus-indica O. monacantha O. salagria O. quimilo O. sulphurea

65(80.4)100 70(79)90 80(86.3)101 75(85.6)100 62.5(67.4)85 126(142)161 62.5(68.9)75 66 (78.86)88 77(93.6)103 56(62.6)70

19 × 15 20 11 15 15 20 x 23 19 × 13 19.5 × 15 22 × 14 13

12–14 14 16 22 12 20 10 12 8–10 12

4 8 6 5 5 5 3 6 5 6

3/1 6/2 5/1 4/1 4/1 4/1 2/1 5/1 4/1 5/1

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apertures, thickness of exine: sexine and nexine of these species are shown in Table 4.

high; at the center of pore membrane these spinules are on little isles of exine (Plate IV, J–L).

4.1.16. Opuntia anacantha Spegazzini SEM: Muri of reticulum are angustimurate, lumina irregularly shaped and smaller around the pores. Exine thickness decreases towards apertures. Very frequently elongated pores, covered by a spinulose membrane, spinules between 0.7 μm and 1.4 μm high (Plate IV, A–C).

4.1.21. Opuntia ficus-indica (L.) Miller SEM: Muri of reticulum from angustimurate to latimurate, lumina irregularly shaped and sizes. Exine thickness decreases towards apertures. Circular pores covered by a spinulose membrane; the spinules are on little isles of exine, spinules ca 0.4 μm high (Plate V, A–C).

4.1.17. Opuntia arechavaletae Spegazzini SEM: Muri of reticulum from angustimurate to latimurate, lumina irregularly shaped and smaller around the pores. Exine thickness decreases towards apertures. Circular pores, covered by a spinulose membrane, spinules ca. 0.6 μm high; in the center of pore membrane these spinules are on little isles of exine (Plate IV, D–F).

4.1.22. Opuntia monacantha Haworth SEM: Pollen grains are perforate-reticulate. Perforate in regions near the pores then reticulate; the perforations are of different sizes and forms, 0.3 μm to 8 μm in diameter. Latimurate, lumina irregularly shaped and of various sizes, muri with spinules 0.5 μm high. Elongated pores; spinules ca. 1 μm high cover the pore membranes (Plate V, G–I).

4.1.18. Opuntia aurantiaca Lindley SEM: Muri of reticulum are angustimurate, lumina irregularly shaped and smaller around the pores. Exine thickness decreases towards apertures. Circular pores, covered by a membrane ornamented with nanospinules b0.1 μm high; these nanospinules are on little isles of exine (Plate V, J–K).

4.1.23. Opuntia quimilo K. Schumann SEM: Muri of reticulum from angustimurate to latimurate, lumina irregularly shaped and of various sizes. Elongated pores, surrounded by a thickening of the exine, ca. 5 μm in width. Pore membranes ornamented with verrucae or gemmas which are sometimes united, 1 μm–2 μm high (Plate III, J–M).

4.1.19. Opuntia chakensis Spegazzini SEM: Muri of reticulum are angustimurate, lumina irregularly shaped and smaller around the pores. Exine thickness decreases towards apertures. Circular pores, covered by a spinulose membrane, spinules ca. 1.5 μm high; in the center of the pore membrane these spinules are on little isles of exine (Plate IV, G–I).

4.1.24. Opuntia salagria Castellanos SEM: Muri of reticulum are angustimurate, lumina irregularly shaped and of various sizes. Exine thickness decreases towards the apertures. Elongated pores covered by a spinulose membrane; these spinules are on little isles of exine, spinules ca. 0.3 μm high (Plate V, L–N).

4.1.20. Opuntia colubrina A. Castellanos SEM: Muri of reticulum are angustimurate, lumina irregularly shaped and smaller around the pores. Exine thickness decreases towards apertures. Circular pores, covered by a spinulose membrane, spinules ca. 1.5 μm

4.1.25. Opuntia sulphurea Gillies ex Salm-Dyck SEM: Muri of reticulum are angustimurate, lumina irregularly shaped and of various sizes. Exine thickness decreases towards apertures. Pores circular with spinulose membrane; spinules ca. 1.5 μm high are on little isles of exine (Plate V, D–F).

Table 5 Comparative measurements of the diameter in μm, size of apertures (μm) and number of apertures of pollen grains from species examined of the genus Tephrocactus Species

Diameter in μm

Size of apertures (μm)

Number of apertures

Exine (μm)

Sex/Nex (μm)

T. alexanderi T. aoracanthus T. articulatus T. geometricus T. halophilus T. weberi

56(64.5)84 73.5(77.7)84 73.5(80.5)84 77(84.5)94.5 56(72.33)84 56(63)70

16 16 10 10 10 10

18 18 28 28 18 18

4 3 3 3 4 3

3/1 2.5/0.5 2.5/0.5 2.5/0.5 3/1 2.5/0.5

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4.1.26. Tephrocactus Lemaire Pollen grains are spheroidal, apolar, radiosymetric. Amb circular. Pantoaperturate, circular pores. Exine tectate with positive elements b 1 μm more or less densely arranged in LM, exine thickness decreases towards the apertures. Tectum is spinulose or nanospinulose, perforate, with circular perforations. Pore membranes covered by spinules or nanospinules in SEM (Plate IV). The diameter of the grain, size of apertures and number of apertures of each species of Tephrocactus are show in Table 5. 4.1.27. Tephrocactus alexanderi (Britton and Rose) Backeberg SEM: Tectum nanospinulose with perforations ca. 0.1 μm in diameter, nanospinules 0.3 μm high. Pore membranes with spinules ca. 0.6 μm high (Plate VI, A–C). 4.1.28. Tephrocactus aoracanthus (Lemaire) Lemaire SEM: Tectum nanospinulose with perforations 0.1 μm–1 μm in diameter, each perforation surrounded by a ring; nanospinules 0.1 μm–0.3 μm high. Pores with nanospinulose membrane, these nanospinules are on little isles of exine, nanospinules ca. 0.2 μm high (Plate VI, D–F). 4.1.29. Tephrocactus articulatus (Pfeiffer) Backeberg SEM: Tectum nanospinulose with perforations 0.1 μm–1 μm in diameter, nanospinules ca. 0.3 μm high. Pores with nanospinulose membrane, these nanospinules are on little isles of exine, nanospinules ca. 0.2 μm high (Plate VI, G–I). 4.1.30. Tephrocactus geometricus (A. Castellanos) Backeberg SEM: Tectum nanospinulose with perforations ca. 0.1 μm in diameter, nanospinules ca. 0.3 μm high. Pores with nanospinulose membrane; these nanospinules are on little isles of exine, nanospinules ca. 0.2 μm high (Plate VI, J–K). 4.1.31. Tephrocactus halophilus (Spegazzini) Backeberg SEM: Tectum spinulose with perforations 0.2 μm in diameter, each perforation surrounded by a ring, spinules ca. 0.5 μm high. Pore membranes with spines ca. 0.8 μm high (Plate VI, L–M). 4.1.32. Tephrocactus weberi (Spegazzini) Backeberg SEM: Tectum spinulose with perforations 0.2 μm– 0.4 μm in diameter, each perforation surrounded by a ring, spinules ca. 0.5 μm high. Pore membrane with spines ca. 0.8 μm high (Plate VI, N).

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5. Discussion and conclusions Based on observations from this study, pollen grains of Argentine species of the genera Austrocylindropuntia, Maihueniopsis, Opuntia and Tephrocactus (Opuntioideae) are spheroid apolar, radio-symmetric and pantoaperturate. Kiesling (1984) also found that the pollen of this subfamily that he described had these characteristics. Pollen grains are large, between 50 and 100 μm; however, the genera or species cannot be differentiated by size alone, since the sizes of the various species overlap. The exine thickness differs among the various species, but the sexine is always thicker than the nexine (Tables 1, 2, 3, 4, and 5). Kiesling (1984), in the key of genera of Argentine Opuntioideae, characterized pollen from species of the genus Opuntia as semitectate and reticulate. This observation is confirmed, but there are also some perforated semitectate grains and perforated tectate grains. Within the perforated tectate pollen grains, it is possible to distinguish Opuntia schickendantzii from the other species of Opuntia, because it is the only species that has circular pore grains, with a larger diameter and greater number of apertures. On the other hand, Leuenberger (1976a) also analyzed O. schickendantzii and Opuntia aurantiaca (cultivated), and indicated the predominance of 12-porate pollen in these two species. According to data published by Ferguson and Kiesling (1997), pollen grains of Puna bonnieae are very similar to those of Tephrocactus halophilus and Tephrocactus weberi. Similar characters identified in all of these taxa include the thickness and type of exine (tectate spinulose, perforated, each perforation surrounded by a ring), size and number of apertures, and pore membranes ornamented with spiniform processes. Kiesling (1984) illustrates pollen of Opuntia quimilo with an SEM photograph, in which the presence of 10 pores (or presumably 12) is shown, and he mentions the semitectate, reticulate condition of the wall. This coincides with the results found in this study, except for the number of apertures (8–10 porate). On the other hand, pollen grains of this species differ from those of other species because of a prominent exine thickness, easily observed under optical microscope. This study allows us to divide the analyzed species into two large groups: (1) species with tectate, imperforate/ perforate, spinulose/nanospinulose grains: Austrocylindropuntia shaferi, Austrocylindropuntia verschaffeltii, Austrocylindropuntia vestita, Maihueniopsis boliviana, Maihueniopsis darwinii, Maihueniopsis glomerata, Maihueniopsis ovata, Maihueniopsis pentlandii (only species with tectate imperforate grains), Opuntia schickendantzii,

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Opuntia schulzii, Opuntia soehrensii, Tephrocactus alexanderi, Tephrocactus aoracanthus, Tephrocactus articulatus, Tephrocactus geometricus, Tephrocactus halophilus, Opuntia schickendantzii, Tephrocactus weberi and Tunilla corrugata, and (2) species with semitectate, reticulate grains: Opuntia anacantha, Opuntia arechavaletae, Opuntia aurantiaca, Opuntia chakensis, Opuntia colubrina, Opuntia ficus-indica, Opuntia monacantha, Opuntia quimilo, Opuntia salagria and Opuntia sulphurea. Opuntia monacantha pollen grains have a reticulate, perforated exine and share this characteristic with species with a perforate tectate exine, as well as with those that have a reticulate exine. According to Walker and Doyle (1975), the structure of the exine evolves from an imperforate tectate exine, to a perforate tectate exine, a semitectate exine, and finally to intectate grains. This suggests an evolutionary development in the analyzed species, from the wall structure point of view, from M. pentlandii (tectate imperforated grains) to species of group 1 (tectate perforated grains) and, finally, to the most evolved ones, species of group 2 (reticulate grains). O. monacantha would occupy a transitional position between the two large groups of species, as it has a reticulate-perforated pollen grain. This hypothesis would be confirmed by analyzing a greater number of species within this family. Fossil records of Argentine Cactaceae from the Miocene (Mautino, 2002), correspond to two types of pollen grain, based on their exine structure and apertures: (1) grains with tectate, echinate, perforated, tricolpate exine, and (2) semitectate, reticulate, pantoaperturate (14–22 pores) grains, comparable to Opuntia aurantiaca and Opuntia chakensis, described in this study. Species of studied groups are palynologically homogeneous; however, we could develop a key that allows us to differentiate most of them. Morphological characteristics of the pollen grain that allows differentiation of the taxa include the type of exine (tectate/ semitectate), shape and number of pores and supratectal elements. On the other hand, the shape, polarity, symmetry and size of the grains and the presence of aperture membrane do not constitute diagnostic characters because they are found in all of the studied taxa. 5.1. Key to the pollen of species of Austrocylindropuntia, Maihueniopsis, Opuntia and Tephrocactus of Argentina 1- Pollen grains tectate imperforate or tectate perforate, spinulose or nanospinulose: 2- Pollen grains tectate imperforate………………… …………………….Maihueniopsis pentlandii

2′- Pollen grains tectate perforate: 3- Circular pores: 4 Pores 14–16: 5- Pores 14. Exine nanospinulose and perforations 0.1–0.5 μm in diameter………. …………………………………Austrocylindropuntia vestita, Maihueniopsis boliviana 5′- Pores 16. Exine spinulose and perforations 1.0–1.5 μm in diameter………………………… …………………Austrocylindropuntia shaferi 4′ Pores 18–28: 6- Pores 18: 7- Pores 13 μm in diameter………...………… …..Tephrocactus weberi, Tephrocactus halophilus 7′ Pores 16 μm in diameter…Tephrocactus aoracanthus, Tephrocactus alexanderi 6′- Pores 24–28: 8- Pores 24; 18 μm in diameter……… …………………Opuntia schickendantzii 8′- Pores 28; 10 μm in diameter…………… Tephrocactus articulatus, Tephrocactus geometricus 3′- Elongated pores: 9- Pores 12–14: 10- Pores 12: 11- Exine nanospinulose……………. ……Maihueniopsis glomerata, Opuntia schulzii 11′- Exine spinulose……………… ……….Austrocylindropuntia verschaffeltii, Tunilla corrugata, Opuntia soehrensii 10′- Pores 14……………………………………. Maihueniopsis ovata 9′- Pores 20…...………………………………………. Maihueniopsis darwinii 1 Pollen grains semitectate, perforate-reticulate or reticulate: 11- Pollen grains perforate-reticulate……Opuntia monacantha 11′- Pollen grains reticulate: 12- Circular pores: 13- Pores 15–16: 1 4 - Pores12………… ……………Opuntia sulphurea, Opuntia colubrina 14′- Pores14–16……… ……………Opuntia are ch a va l e t ae , Opuntia aurantiaca

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13′- Pores 20–22: 15- Pores 20………. …………Opuntia ficus-indica 15′- Pores 22……… ……… Opuntia chakensis 12′- Pores elongate: 16- Pores 8–10 surrounded by a thickening of the exine…….. ………Opuntia quimilo 16′- Pores 12–14 not surrounded by a thickening of the exine………… Opuntia salagria, Opuntia anacantha Acknowledgements This work was financially supported by the National University of the Northeast (UNNE) Argentine. We are grateful to Dr. R. Kiesling for pollen material, and to the Darwinion Botanical Institute for permission to take pollen samples from their herbarium sheets. We thank Prof. Stella Maris Pire and Dr. Konrrad Gajewsky for valuable comments on the manuscript and Eva Acevedo for technical assistance. References Anderson, E.F., 2001. The Cactus Family. Timber Press, Portland. Erdtman, G., 1960. The acetolysis method. Sven. Bot. Tidskr. 54 (4), 561–564.

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Erdtman, G., 1966. Pollen morphology and plant taxonomy. Angiosperms. Hafner Publ. Co, New York. Ferguson, D.J., Kiesling, R., 1997. Punna bonnieae (Cactaceae), a new species from Argentina. Cactus Succul. J. (U.S.) 69 (6), 283–287. Heusser, C.J., 1971. Pollen and spores of Chile. Modern Types of Pteridophyta, Gymnospermae and Angiospermae. The University of Arisona Press, Tucson. Kiesling, R., 1984. Estudios en Cactaceae de Argentina: Maihueniopsis, Tephrocactus y géneros afines (Opuntioideae). Darwiniana 25 (1–4), 171–215. Kiesling, R., 1999. In: Zuloaga, F.O., Morrone, O. (Eds.), Catálogo de las Plantas Vasculares de la República Argentina: Cactaceae, vol. 1. The Missouri Botanical Garden, EE UU, pp. 423–488. Kozar, F., 1974. Ultraestructure of pollen of Opuntia polyacantha. Can. J. Bot. 52, 313–315. Kurtz, E.B., 1948. Pollen grain character of certain Cactaceae. Bull. Torrey Bot. Club 75, 516–522. Kurtz, E.B., 1963. Pollen morphology of the Cactaceae. Grana Palynol. 4 (3), 367–372. Leuenberger, B., 1976a. Die pollen Morphologie der Cactaceae. Diss. Bot. 31, 1–321. Leuenberger, B., 1976b. Pollen morphology of the Cactaceae. Cactus Succul. J. G.B. 38 (4), 79–94. Mautino, L.R., 2002. Palinología de la localidad km 107; Formación San José, Mioceno Medio en la provincia de Tucumán (Argentina). VIII Congreso Argentino de Paleontología y Bioestratigrafía, Corrientes, Argentina. Nilsson, S., Praglowski, J. (Eds.), 1992. Erdtman′s Handbook of Palynology, 2da. ed. Munksgaard, Copenhagen. Nowicke, J.W., 1975. Pollen morphology in the Order Centrospermae. Grana Palynol. 15, 51–77. Nowicke, J.W., Skvarla, J.J., 1979. Pollen morphology: the potential influence in higher Order Systematics. Ann. Missouri Bot. Gar. 66 (4), 33–700. Punt, W., Blackmore, S., Nilsson, S., Le Thomas, A., 1994. Glossary of Pollen and Spore Terminology. LPP Fundation, University of Utrecht, The Netherlands. Ritter, F., 1980. Kakteen in Südamerikka II. Spangenberg. Saenz de Rivas, C., 1978. Polen y Esporas. H. Blume Ediciones, Madrid. 219 pp. Tsukada, M., 1964. Pollen morphology and identification. II. Cactaceae. Pollen Spores 6, 45–84. Walker, J.W., Doyle, J.A., 1975. The bases of angiosperm phylogeny: Palynology. Ann. Miss. Bot. Gard. 62 (3), 664–723.