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Spore morphology and wall ultrastructure of Anemia Swartz species (Anemiaceae) from Argentina
Review of Palaeobotany and Palynology 174 (2012) 27–38
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Research paper
Spore morphology and wall ultrastructure of Anemia Swartz species (Anemiaceae) from Argentina J.P. Ramos Giacosa a,⁎, 1, M.A. Morbelli a, 1, G.E. Giudice b a b
Cátedra de Palinología, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Paseo del Bosque s/n°, B1900FWA, La Plata, Argentina Cátedra de Morfología Vegetal, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Paseo del Bosque s/n°, B1900FWA, La Plata, Argentina
a r t i c l e
i n f o
Article history: Received 30 December 2011 Accepted 24 February 2012 Available online 3 March 2012 Keywords: Anemia Anemiaceae Spores Argentina Morphology Ultrastructure
a b s t r a c t Spores of Anemia species that grow in Argentina are studied. The country corresponds to the southern limit in the distribution of the genus where nine species have been reported: Anemia australis, A. herzogii, A. myriophylla, A. phyllitidis var. phyllitidis, A. phyllitidis var. tweedieana, A. simplicior, A. tomentosa var. anthriscifolia, A. tomentosa var. tomentosa and A. wettsteinii. The material was examined using light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope (TEM). The spores are trilete of 42–114 μm in equatorial diameter and 34–106 μm in polar diameter. The exospore is 1.2–7 μm thick, twolayered; and two different ornamentations are observed: narrow and parallel ridges bearing baculae separated by wide and smooth grooves or parallel wide ridges with several ornamentations separated by narrow and smooth grooves. The perispore is 0.2–1.1 μm thick, two-layered, and it may be plane or echinate with echina of variable sizes and shapes depending on the taxa analyzed. Perforations are also present on the perispore surface. The differences found in exospore and perispore morphology and ultrastructure could be valuable characters for systematic, phylogenetic and paleobotanical purposes. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The genus Anemia Swartz was included, together with Actinostachys, Lygodium, Mohria and Schizaea, within the family Schizaeaceae. This family is an old family with records from the Jurassic, and the living groups are undoubtedly the remnants of a long history of divergence and evolution (Tryon and Tryon, 1982). According to recent phylogenetic analysis based on morphological and molecular data (Smith et al., 2006) three families are accepted Lygodiaceae (Lygodium), Anemiaceae (Anemia and Mohria) and Schizaeaceae (Actinostachys and Schizaea). Mickel (1962, 1981) stated that Anemia is composed of three subgenera: Anemia, Anemiorrhiza and Coptophyllum. Nevertheless, phylogenetic analysis based on morphological and molecular data (Skog et al., 2002; Wikström et al., 2002), has shown that, only two subgenera could be established: subg. Anemiorrhiza and subg. Anemia (including subg. Coptophyllum). There are not enough morphological characters or molecular data to support the separation of the subg. Anemia and Coptophyllum (Skog et al., 2002). Anemia includes about 120 species, most of them in Latin America, ten in Africa and one in southern India. The genus is most abundant in
⁎ Corresponding author. E-mail address: [email protected] (J.P. Ramos Giacosa). 1 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). 0034-6667/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.revpalbo.2012.02.004
Brazil with a secondary center in Mexico (Mickel and Smith, 2004; Smith et al., 2006). Argentina corresponds to the southern limit in the distribution of the genus and nine taxa were reported, all of them included in the subg. Anemia (including subg. Coptophyllum): Anemia australis (Mickel) M. Kessler & A.R. Smith, A. herzogii Rosenstock, A. myriophylla H. Christ, A. phyllitidis (L.) Swartz var. phyllitidis, A. phyllitidis (L.) Sw. var. tweedieana (Hooker) Hassler, A. simplicior (H. Christ) Mickel, A. tomentosa (Savigny) Swartz var. anthriscifolia (Schrader) Mickel, A. tomentosa (Savigny) Sw. var. tomentosa y A. wettsteinii H. Christ (Zuloaga et al., 2008). Several studies deal with the spores of Anemia. Erdtman (1957) illustrated 3 types of ridges internal structure in Anemia spores and compared them with those in Mohria spores. Bolkhovitina (1959) analyzed and illustrated with drawings the spore morphology of the Schizaeaceae and compared the extant and fossil spores. Mickel (1962) studied with LM the spores of the American taxa of subg. Coptophyllum and compared its morphology with other genera within the Schizaeaceae. Erdtman and Sorsa (1971), described the spores of six species from Mexico, USA, Jamaica, Bolivia and Brazil. Lugardon (1974) analyzed the sporoderm ultrastructure of the spores of Anemia phyllitidis from a cultivated plant with TEM. Hill (1977) carried out SEM studies on the spores of some taxa of Anemia subg. Coptophyllum from Brazil. The same author (Hill, 1979) studied the spores of some taxa of Anemia subg. Anemia from Mexico and Brazil using SEM.
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Later, when Mickel (1982) studied twenty-one species and hybrids of Anemia from Mexico, analyzed the ploidal levels and mentioned that morphologically these ploidal races are indistinguishable, except perhaps, on the basis of stoma and spore sizes. Tryon and Tryon (1982) illustrated the spores of some species of Anemia from Brazil and Central America using SEM in a floristic study, and correlated the spore morphology with the systematics of the genus. Tryon and Lugardon (1991) carried out studies of some Anemia taxa that grow in America using SEM, without including species from Argentina. These authors also illustrated the sporoderm ultrastructure of Anemia phyllitidis spores from a cultivated plant. Dettman and Clifford (1991) studied the spores of some species from America and Africa by LM, SEM and TEM. The TEM analysis only included spores of Anemia flexuosa and A. dregeana from Brazil and South Africa respectively. In a comparative study of fossil and extant spores of Schizaeaceae, Van Konijnenburg-van Cittert (1991) recognized evolutionary trends in the different genera of the family. Only a few studies analyzed the spores of the taxa that grow in Argentina. When de la Sota and Mickel (1968) studied the species of Anemia from Argentina in a systematic context, they mentioned some palynological characters as primitive or derivate. Morbelli (1981) carried out preliminary studies of the Anemia spores from northwest Argentina using LM and SEM, and later on, de la Sota and Morbelli (1987) in a comparative analysis of selected aspects of the Schizaeales, illustrated the spores of Anemia australis and A. herzogii by SEM. Skog et al. (2002) carried out a phylogenetic analysis of Anemia using morphological and molecular characters and highlighted the importance of the morphological characters of the spores in helping delimit taxa. These authors also mentioned the need to investigate more species of Anemia and suggested that more morphological characters should be included. The data previously mentioned reveal that the spores of most of the species that grow in Argentina were not analyzed or illustrated using SEM. Additionally, no species of Anemia from Argentina were analyzed with TEM thus, the sporoderm ultrastructure remains still unknown. The aim of the present study is to analyze the spores of Anemia species from Argentina by using LM, SEM and TEM in order to contribute to the knowledge of their morphology and their wall ultrastructure and to give characters that could be useful for future systematic or phylogenetic purposes.
2. Material and methods Spores were obtained from herbarium specimens from the following Argentinean Institutions: Instituto de Botánica del Nordeste (CTES), Instituto Miguel Lillo, Tucumán (LIL), División Plantas Vasculares, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata (LP) and Instituto de Botánica Darwinion (SI). The spores were studied using LM, SEM and TEM. For LM the spores were treated with hot 3% sodium carbonate for 2 min and acetolyzed according to the method of Erdtman (1960). For SEM the material was treated with hot 3% sodium carbonate, washed, dehydrated, suspended in 96% ethanol and then transferred to acetate plates. After drying they were coated with gold. For TEM dry material from herbarium specimens was hydrated following the technique proposed by Rowley and Nilsson (1972) that consists of the use of phosphate buffer and Alcian Blue (AB), then the material was fixed with glutaraldehyde + 1% Alcian Blue in phosphate buffer for 12 h and post-fixed with 1% OsO4 in water plus 1% Alcian Blue. The spores were dehydrated in an acetone series and then embedded in Spurr soft mixture. Sections 3 μm thick were stained with toluidine blue and analyzed with LM. Ultra-thin sections were stained with 1% uranyl acetate for 15 min followed by lead citrate for 3 min.
The observations were performed with Olympus BH2 and BHB LMs, a JEOL JSMT-100 SEM and a Zeiss T-109 TEM. After the LM and SEM analysis, Anemia australis, A. herzogii and A. simplicior were selected as representative for the study with TEM. 2.1. Material studied Anemia australis: ARGENTINA: CATAMARCA, Andalgalá, Yunkasuma Río Chacra, Sleumer 35 (LIL); JUJUY, Capital, Sierra de Zapla, Cabrera et al. 23656 (LP); Capital, Termas de Reyes, Abbiatti & Claps 802 (LP); SALTA, Metán, Dinelli 4483 (LIL); SAN LUIS, Junín, Merlo, Reserva El Tabaquillo, Giudice, Luna & Ramos Giacosa 294 (LP); TUCUMAN, Trancas, San Pedro de Colalao, Cristóbal 162 (LIL). Anemia herzogii: ARGENTINA: JUJUY, Ledesma, Parque Nacional Calilegua, Camino a Valle Grande, Ramos Giacosa 213 (LP); SALTA, General San Martín, Arroyo Tres Quebradas, Krapovickas et al. 19497 (LP). A. myriophylla: ARGENTINA: SALTA, Santa Victoria, Sota 4156 (LP) A. phyllitidis var. phyllitidis: ARGENTINA: JUJUY, Valle Grande, camino a Valle Grande, Mesada de las Colmenas, de la Sota 4482 (LP), MISIONES, Cainguás, Reserva Cuña Pirú, Biganzoli et al. 1373 (LP). A. phyllitidis var. tweedieana: ARGENTINA: BUENOS AIRES, Isla Martín García, Hurrell et al. 2553 (LP); MISIONES, Leandro Alem, Paso Caneta, Krapovickas et al. 15.005 (LP). A. simplicior: MISIONES, General Belgrano, Camino El Dorado a Bernardo de Irigoyen, Katinas 49 (LP); Marquez 180 (LP). A. tomentosa var. anthriscifolia: CATAMARCA, Schunck 9706 (LIL); JUJUY, El Carmen, Dique La Ciénaga, Cabrera et al. 14089 (LP); MISIONES, Cainguás, Puerto Rico, Montes 3993 (LP); SALTA, Cafayate, Santa Teresa, Lourteig 1041 (LIL); Capital, La Lagunilla, Saravia Toledo 2152 (LP); TUCUMAN, Vipos, Dinelli 832 (SI). A. tomentosa var. tomentosa: BUENOS AIRES, Tornquist, Sierra de la Ventana, Reserva Integral “La Blanqueada”, Proyecto Ventania 15 (LP); Tandil, Cerro de las Animas, Fabris & Schwabe 4750 (LP); La Cascada, Cabrera & Torres 17.816 (LP); MISIONES, San Javier, Cabrera et al. 322 (LP). A. wettsteinii: MISIONES, Cataratas del Iguazú, Capurro s/n° BA 55286 (LP); El Dorado, R. P. 17, paraje Cerro 60, Keller 3095 (CTES). 3. Results 3.1. Morphology The palynological characteristics of the analyzed taxa are summarized in Tables 1 and 2. The spores are trilete. The spore shape in equatorial view is convex-hemispheric. They are triangular with straight or convex sides in polar view. The equatorial diameter is 42–114 μm and the polar diameter is 34–106 μm. The laesura is 16–50 μm long. The spores of A. herzogii, A. phyllitidis and A. phyllitidis var. tweediana are triangular with convex sides and rounded angles. The ornamentation is constituted of narrow and parallel ridges of 0.7–2 μm wide. On the ridges, large bacula of 2–7 μm high with rounded apices are seen. Smooth grooves of 3.9–7.9 μm wide separate the ridges (Plate I, 1–4; Plate II). The spores of A. wettsteinii (Plate I, 5–8) are similar to the previous taxa. The differences are basically that the spores of A. wettsteinii have fewer bacula and they are smaller than in the other species. The spores of Anemia australis, A. tomentosa var. anthriscifolia, A. tomentosa var. tomentosa, A. myriophylla and A. simplicior (Plates III, IV, V) are triangular with straight or convex sides and prominent angles. The ornamentation is composed of parallel ridges of 3.3–7 μm wide which are separated by narrow and smooth grooves of 0.5–3.7 μm wide. The ridges are fused near the angles of the spores and they form prominent and thickened angles. On the ridge surfaces spines of variable size and shape are observed, depending on the taxa analyzed. Abundant perforations of variable sizes are also present on the perispore surface.
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Table 1 Spore morphological data for Anemia species from Argentina. Dimensions in μm. Taxa
Shape in polar view
Equatorial diameter
Polar diameter
Laesurae length
A. A. A. A. A. A. A. A. A.
Triangular Triangular Triangular Triangular Triangular Triangular Triangular Triangular Triangular
76–91 52–68 72–88 42–55 51–62 78–89 65–84 86–114 66–82
72–83 39–52 59–67 34–49 41–56 59–65 55–72 72–106 57–72
31–41 22–31 23–31 20–27 16–25 27–34 31–40 36–50 25–41
australis herzogii myriophylla phyllitidis var. phyllitidis phyllitidis var. tweediana simplicior tomentosa var. anthriscifolia tomentosa var. tomentosa wettsteinii
with with with with with with with with with
convex sides. Prominent angles convex sides. Rounded angles convex sides. Prominent angles convex sides. Rounded angles convex sides. Rounded angles straight sides. Prominent angles convex sides. Prominent angles convex sides. Prominent angles convex sides. Rounded angles
In A. australis (Plate III) the spines are low, 0.2–0.4 μm high with truncated or rounded apices. In superficial view, these elements could be wrongly defined as granules. In A. tomentosa var. tomentosa and A. tomentosa var. anthriscifolia (Plate V) the spines are medium high, 0.9–2.2 μm, with acute or rounded apices which are frequently ramified. The ornamentation of the ridge surfaces of A. myriophylla (Plate IV, 1–6) has intermediate characters between A. australis and A. tomentosa since some spines are low with rounded apices while others are high with acute apices; these apices may be ramified or not and similar to those observed in A. tomentosa. In A. simplicior (Plate IV, 7–11) the spines are higher than in the previous species, 1.5–4.5 μm high, filiform with acute apices, and they are frequently ramified and interwoven. Some abnormalities like aborted, immature or spores join in tetrads were observed in all the specimens of Anemia tomentosa var. antrhiscifolia and A. tomentosa var. tomentosa. The same ornamentation that appears on the ridges (i.e. bacula, spines, and perforations) was observed on the laesura surfaces in the nine species analyzed. 3.2. Ultrastructure According to the structural characteristics and ornamentation observed by LM and SEM, Anemia australis, A. herzogii and A. simplicior were selected to be studied by TEM because they summarize the major variations. Two types of sporoderm ultrastructure were observed: 1) In A. herzogii the exospore between the bacula, is 1.2–1.4 μm thick and two-layered showing an inner layer of 60–110 μm thick and an outer layer of 1.2–1.4 μm thick. The outer exospore layer forms the ornamentation of the spores which consists of ridges and bacula. The bacula have wide bases and a rounded apex (Plate VI, 2).
The perispore is 0.5–1.1 μm thick and is composed of two layers: an inner layer (P1) of 50–100 nm thick that is adhered to the exospore and an outer layer (P2) 0.4–1 μm thick with interwoven threads (Plate VI, 1–3). In the bacula apex, the perispore is thicker. In this region, the outer perispore layer has a higher density of threads which are arranged in a lax structure toward the outside of the apex (Plate VI, 2, 4, 5). Spheroids with an inner portion which has the same structure and contrast as the exospore, and an outer layer with the same structure as the perispore, can be observed on the perispore surface (Plate VI, 6). Several small portions of membranes (scales) are observed as immersed within the perispore and below the spheroids (Plate VI, 4–6). 2) In A. australis and A. simplicior the exospore is 2–7 μm thick and is composed of two layers: an inner layer 0.9–1.3 μm thick and an outer layer 1.3–6 μm thick that forms the ornamentation of the spore constituted by ridges. In the outer layer a big amount of cavities with irregular shapes and sizes are evident. These cavities are mainly situated inside the ridges and just a few of them lie between the ridges (Plate VIII, 1). They have a differentiated edge (Plate VIII, 5). Radial channels with contrasted content are mainly observed in the inner exospore layer (Plate VIII, 1). The perispore is 0.2–1 μm thick and has two layers: P1 and P2. The inner layer (P1) is made up of 3 strata: the inner stratum is thin, 50–100 nm thick and is adhered to the exospore. The middle stratum is 60–200 nm thick, has areas of concentrated material and other regions with cavities. This stratum has a laxer structure than the other strata. It is an area of weakness that contributes to the perispore detachment. The outer stratum is 50–100 nm thick (Plate VII, 1–4; Plate VIII 1–4). The outer layer (P2) is 200–600 nm thick, less contrasted than P1 layer and covers the outer surface of P1 layer. P2 layer forms the micro-ornamentation of the spore that consists of spines of variable sizes and shapes as it was described in the Morphology section. (Plate VII, 1–4; Plate VIII, 1–3).
Table 2 Ornamentation and dimensions of ornamental elements in Anemia species from Argentina. Dimensions in μm. Taxa
Exospore ornamentation
Perispore ornamentation
Ridge wide
Grooves width
Bacula/spines height
A. australis
Parallel ridges separated by narrow and smooth grooves. Narrow, parallel ridges with high bacula separated by wide grooves. Parallel ridges separated by narrow and smooth grooves. Narrow and parallel ridges with high baculae separated by wide grooves. Narrow and parallel ridges with high baculae separated by wide grooves. Parallel ridges separated by narrow and smooth grooves. Parallel ridges separated by narrow and smooth grooves Parallel ridges separated by narrow and smooth grooves Narrow and parallel ridges with low baculae separated by wide grooves.
Small spines with truncated or rounded apices. With perforations. Laevigate. Without perforations.
4.2–6.9
0.5–2
0.2–0.4
0.7–1.5
5.0–6.8
2.8–4.6
Spines or spinules with rounded or acute apices. Some ramified. With perforations Laevigate. Without perforations.
4.9–6.3
0.7–2
1.1–1.8
4.3–7.9
2.6–6.7
Laevigate. Without perforations.
1.5–2
3.9–7.8
3.2–8.3
High filiform spines with acute apices, ramified. With perforations. Medium height spines, acute or rounded apices, frequently ramified. Medium height spines, acute or rounded apices, frequently ramified. Laevigate. Without perforations.
3.3–4.8
2.1–3.7
1.5–4.5
3.3–6
0.8–1.8
1.1–2.2
4.6–7
0.8–2.3
0.9–1.8
1.5–2.3
5.1–8.3
1.6–3.7
A. herzogii A. myriophylla A. phyllitidis var. phyllitidis A. phyllitidis var. tweediana A. simplicior A. tomentosa var. anthriscifolia A. tomentosa var. tomentosa A. wettsteinii
1–2.7
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Plate I. Spores of Anemia herzogii and A. wettsteinii with SEM. 1–4. 1. 2. 3. 4. 5–8. 5. 6. 7. 8.
A. herzogii. Proximal view of a spore. The laesurae have the same ornamentation as the ridge surface. Scale bar = 10 μm. Distal view of a triangular spore with convex sides. Scale bar = 10 μm. Equatorial view of a spore. Scale bar = 10 μm. Detail of the distal surface. Narrow and parallel ridges with high baculae separated by wide grooves are observed. The baculae have rounded apices. Scale bar = 5 μm. A. wettsteinii. Proximal view of a spore. Scale bar = 10 μm. Distal view of a triangular spore with convex sides. The ornamentation is composed of narrow and parallel ridges with low baculae separated by wide grooves. Scale bar = 10 μm. Equatorial view of a spore. Scale bar = 10 μm. The baculae are small and has rounded apices. Scale bar = 5 μm.
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Plate II. Spores of Anemia phyllitidis var. phyllitidis and Anemia phyllitidis var. tweediana with SEM. 1–4. 1. 2. 3. 4. 5–8. 5. 6. 7. 8.
A. phyllitidis var. phyllitidis. Proximal view of a triangular spore with convex sides. Scale bar = 10 μm. Distal view of a triangular spore with convex sides. Scale bar = 10 μm. Equatorial view of a spore. Scale bar = 10 μm. Detail of the ornamentation of the spore composed of narrow and parallel ridges with large baculae separated by wide depressions. Scale bar = 5 μm. A. phyllitidis var. tweediana. Proximal view of a triangular spore with convex sides. The laesurae has the same ornamentation as the surface of the ridges. Scale bar = 10 μm. Distal view of a spore. The ornamentation is composed of narrow and parallel ridges with large baculae separated by wide depressions Scale bar = 10 μm. Equatorial view of a convex-hemispheric spore. Scale bar = 10 μm. Detail of the ornamentation. Large baculae with rounded apices are observed. Scale bar = 5 μm.
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Plate III. Spores of Anemia australis with SEM. 1. 2. 3. 4–5.
Proximal view of a triangular spore with convex sides. The angles of the spore are prominent. Scale bar = 10 μm. Distal view of a spore. The ornamentation consists of wide parallel ridges separated by narrow depressions. Scale bar = 10 μm. Equatorial view of a convex-hemispheric spore. Few ridges are dichotomized (arrows). Scale bar = 10 μm. Detail of the perispore surface. The micro-ornamentation is only visible on the ridges and is made up of small spines with rounded apices. Some perforations are also visible (arrowheads). The laesurae (stars) has the same ornamentation as the surfaces of the ridges. Scale bars = 5 μm.
4. Discussion and conclusions According to the observations made in this paper, there exist relevant variations in spore diameter (42–114 μm in equatorial diameter) which coincide with the ones described by Mickel (1962), and may be related to the different ploidal levels found in the genus Anemia (Mickel, 1962, 1982). He also explains that there is a relationship between the size and number of spores per sporangium, including intra-specific variations.
As regards spore morphology, two different morphological types are observed among the taxa analyzed: 1) the Anemia herzogii type (A. herzogii, A. phyllitidis, A. phyllitidis var. tweediana and A. wettsteinii) which shows narrow and parallel ridges with baculae separated by smooth grooves and 2) the A. australis type (A. australis, A. tomentosa var. anthriscifolia, A. tomentosa var. tomentosa, A. myriophylla and A. simplicior) with wide parallel ridges separated by narrow and smooth grooves with prominent and rounded angles. Within this type, variations may be found depending on the different kinds of perispore ornamentation;
Plate IV. Spores of Anemia myriophylla and A. simplicior with SEM. 1–6. 1. 2. 3. 4–6.
7–11. 7. 8. 9. 10. 11.
A. myriophylla. Proximal view of a triangular spore with convex sides. The angles of the spore are prominent. Scale bar = 10 μm. Distal view of a spore. The ornamentation consists of wide parallel ridges separated by narrow depressions. Scale bar = 10 μm. Equatorial view of a convex-hemispheric spore. Scale bar = 10 μm. Detail of the perispore surface. The micro-ornamentation is only visible on the ridges and is composed of some small spines with rounded apices (white arrows) and others which are higher and may be ramified or not (black arrows). The laesurae (star) has the same ornamentation as the surfaces of the ridges. Scale bars = 5 μm. A. simplicior. Proximal view of a triangular spore with straight sides. The angles of the spore are prominent. Scale bar = 10 μm. Distal view of a spore. Parallel ridges separated by narrow and smooth depressed zones are observed. Scale bar = 10 μm. Equatorial view of a convex-hemispheric spore. Scale bar = 10 μm. The micro-ornamentation is only visible on the ridges and consists of abundant large spines. Scale bar = 5 μm. Detail of the perispore surface. The spines are filiforms with acute apices and are frequently ramified and interwoven. Some perforations are visible on the surface (arrowheads). Scale bar = 2 μm.
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spines can go from being small with rounded apices to filiform, ramified, high and interwoven. Besides, abundant perforations are evident in the perispore of all the taxa belonging to this group. The studied taxa of Anemia herzogii type share a homogenous morphology since their spores do not present relevant inter-specific variations. Within this group, only spores of A. wettsteinii are different
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from the rest in the sense that the baculae on their ridges are lower than in the other analyzed taxa. In addition to this, spore diameter is greater than in the other taxa from this group. On the contrary, spores of Anemia australis present variations regarding the perispore ornamentation. Besides, the species belonging to this group have significant variations in spore size, and
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Plate V. Spores of Anemia tomentosa var. anthriscifolia and A. tomentosa var. tomentosa with SEM. 1–5. 1. 2. 3. 4–5. 6–10. 6. 7. 8. 9–10.
Anemia tomentosa var. anthriscifolia. Proximal view of a triangular spore with convex sides. The angles of the spore are prominent. Scale bar = 20 μm. Distal view of a spore. Parallels ridges separated by narrow and smooth depressed zones are observed. Scale bar = 20 μm. Equatorial view of a convex-hemispheric spore. Scale bar = 20 μm. The ornamentation of the perispore is only visible on the ridges and consists of medium-high spines, with acute or rounded apices, they are frequently ramified. The laesurae (star) have the same ornamentation as the ridge surfaces. Some perforations are also evident (arrowheads). Scale bars = 5 μm. A. tomentosa var. tomentosa. Proximal view of a triangular spore with convex sides. The angles of the spore are prominent. Scale bar = 20 μm. Distal view of a spore. Scale bar = 20 μm. Equatorial view of a convex-hemispheric spore. Parallels ridges separated by narrow and smooth depressed zones are observed. Scale bar = 20 μm. Detail of the perispore surface. On the ridges, medium-high spines, with acute or rounded apices are observed. They are frequently ramified. Scale bars 9 = 2.5 μm, 10 = 1 μm.
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Plate VI. TEM images of Anemia herzogii spores. 1. 2. 3. 4–5. 6.
Section through a laesura. The exospore consists of two layers. The inner exospore layer (arrow) is thin and more contrasted than the outer exospore layer (Eo). Scale bar = 1 μm. Section through the sporoderm. The outer exospore layer (Eo) forms the bacula which has a wide base and a rounded apex. On the exospore, the perispore is evident (arrows). Scale bar = 1 μm. Section through the perispore between the baculae. The perispore is two-layered: a thin inner layer P1 (arrow) which is adhered to the exospore (E) and the outer layer P2. Scale bar = 0.25 μm. Detail of the bacula apex. In this region, the perispore outer layer is thicker and the threads are laxly distributed toward the apex. Few scales (arrowheads) are seen on the perispore. Scale bars 4 = 0.5 μm, 5 = 0.25 μm. A spheroid (s) within the perispore are observed. It has a core with the same structure and contrast as the exospore and an outer layer is similar in contrast and structure to the perispore. Scales are seen below the spheroid (arrowhead). Scale bar = 0.25 μm.
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Plate VII. TEM images of Anemia australis spores. 1. 2–4.
Section through the sporoderm. The exospore is two-layered. The inner layer (Ei) has numerous radial channels with contrasted content (arrowhead). The outer exospore layer (Eo) forms the ornamentation which consists of ridges (R). This layer has cavities with irregular shape and size (arrows). Scale bar = 1 μm. Section through the sporoderm. The perispore is two-layered P1 layer has 3 strata: the inner stratum (arrowhead) is adhered to the exospore (E), the middle stratum (P1m) has areas of concentrated material and other regions with cavities and the outer stratum (arrows) is continuous. P2 layer is less contrasted than P1 and forms the perispore micro-ornamentation consisting of spines (double arrows) and low elevations of variable sizes. Scale bars = 0.5 μm.
some taxa even have different levels of abnormalities. The irregularities found in the spores of A tomentosa var. tomentosa were also highlighted by Mickel (1962) and de la Sota and Mickel (1968) when they mentioned spores presenting irregular dimensions and shapes, generally aborted. The explanation for these irregularities may derive from the fact that they are hexaploid with apogamic reproduction. At ultrastructural level, there are also differences worth mentioning regarding the exospore and perispore of the groups mentioned. In the case of A. herzogii, the exospore forms the ornamentation, consisting of baculae. The perispore consists of two strata with a smooth outer surface and frequently presenting scales basally. Lugardon (1974) also observed these scales in the perispore of Anemia phyllitidis, and they were also mentioned among the spores of other families such as Blechnaceae (Ramos Giacosa et al., 2009) and Polypodiaceae (Morbelli and Giudice, 2010). However, both the exospore and the perispore of A. australis have ornamentation. The exospore forms the macro-ornamentation and it consists of ridges which internally have a structure full of cavities.
On the other hand, the perispore presents two layers: P1 layer consisting of 3 strata, and P2 layer. In the outer surface a microornamentation consisting of simple or ramified spines of variable dimensions are observed. The ultrastructural characteristics of the A. herzogii spores analyzed in this study go along with the description of A. phyllitidis made by Lugardon (1974). Besides, the presence of cavities in the inner perispore was previously mentioned by Erdtman (1957) and by Mickel (1962), as well as illustrated for Anemia madagascarensis and A. simii by Tryon and Lugardon (1991) and for A. flexuosa by Dettman and Clifford (1991). From the data collected in this study it can be noted that both exospore and perispore share variations not only as regards morphology but also as regards ultrastructure. In this sense, the exospore is useful in order to differentiate the two groups while the perispore allows us to differentiate taxa within the A. australis group. At the same time, the two morphological spore groups presented in this study go along with the subgenus that Mickel (1962)
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37
Plate VIII. TEM images of Anemia simplicior spores. 1. 2–4.
5.
Section through the sporoderm. The exospore is two-layered: the inner layer (Ei) and the outer layer (Eo) forms the ridges (R). Numerous cavities with irregular shape and size are evident in the outer exospore layer (arrow). Few radial channels with contrasted content are observed in the exospore (arrowheads). Scale bar: 1 μm. Section through the outer exospore and perispore. The perispore is two-layered: P1 and P2. P1 layer has 3 strata: the inner stratum (arrowhead) shows has a high contrast and is adhered to the exospore (E), the middle stratum (P1m) has areas of concentrated material and other regions with cavities, and the outer stratum (arrow) is continuous and, in some cases, difficult to differentiate. P2 layer forms the spines (double arrow) that constitute the micro-ornamentation of the perispore. Scale bars: 0.5 μm. Detail of one exospore outer layer cavity. It has ellipsoidal shape and a differentiated edge (arrow). Scale bar: 0.5 μm.
suggested: subg. Anemia for the A. herzogii, and subg. Coptophyllum for the A. australis group. The spores of Anemia have a great taxonomic value provided not only by the ornamentation of the spores observed with LM and SEM but also for the rich amount of information that the wall ultrastructure provides. These characters are particularly valuable in the A. australis group (previously known as subg. Coptophyllum) whose spores present the most outstanding variations. Moreover, another relevant feature to take into account in the determination of these taxa is the presence of abnormal or aborted spores. The palynological data dealt with here add information of great taxonomic value; some of the taxa presented had not been examined
before in this region. What is more, these morphological characters may be of value in the determination of the taxa and their incorporation into future phylogenetic studies applied to this group. Finally, it is worth mentioning the need to further carry on ultrastructural studies on other species within the genus in order to include this valuable information in the phylogenetic studies. Acknowledgments The authors thanks to Lic. Rafael Urrejola from the SEM Unit, Museo de Ciencias Naturales de La Plata, Lisandro Anton, from the TEM Unit, Instituto de Biología Celular, Facultad de Medicina, Universidad de Buenos Aires and the institutions that very kindly sent the
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herbarium material. This work was supported by grants from the Universidad Nacional de La Plata (project 584; Subs. Jov. Invest. 08, 09, 10) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) for project PICT 0661. References Bolkhovitina, I.A., 1959. The morphology of the spores of the family Schizaeaceae and the history of the family in the geological past. Journal of Paleontology 1, 121–131. de la Sota, E.R., Mickel, J.T., 1968. Sinopsis de las especies argentinas del género Anemia Swartz (Schizaeaceae). Revista Museo de La Plata (nueva serie), Sección Botánica 11, 133–152. de la Sota, E.R., Morbelli, M.A., 1987. Schizaeales. Phytomorphology 37 (4), 365–393. Dettman, M.E., Clifford, H.T., 1991. Spore morphology of Anemia, Mohria and Ceratopteris (Filicales). American Journal of Botany 78 (3), 303–325. Erdtman, G., 1957. Pollen and Spore Morphology, Plant Taxonomy; Gymnospermae, Pteridophyta, Briophyta. Almqvist and Wiksells, Stockholm. Erdtman, G., 1960. The acetolysis method. A revised description. Svensk Botanisk Tidskrift 54, 561–564. Erdtman, G., Sorsa, P., 1971. Pollen and Spore Morphology and Plant Taxonomy. Pteridophyta. Almqvist & Wiksell, Stockholm. Hill, S.R., 1977. Spore morphology of Anemia subgenus Coptophyllum. American Fern Journal 67 (1), 11–17. Hill, S.R., 1979. Spore morphology of Anemia subgenus Anemia. American Fern Journal 69 (3), 71–79. Lugardon, B., 1974. La estructure fine de l'exospore et de la perispore des Filicinées isosporées. II. Filicales. Commentaires. Pollen Spores 16 (2), 161–226. Mickel, J.T., 1962. A monographic study of the fern genus Anemia, subgenus Coptophyllum. Iowa State Journal of Science 36, 349–482. Mickel, J.T., 1981. Revision of Anemia subgenus Anemiorrhiza (Schizaeaceae). Brittonia 33, 413–429.
Mickel, J.T., 1982. The genus Anemia (Schizaeaceae) in Mexico. Brittonia 34 (4), 388–413. Mickel, J.T., Smith, A.R., 2004. The pteridophytes of Mexico. Memoirs of the New York Botanical Garden 88, 48–60. Morbelli, M.A., 1981. Estudio de esporas de las Pteridofitas del Noroeste de Argentina. Ophioglossaceae, Schizaeaceae y Cyatheaceae. XVIII Jornadas Argentinas de Botánica. San Miguel de Tucumán. Libro de Resúmenes, p. 79. Morbelli, M.A., Giudice, G.E., 2010. Spore wall ultrastructure of Polypodiaceae from north-western Argentina. Grana 49, 204–214. Ramos Giacosa, J.P., Morbelli, M.A., Giudice, G.E., 2009. Spore morphology and wall ultrastructure of Blechnum L. species from North West Argentina. Review of Palaeobotany and Palynology 156, 185–197. Rowley, J.R., Nilsson, S., 1972. Structural stabilization for electron microscopy of pollen from herbarium specimens. Grana 12, 23–30. Skog, J.E., Zimmer, E.A., Mickel, J.T., 2002. Additional support for two subgenera of Anemia (Schizaeaceae) from data for the chloroplast intergeneric spacer region trnL-F and morphology. American Fern Journal 92 (2), 119–130. Smith, A.R., Pryer, K.M., Schuettpelz, E., Korall, P., Schneider, H., Wolf, P.G., 2006. A classification for extant ferns. Taxon 55 (3), 705–731. Tryon, A.F., Lugardon, B., 1991. Spores of Pteridophyta. Springer-Verlag, New York. Tryon, R.M., Tryon, A.F., 1982. Fern and allied plants with special reference to tropical America. Springer Verlag, Berlin, Heidelberg, New York. Van Konijnenburg-van Cittert, J.H.A., 1991. Diversification of spores in fossil and extant Schizaeaceae. In: Blackmore, S., Barnes, S.H. (Eds.), Pollen and Spores, Patterns of Diversification: Publ. Syst. Assoc., spec., vol. 44, pp. 103–118. Wikström, N., Kenrick, P., Vogel, J.C., 2002. Schizaeaceae: a phylogenetic approach. Review of Palaeobotany and Palynology 119, 35–50. Zuloaga, F.O., Morrone, O., Belgrano, M. (Eds.), 2008. Catálogo de las plantas vasculares del Cono Sur: Pteridophyta, Gymnospermae y Monocotyledonae. Monogr. Syst.Bot. Missouri Bot. Gard., Vol.1, p. 107.
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Research paper
Spore morphology and wall ultrastructure of Anemia Swartz species (Anemiaceae) from Argentina J.P. Ramos Giacosa a,⁎, 1, M.A. Morbelli a, 1, G.E. Giudice b a b
Cátedra de Palinología, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Paseo del Bosque s/n°, B1900FWA, La Plata, Argentina Cátedra de Morfología Vegetal, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Paseo del Bosque s/n°, B1900FWA, La Plata, Argentina
a r t i c l e
i n f o
Article history: Received 30 December 2011 Accepted 24 February 2012 Available online 3 March 2012 Keywords: Anemia Anemiaceae Spores Argentina Morphology Ultrastructure
a b s t r a c t Spores of Anemia species that grow in Argentina are studied. The country corresponds to the southern limit in the distribution of the genus where nine species have been reported: Anemia australis, A. herzogii, A. myriophylla, A. phyllitidis var. phyllitidis, A. phyllitidis var. tweedieana, A. simplicior, A. tomentosa var. anthriscifolia, A. tomentosa var. tomentosa and A. wettsteinii. The material was examined using light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope (TEM). The spores are trilete of 42–114 μm in equatorial diameter and 34–106 μm in polar diameter. The exospore is 1.2–7 μm thick, twolayered; and two different ornamentations are observed: narrow and parallel ridges bearing baculae separated by wide and smooth grooves or parallel wide ridges with several ornamentations separated by narrow and smooth grooves. The perispore is 0.2–1.1 μm thick, two-layered, and it may be plane or echinate with echina of variable sizes and shapes depending on the taxa analyzed. Perforations are also present on the perispore surface. The differences found in exospore and perispore morphology and ultrastructure could be valuable characters for systematic, phylogenetic and paleobotanical purposes. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The genus Anemia Swartz was included, together with Actinostachys, Lygodium, Mohria and Schizaea, within the family Schizaeaceae. This family is an old family with records from the Jurassic, and the living groups are undoubtedly the remnants of a long history of divergence and evolution (Tryon and Tryon, 1982). According to recent phylogenetic analysis based on morphological and molecular data (Smith et al., 2006) three families are accepted Lygodiaceae (Lygodium), Anemiaceae (Anemia and Mohria) and Schizaeaceae (Actinostachys and Schizaea). Mickel (1962, 1981) stated that Anemia is composed of three subgenera: Anemia, Anemiorrhiza and Coptophyllum. Nevertheless, phylogenetic analysis based on morphological and molecular data (Skog et al., 2002; Wikström et al., 2002), has shown that, only two subgenera could be established: subg. Anemiorrhiza and subg. Anemia (including subg. Coptophyllum). There are not enough morphological characters or molecular data to support the separation of the subg. Anemia and Coptophyllum (Skog et al., 2002). Anemia includes about 120 species, most of them in Latin America, ten in Africa and one in southern India. The genus is most abundant in
⁎ Corresponding author. E-mail address: [email protected] (J.P. Ramos Giacosa). 1 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). 0034-6667/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.revpalbo.2012.02.004
Brazil with a secondary center in Mexico (Mickel and Smith, 2004; Smith et al., 2006). Argentina corresponds to the southern limit in the distribution of the genus and nine taxa were reported, all of them included in the subg. Anemia (including subg. Coptophyllum): Anemia australis (Mickel) M. Kessler & A.R. Smith, A. herzogii Rosenstock, A. myriophylla H. Christ, A. phyllitidis (L.) Swartz var. phyllitidis, A. phyllitidis (L.) Sw. var. tweedieana (Hooker) Hassler, A. simplicior (H. Christ) Mickel, A. tomentosa (Savigny) Swartz var. anthriscifolia (Schrader) Mickel, A. tomentosa (Savigny) Sw. var. tomentosa y A. wettsteinii H. Christ (Zuloaga et al., 2008). Several studies deal with the spores of Anemia. Erdtman (1957) illustrated 3 types of ridges internal structure in Anemia spores and compared them with those in Mohria spores. Bolkhovitina (1959) analyzed and illustrated with drawings the spore morphology of the Schizaeaceae and compared the extant and fossil spores. Mickel (1962) studied with LM the spores of the American taxa of subg. Coptophyllum and compared its morphology with other genera within the Schizaeaceae. Erdtman and Sorsa (1971), described the spores of six species from Mexico, USA, Jamaica, Bolivia and Brazil. Lugardon (1974) analyzed the sporoderm ultrastructure of the spores of Anemia phyllitidis from a cultivated plant with TEM. Hill (1977) carried out SEM studies on the spores of some taxa of Anemia subg. Coptophyllum from Brazil. The same author (Hill, 1979) studied the spores of some taxa of Anemia subg. Anemia from Mexico and Brazil using SEM.
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Later, when Mickel (1982) studied twenty-one species and hybrids of Anemia from Mexico, analyzed the ploidal levels and mentioned that morphologically these ploidal races are indistinguishable, except perhaps, on the basis of stoma and spore sizes. Tryon and Tryon (1982) illustrated the spores of some species of Anemia from Brazil and Central America using SEM in a floristic study, and correlated the spore morphology with the systematics of the genus. Tryon and Lugardon (1991) carried out studies of some Anemia taxa that grow in America using SEM, without including species from Argentina. These authors also illustrated the sporoderm ultrastructure of Anemia phyllitidis spores from a cultivated plant. Dettman and Clifford (1991) studied the spores of some species from America and Africa by LM, SEM and TEM. The TEM analysis only included spores of Anemia flexuosa and A. dregeana from Brazil and South Africa respectively. In a comparative study of fossil and extant spores of Schizaeaceae, Van Konijnenburg-van Cittert (1991) recognized evolutionary trends in the different genera of the family. Only a few studies analyzed the spores of the taxa that grow in Argentina. When de la Sota and Mickel (1968) studied the species of Anemia from Argentina in a systematic context, they mentioned some palynological characters as primitive or derivate. Morbelli (1981) carried out preliminary studies of the Anemia spores from northwest Argentina using LM and SEM, and later on, de la Sota and Morbelli (1987) in a comparative analysis of selected aspects of the Schizaeales, illustrated the spores of Anemia australis and A. herzogii by SEM. Skog et al. (2002) carried out a phylogenetic analysis of Anemia using morphological and molecular characters and highlighted the importance of the morphological characters of the spores in helping delimit taxa. These authors also mentioned the need to investigate more species of Anemia and suggested that more morphological characters should be included. The data previously mentioned reveal that the spores of most of the species that grow in Argentina were not analyzed or illustrated using SEM. Additionally, no species of Anemia from Argentina were analyzed with TEM thus, the sporoderm ultrastructure remains still unknown. The aim of the present study is to analyze the spores of Anemia species from Argentina by using LM, SEM and TEM in order to contribute to the knowledge of their morphology and their wall ultrastructure and to give characters that could be useful for future systematic or phylogenetic purposes.
2. Material and methods Spores were obtained from herbarium specimens from the following Argentinean Institutions: Instituto de Botánica del Nordeste (CTES), Instituto Miguel Lillo, Tucumán (LIL), División Plantas Vasculares, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata (LP) and Instituto de Botánica Darwinion (SI). The spores were studied using LM, SEM and TEM. For LM the spores were treated with hot 3% sodium carbonate for 2 min and acetolyzed according to the method of Erdtman (1960). For SEM the material was treated with hot 3% sodium carbonate, washed, dehydrated, suspended in 96% ethanol and then transferred to acetate plates. After drying they were coated with gold. For TEM dry material from herbarium specimens was hydrated following the technique proposed by Rowley and Nilsson (1972) that consists of the use of phosphate buffer and Alcian Blue (AB), then the material was fixed with glutaraldehyde + 1% Alcian Blue in phosphate buffer for 12 h and post-fixed with 1% OsO4 in water plus 1% Alcian Blue. The spores were dehydrated in an acetone series and then embedded in Spurr soft mixture. Sections 3 μm thick were stained with toluidine blue and analyzed with LM. Ultra-thin sections were stained with 1% uranyl acetate for 15 min followed by lead citrate for 3 min.
The observations were performed with Olympus BH2 and BHB LMs, a JEOL JSMT-100 SEM and a Zeiss T-109 TEM. After the LM and SEM analysis, Anemia australis, A. herzogii and A. simplicior were selected as representative for the study with TEM. 2.1. Material studied Anemia australis: ARGENTINA: CATAMARCA, Andalgalá, Yunkasuma Río Chacra, Sleumer 35 (LIL); JUJUY, Capital, Sierra de Zapla, Cabrera et al. 23656 (LP); Capital, Termas de Reyes, Abbiatti & Claps 802 (LP); SALTA, Metán, Dinelli 4483 (LIL); SAN LUIS, Junín, Merlo, Reserva El Tabaquillo, Giudice, Luna & Ramos Giacosa 294 (LP); TUCUMAN, Trancas, San Pedro de Colalao, Cristóbal 162 (LIL). Anemia herzogii: ARGENTINA: JUJUY, Ledesma, Parque Nacional Calilegua, Camino a Valle Grande, Ramos Giacosa 213 (LP); SALTA, General San Martín, Arroyo Tres Quebradas, Krapovickas et al. 19497 (LP). A. myriophylla: ARGENTINA: SALTA, Santa Victoria, Sota 4156 (LP) A. phyllitidis var. phyllitidis: ARGENTINA: JUJUY, Valle Grande, camino a Valle Grande, Mesada de las Colmenas, de la Sota 4482 (LP), MISIONES, Cainguás, Reserva Cuña Pirú, Biganzoli et al. 1373 (LP). A. phyllitidis var. tweedieana: ARGENTINA: BUENOS AIRES, Isla Martín García, Hurrell et al. 2553 (LP); MISIONES, Leandro Alem, Paso Caneta, Krapovickas et al. 15.005 (LP). A. simplicior: MISIONES, General Belgrano, Camino El Dorado a Bernardo de Irigoyen, Katinas 49 (LP); Marquez 180 (LP). A. tomentosa var. anthriscifolia: CATAMARCA, Schunck 9706 (LIL); JUJUY, El Carmen, Dique La Ciénaga, Cabrera et al. 14089 (LP); MISIONES, Cainguás, Puerto Rico, Montes 3993 (LP); SALTA, Cafayate, Santa Teresa, Lourteig 1041 (LIL); Capital, La Lagunilla, Saravia Toledo 2152 (LP); TUCUMAN, Vipos, Dinelli 832 (SI). A. tomentosa var. tomentosa: BUENOS AIRES, Tornquist, Sierra de la Ventana, Reserva Integral “La Blanqueada”, Proyecto Ventania 15 (LP); Tandil, Cerro de las Animas, Fabris & Schwabe 4750 (LP); La Cascada, Cabrera & Torres 17.816 (LP); MISIONES, San Javier, Cabrera et al. 322 (LP). A. wettsteinii: MISIONES, Cataratas del Iguazú, Capurro s/n° BA 55286 (LP); El Dorado, R. P. 17, paraje Cerro 60, Keller 3095 (CTES). 3. Results 3.1. Morphology The palynological characteristics of the analyzed taxa are summarized in Tables 1 and 2. The spores are trilete. The spore shape in equatorial view is convex-hemispheric. They are triangular with straight or convex sides in polar view. The equatorial diameter is 42–114 μm and the polar diameter is 34–106 μm. The laesura is 16–50 μm long. The spores of A. herzogii, A. phyllitidis and A. phyllitidis var. tweediana are triangular with convex sides and rounded angles. The ornamentation is constituted of narrow and parallel ridges of 0.7–2 μm wide. On the ridges, large bacula of 2–7 μm high with rounded apices are seen. Smooth grooves of 3.9–7.9 μm wide separate the ridges (Plate I, 1–4; Plate II). The spores of A. wettsteinii (Plate I, 5–8) are similar to the previous taxa. The differences are basically that the spores of A. wettsteinii have fewer bacula and they are smaller than in the other species. The spores of Anemia australis, A. tomentosa var. anthriscifolia, A. tomentosa var. tomentosa, A. myriophylla and A. simplicior (Plates III, IV, V) are triangular with straight or convex sides and prominent angles. The ornamentation is composed of parallel ridges of 3.3–7 μm wide which are separated by narrow and smooth grooves of 0.5–3.7 μm wide. The ridges are fused near the angles of the spores and they form prominent and thickened angles. On the ridge surfaces spines of variable size and shape are observed, depending on the taxa analyzed. Abundant perforations of variable sizes are also present on the perispore surface.
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Table 1 Spore morphological data for Anemia species from Argentina. Dimensions in μm. Taxa
Shape in polar view
Equatorial diameter
Polar diameter
Laesurae length
A. A. A. A. A. A. A. A. A.
Triangular Triangular Triangular Triangular Triangular Triangular Triangular Triangular Triangular
76–91 52–68 72–88 42–55 51–62 78–89 65–84 86–114 66–82
72–83 39–52 59–67 34–49 41–56 59–65 55–72 72–106 57–72
31–41 22–31 23–31 20–27 16–25 27–34 31–40 36–50 25–41
australis herzogii myriophylla phyllitidis var. phyllitidis phyllitidis var. tweediana simplicior tomentosa var. anthriscifolia tomentosa var. tomentosa wettsteinii
with with with with with with with with with
convex sides. Prominent angles convex sides. Rounded angles convex sides. Prominent angles convex sides. Rounded angles convex sides. Rounded angles straight sides. Prominent angles convex sides. Prominent angles convex sides. Prominent angles convex sides. Rounded angles
In A. australis (Plate III) the spines are low, 0.2–0.4 μm high with truncated or rounded apices. In superficial view, these elements could be wrongly defined as granules. In A. tomentosa var. tomentosa and A. tomentosa var. anthriscifolia (Plate V) the spines are medium high, 0.9–2.2 μm, with acute or rounded apices which are frequently ramified. The ornamentation of the ridge surfaces of A. myriophylla (Plate IV, 1–6) has intermediate characters between A. australis and A. tomentosa since some spines are low with rounded apices while others are high with acute apices; these apices may be ramified or not and similar to those observed in A. tomentosa. In A. simplicior (Plate IV, 7–11) the spines are higher than in the previous species, 1.5–4.5 μm high, filiform with acute apices, and they are frequently ramified and interwoven. Some abnormalities like aborted, immature or spores join in tetrads were observed in all the specimens of Anemia tomentosa var. antrhiscifolia and A. tomentosa var. tomentosa. The same ornamentation that appears on the ridges (i.e. bacula, spines, and perforations) was observed on the laesura surfaces in the nine species analyzed. 3.2. Ultrastructure According to the structural characteristics and ornamentation observed by LM and SEM, Anemia australis, A. herzogii and A. simplicior were selected to be studied by TEM because they summarize the major variations. Two types of sporoderm ultrastructure were observed: 1) In A. herzogii the exospore between the bacula, is 1.2–1.4 μm thick and two-layered showing an inner layer of 60–110 μm thick and an outer layer of 1.2–1.4 μm thick. The outer exospore layer forms the ornamentation of the spores which consists of ridges and bacula. The bacula have wide bases and a rounded apex (Plate VI, 2).
The perispore is 0.5–1.1 μm thick and is composed of two layers: an inner layer (P1) of 50–100 nm thick that is adhered to the exospore and an outer layer (P2) 0.4–1 μm thick with interwoven threads (Plate VI, 1–3). In the bacula apex, the perispore is thicker. In this region, the outer perispore layer has a higher density of threads which are arranged in a lax structure toward the outside of the apex (Plate VI, 2, 4, 5). Spheroids with an inner portion which has the same structure and contrast as the exospore, and an outer layer with the same structure as the perispore, can be observed on the perispore surface (Plate VI, 6). Several small portions of membranes (scales) are observed as immersed within the perispore and below the spheroids (Plate VI, 4–6). 2) In A. australis and A. simplicior the exospore is 2–7 μm thick and is composed of two layers: an inner layer 0.9–1.3 μm thick and an outer layer 1.3–6 μm thick that forms the ornamentation of the spore constituted by ridges. In the outer layer a big amount of cavities with irregular shapes and sizes are evident. These cavities are mainly situated inside the ridges and just a few of them lie between the ridges (Plate VIII, 1). They have a differentiated edge (Plate VIII, 5). Radial channels with contrasted content are mainly observed in the inner exospore layer (Plate VIII, 1). The perispore is 0.2–1 μm thick and has two layers: P1 and P2. The inner layer (P1) is made up of 3 strata: the inner stratum is thin, 50–100 nm thick and is adhered to the exospore. The middle stratum is 60–200 nm thick, has areas of concentrated material and other regions with cavities. This stratum has a laxer structure than the other strata. It is an area of weakness that contributes to the perispore detachment. The outer stratum is 50–100 nm thick (Plate VII, 1–4; Plate VIII 1–4). The outer layer (P2) is 200–600 nm thick, less contrasted than P1 layer and covers the outer surface of P1 layer. P2 layer forms the micro-ornamentation of the spore that consists of spines of variable sizes and shapes as it was described in the Morphology section. (Plate VII, 1–4; Plate VIII, 1–3).
Table 2 Ornamentation and dimensions of ornamental elements in Anemia species from Argentina. Dimensions in μm. Taxa
Exospore ornamentation
Perispore ornamentation
Ridge wide
Grooves width
Bacula/spines height
A. australis
Parallel ridges separated by narrow and smooth grooves. Narrow, parallel ridges with high bacula separated by wide grooves. Parallel ridges separated by narrow and smooth grooves. Narrow and parallel ridges with high baculae separated by wide grooves. Narrow and parallel ridges with high baculae separated by wide grooves. Parallel ridges separated by narrow and smooth grooves. Parallel ridges separated by narrow and smooth grooves Parallel ridges separated by narrow and smooth grooves Narrow and parallel ridges with low baculae separated by wide grooves.
Small spines with truncated or rounded apices. With perforations. Laevigate. Without perforations.
4.2–6.9
0.5–2
0.2–0.4
0.7–1.5
5.0–6.8
2.8–4.6
Spines or spinules with rounded or acute apices. Some ramified. With perforations Laevigate. Without perforations.
4.9–6.3
0.7–2
1.1–1.8
4.3–7.9
2.6–6.7
Laevigate. Without perforations.
1.5–2
3.9–7.8
3.2–8.3
High filiform spines with acute apices, ramified. With perforations. Medium height spines, acute or rounded apices, frequently ramified. Medium height spines, acute or rounded apices, frequently ramified. Laevigate. Without perforations.
3.3–4.8
2.1–3.7
1.5–4.5
3.3–6
0.8–1.8
1.1–2.2
4.6–7
0.8–2.3
0.9–1.8
1.5–2.3
5.1–8.3
1.6–3.7
A. herzogii A. myriophylla A. phyllitidis var. phyllitidis A. phyllitidis var. tweediana A. simplicior A. tomentosa var. anthriscifolia A. tomentosa var. tomentosa A. wettsteinii
1–2.7
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Plate I. Spores of Anemia herzogii and A. wettsteinii with SEM. 1–4. 1. 2. 3. 4. 5–8. 5. 6. 7. 8.
A. herzogii. Proximal view of a spore. The laesurae have the same ornamentation as the ridge surface. Scale bar = 10 μm. Distal view of a triangular spore with convex sides. Scale bar = 10 μm. Equatorial view of a spore. Scale bar = 10 μm. Detail of the distal surface. Narrow and parallel ridges with high baculae separated by wide grooves are observed. The baculae have rounded apices. Scale bar = 5 μm. A. wettsteinii. Proximal view of a spore. Scale bar = 10 μm. Distal view of a triangular spore with convex sides. The ornamentation is composed of narrow and parallel ridges with low baculae separated by wide grooves. Scale bar = 10 μm. Equatorial view of a spore. Scale bar = 10 μm. The baculae are small and has rounded apices. Scale bar = 5 μm.
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Plate II. Spores of Anemia phyllitidis var. phyllitidis and Anemia phyllitidis var. tweediana with SEM. 1–4. 1. 2. 3. 4. 5–8. 5. 6. 7. 8.
A. phyllitidis var. phyllitidis. Proximal view of a triangular spore with convex sides. Scale bar = 10 μm. Distal view of a triangular spore with convex sides. Scale bar = 10 μm. Equatorial view of a spore. Scale bar = 10 μm. Detail of the ornamentation of the spore composed of narrow and parallel ridges with large baculae separated by wide depressions. Scale bar = 5 μm. A. phyllitidis var. tweediana. Proximal view of a triangular spore with convex sides. The laesurae has the same ornamentation as the surface of the ridges. Scale bar = 10 μm. Distal view of a spore. The ornamentation is composed of narrow and parallel ridges with large baculae separated by wide depressions Scale bar = 10 μm. Equatorial view of a convex-hemispheric spore. Scale bar = 10 μm. Detail of the ornamentation. Large baculae with rounded apices are observed. Scale bar = 5 μm.
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Plate III. Spores of Anemia australis with SEM. 1. 2. 3. 4–5.
Proximal view of a triangular spore with convex sides. The angles of the spore are prominent. Scale bar = 10 μm. Distal view of a spore. The ornamentation consists of wide parallel ridges separated by narrow depressions. Scale bar = 10 μm. Equatorial view of a convex-hemispheric spore. Few ridges are dichotomized (arrows). Scale bar = 10 μm. Detail of the perispore surface. The micro-ornamentation is only visible on the ridges and is made up of small spines with rounded apices. Some perforations are also visible (arrowheads). The laesurae (stars) has the same ornamentation as the surfaces of the ridges. Scale bars = 5 μm.
4. Discussion and conclusions According to the observations made in this paper, there exist relevant variations in spore diameter (42–114 μm in equatorial diameter) which coincide with the ones described by Mickel (1962), and may be related to the different ploidal levels found in the genus Anemia (Mickel, 1962, 1982). He also explains that there is a relationship between the size and number of spores per sporangium, including intra-specific variations.
As regards spore morphology, two different morphological types are observed among the taxa analyzed: 1) the Anemia herzogii type (A. herzogii, A. phyllitidis, A. phyllitidis var. tweediana and A. wettsteinii) which shows narrow and parallel ridges with baculae separated by smooth grooves and 2) the A. australis type (A. australis, A. tomentosa var. anthriscifolia, A. tomentosa var. tomentosa, A. myriophylla and A. simplicior) with wide parallel ridges separated by narrow and smooth grooves with prominent and rounded angles. Within this type, variations may be found depending on the different kinds of perispore ornamentation;
Plate IV. Spores of Anemia myriophylla and A. simplicior with SEM. 1–6. 1. 2. 3. 4–6.
7–11. 7. 8. 9. 10. 11.
A. myriophylla. Proximal view of a triangular spore with convex sides. The angles of the spore are prominent. Scale bar = 10 μm. Distal view of a spore. The ornamentation consists of wide parallel ridges separated by narrow depressions. Scale bar = 10 μm. Equatorial view of a convex-hemispheric spore. Scale bar = 10 μm. Detail of the perispore surface. The micro-ornamentation is only visible on the ridges and is composed of some small spines with rounded apices (white arrows) and others which are higher and may be ramified or not (black arrows). The laesurae (star) has the same ornamentation as the surfaces of the ridges. Scale bars = 5 μm. A. simplicior. Proximal view of a triangular spore with straight sides. The angles of the spore are prominent. Scale bar = 10 μm. Distal view of a spore. Parallel ridges separated by narrow and smooth depressed zones are observed. Scale bar = 10 μm. Equatorial view of a convex-hemispheric spore. Scale bar = 10 μm. The micro-ornamentation is only visible on the ridges and consists of abundant large spines. Scale bar = 5 μm. Detail of the perispore surface. The spines are filiforms with acute apices and are frequently ramified and interwoven. Some perforations are visible on the surface (arrowheads). Scale bar = 2 μm.
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spines can go from being small with rounded apices to filiform, ramified, high and interwoven. Besides, abundant perforations are evident in the perispore of all the taxa belonging to this group. The studied taxa of Anemia herzogii type share a homogenous morphology since their spores do not present relevant inter-specific variations. Within this group, only spores of A. wettsteinii are different
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from the rest in the sense that the baculae on their ridges are lower than in the other analyzed taxa. In addition to this, spore diameter is greater than in the other taxa from this group. On the contrary, spores of Anemia australis present variations regarding the perispore ornamentation. Besides, the species belonging to this group have significant variations in spore size, and
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Plate V. Spores of Anemia tomentosa var. anthriscifolia and A. tomentosa var. tomentosa with SEM. 1–5. 1. 2. 3. 4–5. 6–10. 6. 7. 8. 9–10.
Anemia tomentosa var. anthriscifolia. Proximal view of a triangular spore with convex sides. The angles of the spore are prominent. Scale bar = 20 μm. Distal view of a spore. Parallels ridges separated by narrow and smooth depressed zones are observed. Scale bar = 20 μm. Equatorial view of a convex-hemispheric spore. Scale bar = 20 μm. The ornamentation of the perispore is only visible on the ridges and consists of medium-high spines, with acute or rounded apices, they are frequently ramified. The laesurae (star) have the same ornamentation as the ridge surfaces. Some perforations are also evident (arrowheads). Scale bars = 5 μm. A. tomentosa var. tomentosa. Proximal view of a triangular spore with convex sides. The angles of the spore are prominent. Scale bar = 20 μm. Distal view of a spore. Scale bar = 20 μm. Equatorial view of a convex-hemispheric spore. Parallels ridges separated by narrow and smooth depressed zones are observed. Scale bar = 20 μm. Detail of the perispore surface. On the ridges, medium-high spines, with acute or rounded apices are observed. They are frequently ramified. Scale bars 9 = 2.5 μm, 10 = 1 μm.
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Plate VI. TEM images of Anemia herzogii spores. 1. 2. 3. 4–5. 6.
Section through a laesura. The exospore consists of two layers. The inner exospore layer (arrow) is thin and more contrasted than the outer exospore layer (Eo). Scale bar = 1 μm. Section through the sporoderm. The outer exospore layer (Eo) forms the bacula which has a wide base and a rounded apex. On the exospore, the perispore is evident (arrows). Scale bar = 1 μm. Section through the perispore between the baculae. The perispore is two-layered: a thin inner layer P1 (arrow) which is adhered to the exospore (E) and the outer layer P2. Scale bar = 0.25 μm. Detail of the bacula apex. In this region, the perispore outer layer is thicker and the threads are laxly distributed toward the apex. Few scales (arrowheads) are seen on the perispore. Scale bars 4 = 0.5 μm, 5 = 0.25 μm. A spheroid (s) within the perispore are observed. It has a core with the same structure and contrast as the exospore and an outer layer is similar in contrast and structure to the perispore. Scales are seen below the spheroid (arrowhead). Scale bar = 0.25 μm.
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Plate VII. TEM images of Anemia australis spores. 1. 2–4.
Section through the sporoderm. The exospore is two-layered. The inner layer (Ei) has numerous radial channels with contrasted content (arrowhead). The outer exospore layer (Eo) forms the ornamentation which consists of ridges (R). This layer has cavities with irregular shape and size (arrows). Scale bar = 1 μm. Section through the sporoderm. The perispore is two-layered P1 layer has 3 strata: the inner stratum (arrowhead) is adhered to the exospore (E), the middle stratum (P1m) has areas of concentrated material and other regions with cavities and the outer stratum (arrows) is continuous. P2 layer is less contrasted than P1 and forms the perispore micro-ornamentation consisting of spines (double arrows) and low elevations of variable sizes. Scale bars = 0.5 μm.
some taxa even have different levels of abnormalities. The irregularities found in the spores of A tomentosa var. tomentosa were also highlighted by Mickel (1962) and de la Sota and Mickel (1968) when they mentioned spores presenting irregular dimensions and shapes, generally aborted. The explanation for these irregularities may derive from the fact that they are hexaploid with apogamic reproduction. At ultrastructural level, there are also differences worth mentioning regarding the exospore and perispore of the groups mentioned. In the case of A. herzogii, the exospore forms the ornamentation, consisting of baculae. The perispore consists of two strata with a smooth outer surface and frequently presenting scales basally. Lugardon (1974) also observed these scales in the perispore of Anemia phyllitidis, and they were also mentioned among the spores of other families such as Blechnaceae (Ramos Giacosa et al., 2009) and Polypodiaceae (Morbelli and Giudice, 2010). However, both the exospore and the perispore of A. australis have ornamentation. The exospore forms the macro-ornamentation and it consists of ridges which internally have a structure full of cavities.
On the other hand, the perispore presents two layers: P1 layer consisting of 3 strata, and P2 layer. In the outer surface a microornamentation consisting of simple or ramified spines of variable dimensions are observed. The ultrastructural characteristics of the A. herzogii spores analyzed in this study go along with the description of A. phyllitidis made by Lugardon (1974). Besides, the presence of cavities in the inner perispore was previously mentioned by Erdtman (1957) and by Mickel (1962), as well as illustrated for Anemia madagascarensis and A. simii by Tryon and Lugardon (1991) and for A. flexuosa by Dettman and Clifford (1991). From the data collected in this study it can be noted that both exospore and perispore share variations not only as regards morphology but also as regards ultrastructure. In this sense, the exospore is useful in order to differentiate the two groups while the perispore allows us to differentiate taxa within the A. australis group. At the same time, the two morphological spore groups presented in this study go along with the subgenus that Mickel (1962)
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Plate VIII. TEM images of Anemia simplicior spores. 1. 2–4.
5.
Section through the sporoderm. The exospore is two-layered: the inner layer (Ei) and the outer layer (Eo) forms the ridges (R). Numerous cavities with irregular shape and size are evident in the outer exospore layer (arrow). Few radial channels with contrasted content are observed in the exospore (arrowheads). Scale bar: 1 μm. Section through the outer exospore and perispore. The perispore is two-layered: P1 and P2. P1 layer has 3 strata: the inner stratum (arrowhead) shows has a high contrast and is adhered to the exospore (E), the middle stratum (P1m) has areas of concentrated material and other regions with cavities, and the outer stratum (arrow) is continuous and, in some cases, difficult to differentiate. P2 layer forms the spines (double arrow) that constitute the micro-ornamentation of the perispore. Scale bars: 0.5 μm. Detail of one exospore outer layer cavity. It has ellipsoidal shape and a differentiated edge (arrow). Scale bar: 0.5 μm.
suggested: subg. Anemia for the A. herzogii, and subg. Coptophyllum for the A. australis group. The spores of Anemia have a great taxonomic value provided not only by the ornamentation of the spores observed with LM and SEM but also for the rich amount of information that the wall ultrastructure provides. These characters are particularly valuable in the A. australis group (previously known as subg. Coptophyllum) whose spores present the most outstanding variations. Moreover, another relevant feature to take into account in the determination of these taxa is the presence of abnormal or aborted spores. The palynological data dealt with here add information of great taxonomic value; some of the taxa presented had not been examined
before in this region. What is more, these morphological characters may be of value in the determination of the taxa and their incorporation into future phylogenetic studies applied to this group. Finally, it is worth mentioning the need to further carry on ultrastructural studies on other species within the genus in order to include this valuable information in the phylogenetic studies. Acknowledgments The authors thanks to Lic. Rafael Urrejola from the SEM Unit, Museo de Ciencias Naturales de La Plata, Lisandro Anton, from the TEM Unit, Instituto de Biología Celular, Facultad de Medicina, Universidad de Buenos Aires and the institutions that very kindly sent the
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herbarium material. This work was supported by grants from the Universidad Nacional de La Plata (project 584; Subs. Jov. Invest. 08, 09, 10) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) for project PICT 0661. References Bolkhovitina, I.A., 1959. The morphology of the spores of the family Schizaeaceae and the history of the family in the geological past. Journal of Paleontology 1, 121–131. de la Sota, E.R., Mickel, J.T., 1968. Sinopsis de las especies argentinas del género Anemia Swartz (Schizaeaceae). Revista Museo de La Plata (nueva serie), Sección Botánica 11, 133–152. de la Sota, E.R., Morbelli, M.A., 1987. Schizaeales. Phytomorphology 37 (4), 365–393. Dettman, M.E., Clifford, H.T., 1991. Spore morphology of Anemia, Mohria and Ceratopteris (Filicales). American Journal of Botany 78 (3), 303–325. Erdtman, G., 1957. Pollen and Spore Morphology, Plant Taxonomy; Gymnospermae, Pteridophyta, Briophyta. Almqvist and Wiksells, Stockholm. Erdtman, G., 1960. The acetolysis method. A revised description. Svensk Botanisk Tidskrift 54, 561–564. Erdtman, G., Sorsa, P., 1971. Pollen and Spore Morphology and Plant Taxonomy. Pteridophyta. Almqvist & Wiksell, Stockholm. Hill, S.R., 1977. Spore morphology of Anemia subgenus Coptophyllum. American Fern Journal 67 (1), 11–17. Hill, S.R., 1979. Spore morphology of Anemia subgenus Anemia. American Fern Journal 69 (3), 71–79. Lugardon, B., 1974. La estructure fine de l'exospore et de la perispore des Filicinées isosporées. II. Filicales. Commentaires. Pollen Spores 16 (2), 161–226. Mickel, J.T., 1962. A monographic study of the fern genus Anemia, subgenus Coptophyllum. Iowa State Journal of Science 36, 349–482. Mickel, J.T., 1981. Revision of Anemia subgenus Anemiorrhiza (Schizaeaceae). Brittonia 33, 413–429.
Mickel, J.T., 1982. The genus Anemia (Schizaeaceae) in Mexico. Brittonia 34 (4), 388–413. Mickel, J.T., Smith, A.R., 2004. The pteridophytes of Mexico. Memoirs of the New York Botanical Garden 88, 48–60. Morbelli, M.A., 1981. Estudio de esporas de las Pteridofitas del Noroeste de Argentina. Ophioglossaceae, Schizaeaceae y Cyatheaceae. XVIII Jornadas Argentinas de Botánica. San Miguel de Tucumán. Libro de Resúmenes, p. 79. Morbelli, M.A., Giudice, G.E., 2010. Spore wall ultrastructure of Polypodiaceae from north-western Argentina. Grana 49, 204–214. Ramos Giacosa, J.P., Morbelli, M.A., Giudice, G.E., 2009. Spore morphology and wall ultrastructure of Blechnum L. species from North West Argentina. Review of Palaeobotany and Palynology 156, 185–197. Rowley, J.R., Nilsson, S., 1972. Structural stabilization for electron microscopy of pollen from herbarium specimens. Grana 12, 23–30. Skog, J.E., Zimmer, E.A., Mickel, J.T., 2002. Additional support for two subgenera of Anemia (Schizaeaceae) from data for the chloroplast intergeneric spacer region trnL-F and morphology. American Fern Journal 92 (2), 119–130. Smith, A.R., Pryer, K.M., Schuettpelz, E., Korall, P., Schneider, H., Wolf, P.G., 2006. A classification for extant ferns. Taxon 55 (3), 705–731. Tryon, A.F., Lugardon, B., 1991. Spores of Pteridophyta. Springer-Verlag, New York. Tryon, R.M., Tryon, A.F., 1982. Fern and allied plants with special reference to tropical America. Springer Verlag, Berlin, Heidelberg, New York. Van Konijnenburg-van Cittert, J.H.A., 1991. Diversification of spores in fossil and extant Schizaeaceae. In: Blackmore, S., Barnes, S.H. (Eds.), Pollen and Spores, Patterns of Diversification: Publ. Syst. Assoc., spec., vol. 44, pp. 103–118. Wikström, N., Kenrick, P., Vogel, J.C., 2002. Schizaeaceae: a phylogenetic approach. Review of Palaeobotany and Palynology 119, 35–50. Zuloaga, F.O., Morrone, O., Belgrano, M. (Eds.), 2008. Catálogo de las plantas vasculares del Cono Sur: Pteridophyta, Gymnospermae y Monocotyledonae. Monogr. Syst.Bot. Missouri Bot. Gard., Vol.1, p. 107.