Abstract

Isoetes candelariensis is a new species of Isoetaceae from Misiones, Argentina. This species is ephemeral, and grows on basaltic bedrock outcrop pools, in Urutau Reserve from Candelaria Department. This taxon differs from other aquatic Isoetes in this region by the unique combination of characters of its leaves, ligule, labium and megaspore ornamentation.

Key words
Isoetes; new species; lycophyte; Argentina

INTRODUCTION

Isoetes L., the only genus of Isoetaceae, constitutes a cosmopolitan genus of approximately 200 species (Troia et al. 2016TROIA A, PEREIRA JB, KIM C & TAYLOR WC. 2016. The genus Isoetes (Isoetaceae): a provincial checklist of the accepted and unresolved taxa. Phytotaxa 277: 101-145.). Isoetes species occur on all continents but Antarctica, found in a wide variety of permanent and ephemeral wetlands habitats from sea level to 4200 m (Pfeiffer 1922PFEIFFER NE. 1922. Monograph on the Isoetaceae. Trans Acad Sci St Louis 9: 79-233., Tryon & Tryon 1982TRYON RM & TRYON AF. 1982. Isoetaceae. In: TRYON RM & TRYON AF (Eds), Ferns and allied plants with special reference to Tropical America, Springer, New York, p. 826-834., Jermy 1990JERMY AC. 1990. Isoetaceae. In: KRAMER KU & GREEN PS (Eds), p. 24-31, The Families and Genera of Vascular Plants Volume 1: Pteridophyes and Gymnosperms, Springer-Verlag, Berlin., Brunton & Troia 2018BRUNTON DF & TROIA A. 2018. Global review of recent taxonomic research into Isoetes (ISOETACEAE), with implications for biogeography and conservation. Fern Gaz 20(8): 309-333.). Many species are terrestrial, while others are submerged in shallow water along lake margins, or grow as true aquatics to depths of 4-5 m in lakes or streams. Under optimum conditions, the plants may form dense lawns. While typically reproducing sexually, some Isoetes taxa reproduce vegetatively by gemmae (Hickey 1986aHICKEY RJ. 1986a. The early evolutionary and morphological diversity of Isoetes, with descriptions of two new Neotropical species. Syst Bot 11: 304-321.). This last condition is rare, if not unknown, in South America.

Isoetes is characterised by leaves with four air-chambers, and single micro- or mega sporangia inserted into inner leaf bases, which typically are covered by the velum (Hickey et al. 2009HICKEY RJ, MACLUF CC & LINK-PÉREZ M. 2009. Isoetes maxima, a New Species from Brazil. Amer Fern J 99(3): 194-199. https://doi.org/10.1640/0002-8444-99.3.194.
https://doi.org/10.1640/0002-8444-99.3.1... , Pereira et al. 2012PEREIRA JBS, WINDISCH PG, LORSCHEITTER ML & LABIAK PH. 2012. Isoetes mourabaptistae, a New Species from Southern Brazil. Amer Fern J 102(2): 174-180. https://doi.org/10.1640/0002-8444-102.2.174.
https://doi.org/10.1640/0002-8444-102.2.... , 2019). The megaspores and microspores ornamentation is very important in taxonomic identification within the genus; scanning electron microscopy (SEM) images have proven very helpful in the interpretation of subtle but informative spore variations (Kott & Britton 1983KOTT LS & BRITTON DM. 1983. Spore morphology and taxonomy of Isoetes in northeastern North America. Can J Bot 61: 3140-3164. doi.org/10.1139/b83-353., Brunton & Troia 2018BRUNTON DF & TROIA A. 2018. Global review of recent taxonomic research into Isoetes (ISOETACEAE), with implications for biogeography and conservation. Fern Gaz 20(8): 309-333.). Cytology and more recently DNA sequencing, have provided invaluable insights into Isoetes diversity, evolution and taxonomic affinities (Hoot et al. 2006HOOT SB, TAYLOR WC & NAPIER NS. 2006. Phylogeny and biogeography of Isoetes (Isoetaceae) Based on Nuclear and Chloroplast DNA Sequence Data. Sys Bot 31: 449-460., Wood et al. 2020WOOD D, BESNARD G, BEERLING DJ, OSBORNE CP & CHRISTIN PA. 2020. Phylogenomics indicates the “living fossil” Isoetes diversified in the Cenozoic. PLoS ONE 15(6): e0227525. https://doi.org/10.1371/journal.pone.0227525.
https://doi.org/10.1371/journal.pone.022... ).

Eight Isoetes species are reported in Argentina from the provinces of Buenos Aires, Misiones, Corrientes, Córdoba, Tucumán, Catamarca and the Patagonian region (Weber 1922WEBER U. 1922. Zur Anatomie und Systematik der Gattung Isoetes L. Hedwigia 63: 219-262., Macluf 2016MACLUF CC. 2016. Isoëtaceae. In: ANTON AM & ZULOAGA FO (Eds), Flora Argentina: flora vascular de la República Argentina, Vol. 2, Licofitas y Helechos, Instituto de Botánica Darwinion, Argentina, p. 3-9. https://doi.org/10.2307/j.ctt1p0vjr5.
https://doi.org/10.2307/j.ctt1p0vjr5... ).

During a vascular flora survey in the Urutaú Reserve and surrounding areas, near the city of Candelaria in Misiones Province, Argentina, some populations of Isoetes were found in small pools on a basaltic bedrock outcrop (Figures 1, 2). Investigation about that material revealed a combination of morphological attributes inconsistent with species described to date.

On the basis of a comparative study of closely associated species from the region, the present investigation provides evidence that this population constitutes a previously undescribed species.

MATERIALS AND METHODS

Dried herbarium material was studied with a Wild M5 stereoscopic microscope (SM) and a scanning electron microscope (SEM). For studies with SEM, the spores were handled without any chemical treatment and placed with a moist brush on doublestick tape attached to bronze stubs. Samples were coated with gold and examined under a Jeol JSM-35 CF at the SEM laboratory of the Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata, Argentina. To estimate the average measurements of the spores, we used a minimum of 20 spores per sporangium, from at least three sporangia. Terminology for the discussion regarding spore morphology follows Hickey (1986b)HICKEY RJ. 1986b. Isoetes Megaspore Surface Morphology: Nomenclature, Variation, and Systematic Importance. Amer Fern J 76: 1-16..

RESULTS

Taxonomic treatment

Isoetes candelariensis C.C. Macluf & G.J. Marquez, sp. nov. TYPE Argentina, Misiones province: Department Candelaria, Reserva Urutaú, 27° 29´ 20.33´´ S, 55° 43´ 47.15´´ W, Charcos de agua en basalto aflorante [pools on a basaltic bedrock outcrop], 5 july 2019, H. A. Keller & G. Ruiz Díaz 13900 (Holotype LP!, Isotype CTES!). (Figures 1, 2, 3).

Plants herbaceous (less than 15 cm tall). Roots numerous, dichotomously branched. Corm bilobed, globose, 4-5 mm wide, 5 mm long. Leaves 45-50, spirally arranged, erect and recurved distally, 9-14cm long, 0.5-2.5 mm wide at the base, 0.2-1 mm wide at mid length; alae hyaline and chartaceous proximally, dark green and membranaceous distally, 1 mm wide at the height of the sporangium, 12-14 cm long (extending to 80% of the leaf length); subula acute, dark green, their apex long acuminate; scale leaves present. Sporangia elliptic, hyaline, concolorous, 4-5 mm long, 3-4 mm wide. Velum incomplete, extending 60 % across sporangium from the top. Ligule 1-1.5 mm high, 0.5 mm wide; black, delicate, ephemeral; deltate to widely ovate. Megaspores white, 342 ([387.75] µm (N=20, SD=29.47, 3 plants) 425 µm in diameter, laevigate to obscurely rugulate (in distal surface) with velutinous perispore (spore surface) and no equatorial band (girdle); equatorial and proximal ridges straight, as high as broad (Figures 4a, 4b, 4c). Microspores dark brown en masse, 28 ([30] µm (N=20, SD=1.45, 3 plants)) 32 µm long, densely echinate with broad-based spines (Figures 4d, 4e, 4f).

Etymology

The proposed epithet refers to the Department Candelaria locality where the type material was collected.

Additional specimens examined

Argentina, Misiones province: Department Candelaria, Reserva Urutaú, 27° 30´ 25.4´´ S, 55° 47´ 20.6´´ W, 9 june 2022, H. A. Keller & G. Ruiz Díaz 15116 (LP, CTES); ídem, 27° 30´ 20.34´´ S, 55° 47´ 14.12´´ W, 5 August 2022, H. A. Keller & G. Ruiz Díaz 15231 (LP, CTES); ídem, 27° 30´ 28.33´ S, 55° 47´ 16.84´´ W, 5 August 2022, H. A. Keller & G. Ruiz Díaz 15232 (LP, CTES); ídem, 27° 30´ 26.24´ S, 55° 47´ 21.01´´ W, 5 August 2022, H. A. Keller & G. Ruiz Díaz 15233 (LP, CTES); ídem, 27° 29´ 20´ S, 55° 48´ 47´´ W, 5 August 2022, H. A. Keller et al. 15234 (LP, CTES); ídem, 27° 29´ 24´ S, 55° 43´ 44´´ W, 5 August 2022, H. A. Keller & G. Ruiz Díaz 15235 (LP, CTES).

Distribution and habitat

This species occurs in Reserva Urutaú (Misiones, Argentina) (Figure 1), and grows in shallow, ephemeral pools and seepage areas on basalt bedrock outcropping (Figures 2a, 2b). The individuals are small grasses that grow on the waterlogged basalt, with the leaves grow outside water, and can be seen together with other swamp species (Figures 2c, 2d). Exist three populations, and each has less than 50 mature individuals, which appears during a short annual period (June to August).

DISCUSSION

Eight species of Isoetes grow in Argentina: I. alcalophila S. Halloy (Halloy, 1979), I. chubutiana R.J. Hickey, C.C. Macluf & W.C. Taylor (Hickey et al. 2003HICKEY RJ, MACLUF CC & TAYLOR WC. 2003. A re-evaluation of Isoetes savatieri Franchet in Argentina and Chile. Amer Fern J 93(3): 126-136.), I. ekmanii U. Weber (Weber, 1922), I. escondidensis S. Halloy (Halloy, 1979), I. gardneriana Kunze ex Mett. (Mettenius, 1859). I. hieronymii Weber (Weber, 1922, Macluf et al. 2009MACLUF CC, MEZA TORRES EI & MARQUEZ GJ. 2009. Isoëtes hieronymi (Isoetaceae-Lycophyta) endémica de Pampa de Achala, Córdoba, Argentina. Bol Soc Argent Bot (Supl.) 44: 210.), I. pedersenii H.P. Fuchs ex E. I. Meza T. & C.C. Macluf (Macluf et al. 2010MACLUF CC, MEZA TORRES EI & SOLÍS SM. 2010. Isoetes pedersenii, a new species from Southern South America. An Acad Bras Cienc 82: 353-359. https://doi.org/10.1590/S0001-37652010000200011.
https://doi.org/10.1590/S0001-3765201000... ) and I. savatieri Franchet (Franchet, 1884). I. candelariensis is closest in geographic distribution and habitat with I. ekmanii and I. pedersenii (Figure 1).

Isoetes ekmanii, which occurs in lowland from Argentina and Brazil (Giudice et al. 2011GIUDICE GE, RAMOS GIACOSA JP, LUNA ML, MACLUF CC, PONCE M, MARQUEZ GJ & DE LA SOTA ER. 2011. Evaluación preliminar del grado de amenaza de los helechos y licofitas de Argentina. Bol Soc Argent Bot 46(1-2): 151-161.), grows both as an emergent in silty river banks and as a permanently submerged aquatic (Macluf 2012MACLUF CC. 2012. Análisis morfológico, palinológico y sistemático de las Isoetales del Cono Sur de América del Sur. Doctoral thesis. Universidad Nacional de La Plata, Argentina, 259 p. https://doi.org/10.35537/10915/25302.). I. candelariensis has similar leaf features with I. ekmanii and both of them grow in the Candelaria department (Misiones). However, I. ekmanii have megaspores typically reticulate (Table I, Figures 5a, b). Microspores are similar in I. candelariensis, I. ekmanii and I. pedersenii (Figures 4d, e, f, 5c, f).

I. pedersenii grows in flooded land of Dep. Mburucuyá, Corrientes province, and probably it is found in the adjacent regions of Argentina, Paraguay and Brazil (Macluf et al. 2010MACLUF CC, MEZA TORRES EI & SOLÍS SM. 2010. Isoetes pedersenii, a new species from Southern South America. An Acad Bras Cienc 82: 353-359. https://doi.org/10.1590/S0001-37652010000200011.
https://doi.org/10.1590/S0001-3765201000... ). The major differences with I. candelariensis is the megaspores with verrucae ornamentation (Table I, Figures 5d, e).

The megaspore and microspore ornamentation of Isoetes candelariensis is somewhat similar to that in Isoetes maxima Hickey, Macluf & Link-Perez, from southeastern Brazil. However, megaspore ornamentation of I. maxima is rugulate to tuberculate in distal surface (Hickey et al. 2009HICKEY RJ, MACLUF CC & LINK-PÉREZ M. 2009. Isoetes maxima, a New Species from Brazil. Amer Fern J 99(3): 194-199. https://doi.org/10.1640/0002-8444-99.3.194.
https://doi.org/10.1640/0002-8444-99.3.1... , Pereira et al. 2017PEREIRA JBS, STÜTZEL T & SCHULZ C. 2017. Isoetes nana, a new species from the coastal mountains of southeastern Brazil. PhytoKeys 89: 91-105. https://doi.org/10.3897/phytokeys.89.20171.
https://doi.org/10.3897/phytokeys.89.201... ), and the equatorial diameter size is higher (525-650 µm) than I. candelariensis (342-425 µm). In addition, other characters, like leaves, alae, velum and sporangium (Table I), differentiate the species. Another ecological characteristic to differentiate both species is that I. maxima grows submerged in streams at high elevations (900 to 1200 m. a.s.l.) while I. candelariensis live in seepage areas on basalt bedrock outcropping to no more than 150 m. a.s.l. (Hickey et al. 2009HICKEY RJ, MACLUF CC & LINK-PÉREZ M. 2009. Isoetes maxima, a New Species from Brazil. Amer Fern J 99(3): 194-199. https://doi.org/10.1640/0002-8444-99.3.194.
https://doi.org/10.1640/0002-8444-99.3.1... ).

As it has been reported with Isoetes pedersenii (Macluf et al. 2010MACLUF CC, MEZA TORRES EI & SOLÍS SM. 2010. Isoetes pedersenii, a new species from Southern South America. An Acad Bras Cienc 82: 353-359. https://doi.org/10.1590/S0001-37652010000200011.
https://doi.org/10.1590/S0001-3765201000... ), some microspore spines on I. candelariensis are hollow (arrowheads and detail in Fig. 4d). This characteristic has also been reported from India (Srivastava 1995SRIVASTAVA GK. 1995. The microspore structure of Indian species of Isoetes. Palynology 31: 35-44.) as well as in Isoetes plants in Canada, the United States, Australia and several countries in eastern Mediterranean (D. F. Brunton pers. comm.). The taxonomic and/or physiological significance of this is unknown.

Isoetes weberi Herter, I. maxima and I. quiririensis J. B. S. Pereira & P.H. Labiak (Pereira & Labiak 2013PEREIRA JBS & LABIAK PH. 2013. A New Species of Isoetes with tuberculate spores from Southeastern Brazil (Isoetaceae). Syst Bot 38(4): 869-874. Available from: http://www.bioone.org/doi/full/10.1600/036364413X674742.) are the only species from southern Brazil with non-reticulate megaspores like I. candelariensis. Accordingly, I. candelariensis may be considered to have at least some morphological affinity with these Brazilian species, which live in the same biogeographical region, the Atlantic Forest.

Conservation status

Isoetes candelariensis is currently known only from type locality. According to IUCN Red List Criteria (IUCN 2012IUCN - INTERNATIONAL UNION FOR CONSERVATION OF NATURE. 2012. IUCN Red List Categories and Criteria, version 3.1. Second edition. IUCN Red List Unit, Cambridge U.K. Available from: https://portals.iucn.org/library/sites/library/files/documents/RL-2001-001-2nd.pdf.
https://portals.iucn.org/library/sites/l... , 2022) this species may qualify Data Deficient (DD), because the geographic distribution and possible threats are so imperfectly known. Regardless, the limited availability of potential habitat suggests this will be a taxon of significant conservation concern at some level. For example, in 2011 the elevation of the Yacyreta dam to 83m changed the dynamics of the Paraná River, flooding many sectors of the Department of Candelaria. Future environmental changes associated with the reservoir of this dam may be a potential hazard.

ACKNOWLEDGMENTS

D. F. Brunton, for your important contribution in improving of the manuscript. The Temaiken Foundation, and especially Maria Paula Bertolini and Jerónimo Torresín, are thanked for promoting the studies that allowed us to find the subject population. Depimiel S. A. Company gave permission for us to study the vegetation on their property adjacent to the Urutau Reserve. Ranger Gabriel Ruiz Diaz provided valuable assistance during the field investigations. Funding was provided from the National Agency for the Promotion of Research, Technological Development and Innovation (PICT 0043/17).

REFERENCES

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