Available online at www.sciencedirect.com South African Journal of Botany 79 (2011) 32 – 38 www.elsevier.com/locate/sajb Short communication Rediscovery of Riella alatospora (Riellaceae, Sphaerocarpales), an aquatic, South African endemic liverwort previously known from a now largely transformed type locality J.G. Segarra-Moragues a,⁎, F. Puche b , M. Sabovljević a b c Centro de Investigaciones sobre Desertificación (CIDE-CSIC-UV-GV), C/ Carretera de Moncada-Náquera Km 4.5, Apartado Oficial, E-46113, Moncada (Valencia), Spain Departamento de Botánica, Facultad de Ciencias Biológicas, Universitat de València. C/ Dr. Moliner s/n. E-46100, Burjassot (Valencia), Spain c Institute of Botany and Garden, Faculty of Biology, University of Belgrade, Takovska 43, RS-11000, Belgrade, Serbia Received 5 September 2011; received in revised form 21 November 2011; accepted 23 November 2011 Abstract Riella alatospora is reported from cultured sediments from a salt pan near Springfield (Western Cape), South Africa. This species was previously known only from the type locality near Cape Town, and has not been collected since 1932. Because the original locality has been severely affected by urbanization in the past decades, the species is unlikely to survive there. The identity of the specimens obtained from the cultured sediments were confirmed by comparison with the type material of R. alatospora. A detailed description of R. alatospora including lectotypification, illustrations, light and scanning electron microscopy micrographs of spores, and an updated distribution map is presented. Our finding suggests that the genus Riella is likely to be more widespread in South Africa than previously thought and that extensive sampling is required before an accurate distribution of the species can be ascertained. This study also highlights the value of culture sediments in revealing the presence of Riella. © 2011 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Aquatic liverworts; Bryophytes; South Africa; Southern Hemisphere; Spore morphology 1. Introduction The genus Riella Mont. (Riellaceae, Sphaerocarpales) includes some 22 species of aquatic thallose liverworts and is distributed in areas of Mediterranean or subdesertic climate types on all continents except Antarctica. Their small size, ephemeral aquatic habitat, and dependence on the nature of the flooding season makes finding populations somewhat difficult. Populations are generally demographically fluctuant, with some years experiencing sudden demographic blooms and other of complete absence, as population growth may be inhibited both by too high or too low water levels. Furthermore, their specialised aquatic habitat offers very little of interest to bryologists, apart from Riella, and so they are often overlooked. It is not surprising then that some populations and even species have been discovered from cultures aimed at sampling ponds and lagoons for other organisms like crustacean (Cavers, 1903; Hässel de Menéndez, 1979; Perold, 2000; Porsild, 1902). Five species of Riella (R. affinis M. Howe & Underw., R. alatospora Wigglesworth, R. capensis Cavers, R. echinospora Wigglesworth and R. purpureospora Wigglesworth) are present in southern Africa (Perold, 2000; Wigginton and Grolle, 1996; Wigginton, 2009). Of these five species, R. affinis a monoecious species of subgenus Trabutiella has the widest distribution range, and is the only species reported outside southern Africa (Howe and Underwood, 1903; Patel, 1977; Puche and Boisset, 2009; Thompson, 1940). However, within South Africa this species is ⁎ Corresponding author. Tel.: +34 963424126; fax: +34 963424160. E-mail address: j.gabriel.segarra@uv.es (J.G. Segarra-Moragues). 0254-6299/$ -see front matter © 2011 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2011.11.006 J.G. Segarra-Moragues et al. / South African Journal of Botany 79 (2011) 32–38 still known only from a single reported locality (Proskauer, 1955). The remaining four species belong to subgenus Riella, sharing the smooth female involucres. Riella echinospora is known from only two isolated localities, the one in South Africa (Wigglesworth, 1937) and the other in Namibia (Arnell, 1957); R. purpureospora is endemic to South Africa and is the only species reported in recent times (Harding et al., 2000; Perold, 2000) around the Cape Town area. The two remaining species R. capensis Cavers and R. alatospora Wigglesworth were both known only from one locality (Cavers, 1903; Wigglesworth, 1937). Some other existing reports of Riella from South Africa (Perold, 2000; Wigglesworth, 1937) and Namibia (Arnell, 1957) have not been assigned to any of these species because of the absence of mature spores which are essential for species identification whereas others have been tentatively assigned to R. capensis (Coetzer, 1987), but have not been verified in recent revisions of the genus (Perold, 2000). Riella alatospora was originally collected by Dr. E.L. Stephens from a vlei (pond) in Salt River, Cape Town in 1932 and sent to Manchester where it was studied by Dr. G. Wigglesworth who described it after the presence of a wing along the equatorial plane of the spores. This original locality has been severely altered by urbanizing activities, as have many parts of the city area (Rebelo et al., 2011), to the point that it is unlikely that populations of this species are currently developing there. As a result, R. alatospora has not been collected or reported for more than 80 years (Perold, 2000). As part of a world-wide study of the genus Riella, sediments from a salt pan at Springfield, near Cape Agulhas were collected by the first author during February 2010. Cultures of those sediments rendered plants of Riella in an area not previously included in the known distribution ranges for any of the South African species. 2. Materials and methods About 0.5 kg of dried sediments were collected from several points in one side of the salt pan and included the first 5 cm depth of soil. Collection points were located just a few meters from the shore line and where visible dried debris of plants and algae had accumulated. Sediments were kept on a zip plastic bag in the laboratory at room temperature. One year after collection of soil samples the sediments were flooded with distilled water in the laboratory. The cultures were turbid after this initial flooding and two subsequent water changes were required until they were transparent. These changes were conducted without disturbing the sediments and any visible germination that could have occurred. Measurements of vegetative and spore characters were taken using the interactive measurement module of Leica Application Suite (LAS) v. 3.8 (Leica microsystems, Barcelona, Spain) calibrated to the nearest 0.01 μm on digital images. All measurements were taken under a Leica DMLB 100S light microscope, except for the size of gametophytes and width of wing from the thallus that were taken under a Leica M76 stereomicroscope. A Leica DFC425 digital camera was used for producing high resolution images. Mature spores were mounted directly on stubs using double-sided adhesive tape and coated with gold/palladium in a 33 BIORAD SC-500 ion sputtering coater. Morphological observations were carried out in a Hitachi S-4100 field emission scanning electron microscope (SEM) at the University of Valencia (SCSIEUV). Terminology for spore characters follows Perold (2000). 3. Results In May 2011 the first germlings of Riella appeared in the cultures, just two months after the flooding of the sediments with distilled water. Gametophyte development continued during the two following months until the first reproductive organs were visible. The individuals corresponded to a dioecious species and males were the first to reach maturity. Two weeks later female individuals began to develop involucres after the fertilization of the archegonia and sporophytes contained ripe spores by the end of August 2011. The spores of these individuals were compared to those present in the original cultured material used for the description of R. alatospora (Wigglesworth, 1937). Both samples showed spores triangular in outline and with a conspicuous wing around the equatorial plane, short truncate spines on the distal face and short acute spines over a flattened central dome on the proximal face. These distinctive spore characters allowed for the unambiguous placement of the cultured specimens with that of R. alatospora (Fig. 1). A detailed description of R. alatospora was provided by Wigglesworth (1937) including developmental stages observed in the laboratory and later by Perold (2000) from herbarium material. Here we supplement those descriptions with individual characters from this new collection. 3.1. Riella alatospora Wigglesworth in J. Linn. Soc., Bot., 5: 317 (1937). Type: South Africa. Western Cape, Cape Town (3318), ‘vlei at Salt River between main road and railway line’ (−CD), 1932, E. L. Stephens s.n. (CC 1627 MANCH! lecto.; BOL, iso.). [Note: Grace Wigglesworth received a specimen of R. alatospora from E. L. Stephens and then cultured the plant further from spores. Apparently, both wild and cultivated materials were used in the original description of this species with no explicit designation of a holotype in Wigglesworth (1937). Contrary to other South African specimens of Riella reared in the laboratory by Wigglesworth, the material of R. alatospora (CC 1627) was not labelled as having been derived from cultures and rather represents the duplicate of the original Stephens' collection in BOL (not located). As such the specimen in MANCH is here designated as the lectotype.] Plants 11–18 (20) mm tall, erect, caespitose, usually bifurcate from the base and 2 to 3 branched above, becoming shrubby, rarely unbranched (Figs. 2, 3B, C). Thallus apex falciform. Axis slightly flattened, 0.15–0.22 mm wide. Dorsal wing 0.8–2.1 mm wide, undulate, margin entire, marginal cells quadrate or oblate, the first 5 to 7 rows of cells hyaline (Figs. 2D, 3F), 15–41 × 14–34 μm; cells from middle part of wing polygonal 38–70× 30–55 μm; cells from wing near axis rectangular or hexagonal 58–123 × 28–75 μm; oil cells 14–26 × 17–28 μm, oil bodies 12–20 × 14–22 μm spherical or ovoid, opaque, smooth. Vegetative scales lanceolate-triangular 313–1015 × 185–805 μm, arranged in two lateral rows, opposite (Figs. 2C, 3E). Propaguliferous scales not 34 J.G. Segarra-Moragues et al. / South African Journal of Botany 79 (2011) 32–38 Fig. 1. Light and scanning electron microscopy photographs of spores from Riella alatospora. (A, D) Equatorial plane; (B, G) distal face; (E, H) proximal face; (C, I) detail of wing; (F, J) detail of centre of distal face showing the reticulation formed by basal membranes joining spines; (K) detail of spines from distal face; (L) detail of spines from proximal face.. Vouchers: A, C, D, Stephens s.n. sub comp. cat. 1627 (MANCH); B, E–L, Segarra-Moragues s.n. sub VAL-Briof. 9196 (VAL). seen. Dioicous; male plants equal in size to female plants or larger. Antheridia numerous, continuous, in a single linear series in pockets along thickened wing margin or in discontinuous groups of 12 to 18 to 60, rarely solitary (Figs. 2A, 3B); antheridial body 190–230 × 95–135 μm. Archegonia on axis, produced in acropetal sequence. Archegonial involucre sessile to shortly pedunculate, globose, ovoid, abruptly narrowed above capsule, rostrate, beaked, 1.5–2.0× 0.9–1.2 mm, smooth to slightly papillose (Figs. 2E, 3C,D); mouth of involucre with apical pore not occluded. Sporophyte with seta of 0.1 mm. Capsule globose, 0.7–0.8 mm in diameter. Spores 102–127 × 100–118 μm including spines, orangebrown to golden brown, triangular, winged (Figs. 1A–E, G–I, 2F–G). Distal face covered with 14 to 18 irregular rows of spines across diameter and 24 to 32 projecting spines at periphery at the equatorial plane (Fig. 1A–B, G, J). Spines (4) 6 (7.1) μm long (2) 3.6 (5.9) μm wide, apices truncate, and some dilated at the apices (Fig. 1K). Basal membranes interconnecting spines of centre of distal face, forming imperfect reticulation and becoming indistinct towards marginal wing (Fig. 1B, G, J). Wing (10.9) 18.2 (25) μm wide, furrowed (Fig. 1A, C, H-I). Proximal face concave with spore body appearing as a raised central dome (Fig. 1E, H-I), J.G. Segarra-Moragues et al. / South African Journal of Botany 79 (2011) 32–38 35 Fig. 2. Riella alatospora. (A, B) General habit of female and male plants, respectively; (C) vegetative scale; (D) detail of cells from margin of thallus wing; (E) female involucre; (F, G) spores on distal and proximal view, respectively. Voucher: Segarra-Moragues s.n. sub VAL-Briof. 9196 (VAL). granulose (Fig. 1E, H–I, L). Triradiate mark indistinct; spines of proximal face (2) 2.7 (4.1) μm long (1) 1.6 (2.6) μm wide μm, with papillose-ganulose apex (Fig. 1L). 3.2. Habitat and distribution Known only from two seasonal salt pans in Western Cape, together with species of Characeae and Zannichelliacae (Fig. 4). 3.3. Conservation status The lack of collections of R. alatospora since its discovery in the Cape Town area in 1932 justified its inclusion in the IUCN Red List of threatened species under the category of Data Deficient (DD) by Cholo and Foden (2006). It is probable that because of severe urban transformations of the type locality (Rebelo et al., 2011) the species no longer exists there. Nonetheless, other potential sites around the Cape Town area should be screened for the presence of 36 J.G. Segarra-Moragues et al. / South African Journal of Botany 79 (2011) 32–38 Fig. 3. Habitat and appearance of Riella alatospora from Springfield. (A) dried salt pan where the sediments were collected. A whitish line a few meters from the shore-line composed of the accumulation of dried debris of plants and algae is visible; (B) male plant; (C) female plant; (D) detail of female involucre; (E) vegetative scale; (F) detail of cells from margin of wing showing the conspicuous rows of hyaline cells from margin. B–F from VAL-Briof. 9196 (VAL). this species as well as the endemic R. purpureospora, that inhabits similar brackish habitats (Harding et al., 2000). The new Springfield population of R. alatospora reported herein provides an excellent opportunity to initiate conservation activities aimed at deciphering actual extension and abundance of individuals and mitigate against alterations to the habitat that could compromise the survival of the plants. The new population occurs within the boundary of the Cape Agulhas National Park which should confer habitat protection and conservation. The abundance of seemingly suitable habitats in the surrounding area, including the Cape J.G. Segarra-Moragues et al. / South African Journal of Botany 79 (2011) 32–38 Fig. 4. Map showing the known distribution of Riella alatospora. Black circle=type locality, black star=new locality. Agulhas National Park boundary and private-owned lands, suggests that further searches may reveal additional populations. Alternatively, translocation of sediments from this locality to other potentially suitable salt pans within the protected area would allow the establishment of reservoir populations for R. alatospora. 3.4. Additional specimens examined South Africa. WESTERN CAPE. 3419 (Caledon), Springfield, near Cape Agulhas, on a salt pan (− DD), 24 Feb 2010, J.G. Segarra-Moragues s.n. sub VAL-Briof. 9196 (VAL). 4. Discussion Our study has revealed another locality for R. alatospora and the rediscovery of this species, which has not been reported since its description in 1937 (Perold, 2000; Wigglesworth, 1937). The new locality is within the Agulhas National Park and represents a considerable extension of the known distribution of this species. It is likely that, as in other species of the genus, R. alatospora only develops in favourable years and thus, the plants obtained in culture may have been derived from spores produced during the previous growing season or even from older seasons, owing to their resistance to long 37 periods of desiccation (Harding et al., 2000; Proctor, 1972). Riella alatospora must have been very abundant as many plants were reared from as little as 0.5 kg dried sediment collected from one side of the salt pan (Fig. 3A). However, some cultures of sediments may not be as successful as others (Hässel de Menéndez, 1987; Proctor, 1972) and care should be taken in the selection of the place they are collected from (Proctor, 1972). Usually the most promising place is just a few meters from the shore line and where visible dried debris of plants and algae have accumulated (Fig. 3A). Our finding of R. alatospora from cultures of dried sediments from a South African salt pan in an area far apart from the type locality suggests that the genus and species must be more widespread than previously thought. Similarly, other species and populations of Riella have also been discovered accidentally following this same procedure (Marín, 1982) from cultures either specifically designed for the study of Riella (Wigglesworth, 1937) or for the study of other organisms such as Crustacea (Hässel de Menéndez, 1979; Porsild, 1902). Indeed, some species of Riella have still not been discovered growing in the wild and are known only from laboratory reared specimens. This is true for the Argentinian R. pampae Hässel de Menéndez and R. undulata Hässel de Menéndez (Hässel de Menéndez, 1979, 1987) but also for some South African species such as R. capensis (Cavers, 1903) or R. purpureospora (Wigglesworth, 1937) and the Asian R. paulsenii Porsild (Porsild, 1902) for which their presence in the wild has been only sporadically confirmed in relatively recent times (Coetzer, 1987; Harding et al., 2000; Ladyzhenskaja and Obuchova, 1956). While this lack of knowledge may indicate the need for more specific sampling, the strong demographic fluctuations of Riella populations (Griffin, 1961; Proctor, 1972; Studhalter, 1933) represent a major challenge in establishing their precise distributions and abundance. Acknowledgements We are indebted to F. Ojeda (University of Cádiz) for his help in the field work and L. Loughtman (MANCH) for the loan of the specimens of R. alatospora. A.J. Ibáñez, P. Gómez, M.T. Mínguez, and E. Navarro (SCSIE-UV), for their help in SEM analysis; two anonymous referees and the editor A.R. Magee for their useful comments on a previous version of the manuscript. JGS-M and MS were supported by a Spanish Ministry of Science and Innovation Ramón y Cajal postdoctoral contract and a research stay from Serbian Ministry of Science and Technological Development (Grant 173030), respectively. References Arnell, S.W., 1957. Hepaticae collected in South West Africa by Prof. Dr. O.H. Volk. Mitteilungen der Botanischen Staatssammlung München 16, 262–272. Cavers, F., 1903. A new species of Riella (R. capensis) from South Africa. Revue Bryologique 30, 81–84. Cholo, F., Foden, W., 2006. Riella alatospora. IUCN 2011. IUCN Red List of Threatened Species. Version 2011.1www.iucnredlist.org. Coetzer, A.H., 1987. Succession in zooplankton and hydrophytes of a seasonal water on the West coast of South Africa. Hydrobiologia 148, 193–210. 38 J.G. 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