Isoetes dubsii and Isoetes santacruzensis, two new species from lowland areas in South America

Abstract Isoetes dubsiisp. nov. and I. santacruzensissp. nov., two new species from lowland areas in South America, are described, illustrated and compared to similar species. Isoetes dubsii can be distinguished from other species of the Brazilian Pantanal wetlands by a set of characters including leaves that are long, flexuous and trigonal in transverse section, tri-lobate stems, rudimentary velum, pustulate megaspores of 310‒390 µm diameter and laesurae of the megaspore at least four times wider than high. Isoetes santacruzensis has flexuous, filiform leaves, 0.4–0.8 mm wide at mid length and reaching up to 15 cm long, black or reddish-black sporangia, sclerified phyllopodia and sparsely verrucate megaspores of 320‒390 µm in diameter. We also include a key for species from the Brazilian Pantanal wetlands and Bolivia and spore images for all species that are discussed. Isoetes dubsii and I. santacruzensis are only known from their type localities and they may deserve special attention concerning their conservation status. However, based on our current knowledge on these species and according to IUCN Red List criteria, they are assessed here as data deficient (DD).


Introduction
Herbarium collections are amongst the most important tools for obtaining information about the composition, distribution and content of plant diversity in a given region (Nualart et al. 2017). They represent a cumulative body of knowledge, which has been generated over time. It is also well known that many undescribed species reside in existing herbarium collections (Bebber et al. 2010).
Isoetes L. is the unique extant genus of heterosporous lycophytes in the Isoetales (PPG I 2016). Isoetes is morphologically well defined and readily distinguishable from any other group of vascular plant by its narrow leaves containing four air-chambers, a single sunken adaxial sporangium covered by a velum and sporangial trabeculae . The genus comprises about 250 species (Troia et al. 2016). South America is one of its centres of taxonomic diversity (Troia et al. 2016) with an estimated 64 species (Hickey et al. 2003). Most of the species are narrowly endemic and they occur as aquatic or terrestrial plants in wet soils (Pfeiffer 1922). However, Isoetes species are notorious for the difficulties they present in identification, which are partially associated with morphological simplicity (Taylor and Hickey 1992).
Difficulties in the identification have frequently led many Isoetes specimens to be deposited in herbaria without determinations or with wrong determinations (Troia and Rouhan 2018). The sculpture of the megaspore is one of the most important characters in the taxonomy of the genus (Pfeiffer 1922) and, in many cases, scanning electron microscopy images (SEM) of megaspores are needed to identify species (Hickey 1986a). The use of SEM adds logistic difficulties in the taxonomy of the genus and, as a result, many Isoetes species remain unidentified and/or undescribed in herbarium collections.
Our recent efforts to access the diversity of Isoetes in South America has led us to consult herbarium collections where we discovered two species that we recognised as undescribed. One of the new species is from Bolivia and the other is from Pantanal wetlands in Brazil. We provide descriptions to distinguish these species, SEM images of mega-and microspores of species of these regions and a key for the identification of these new species.

Material and methods
Fieldwork was carried out by Balthasar Dubs, a Swiss botanist and ornithologist who intensively collected plants in the Pantanal wetlands in Brazil and found Isoetes dubsii on 3 June 1988 in the Pantanal do Rio Negro (currently belonging to the municipalities of Aquidauana and Corumbá), in the state of Mato Grosso do Sul, mid-western Brazil. We also tried to locate this new species in the same area in November 2017. For I. santacruzensis, fieldwork was carried out by Timothy J. Killeen on 11 November 1994 in the province of Ñuflo de Chávez, Department of Santa Cruz, Bolivia. Spore images were generated by transferring the spores to aluminium scanning electron microscope (SEM) stubs coated with a carbon adhesive. The stubs were then coated with gold-palladium-alloy in a sputter-coater for 180 sec, after which the spores were digitally imaged using a Zeiss SIGMA VP. The resulting images were adjusted in Photoshop for contrast and the background was altered to black. To measure the spores, we used a minimum of 20 spores per sporangium, from at least two sporangia. The spore measurements were taken using SEM. The terminology used for the description of the spores follows that of Punt et al. (2007), with some modification using Hickey (1986a).

Taxonomic treatment
Isoetes dubsii J.B.S.Pereira, sp. nov. urn:lsid:ipni.org:names:77201652-1 Figs 1, 2 Diagnosis. Isoetes dubsii is distinguished from other species from the Brazilian Pantanal wetlands by a set of characters that include leaves that are long (90-100 cm), flexuous and trigonal in the transverse section, tri-lobated stems, rudimentary velum, pustulate and small megaspores of 310-390 µm in diameter (average 350 µm) and laesurae of the megaspore at least four times wider than high.
Distribution and habitat. This species is only known from its type locality (state of Mato Grosso do Sul, Brazil; Fig. 3), where it grows in a fresh water pond at ca. 100 m a.s.l. Although we tried to re-collect Isoetes dubsii in the area indicated by Balthasar Dubs, no additional collections have been made.
Etymology. The specific epithet honours the Swiss ornithologist and botanist B. Dubs, for his valuable contributions to the knowledge of the flora and fauna of the Pantanal wetlands of Brazil. He was also the first naturalist to collect Isoetes dubsii.
Distribution and Habitat. Isoetes santacruzensis is only known from its type locality, where it grows as aquatic to ephemeral terrestrial in rocky granite outcrops, at elevations of about 450 m.
Etymology. The specific epithet refers to the type region, the Department of Santa Cruz in Bolivia (Fig. 3).
Notes. Until now, six species of Isoetes were known from Bolivia, although the presence of unpublished species has already been mentioned (Kessler and Smith 2018). Most of the known Bolivian species are from Andean habitats (Kessler and Smith 2018) and have rugulate, laevigate (Fig. 5) or tuberculate megaspores (see Hickey 1986b, Fig. 2) and laevigate, echinate or tuberculate microspores (Fig. 5).  Besides habitat, the macrosculpture of at least one of the spore types, megaspore or microspore, helps to differentiate I. santacruzensis from the Andean Isoetes species (Figs  2, 4). Additionally, I. santacruzensis is similar to I. pedersenii by its small and verrucate megaspores. However, I. santacruzensis can be readily distinguished by its erect and flexuous leaves (vs. ascending, linear and straight; Fig. 6), as well as by the characters present in the taxonomic key.
Conservation status. Isoetes santacruzensis is currently known from a single locality. The expansion of agricultural activities and cattle farming in this area show that this species may be prone to the effects of human activities within a very short time. However, given its potential occurrence in other areas and the lack of current knowledge about its distribution range, I. santacruzensis should be assessed as data deficient (DD), according to IUCN criteria (IUCN 2012).
Key to the species from the Brazilian Pantanal wetlands and Bolivia