Isoetes nana, a new species from the coastal mountains of southeastern Brazil

Abstract Isoetes nana, a new species from the coastal mountains of southeastern Brazil (Serra de Itatiaia), is described, illustrated and compared to similar species. This species can be distinguished from similar species by a set of characters that include 5–15 small erect leaves reaching only up to 3.5cm long, megaspores rugulate (rarely laevigate or obscurely cristate) and microspores sparsely echinate. We include a key to identify this new species and spore images for all species that are discussed in this study. Isoetes nana is known only from the type locality, where it was reported to occur in small ponds on rocky outcrops at high elevations. We suggest it should be classified as a data deficient species based on the IUCN criteria.


Introduction
The lycophytes genus Isoetes is a heterosporous plant group occurring as aquatic in lakes, temporary ponds, streams, or terrestrial in wet soils (Pfeiffer 1922;Taylor and Hickey 1992). The genus is distributed worldwide and comprises approximately 250 species (Troia et al. 2016). In South America, Isoetes is represented by 64 species (Troia et al. 2016). The coastal mountains of southeastern Brazil represent one of the major centres of diversity and endemism in South America (Prado et al. 2015).
Isoetes is readily distinguished by its leaves containing a central vascular strand surrounded by four transversely septate lacunae, a single sunken sporangium at the base of the leaves, sporangial trabeculae and a ligule with a basal glossopodium (Gifford and Foster 1987;Pigg 1992;Moran 2004). However, species identification is a complicated task due to the morphological simplicity of the genus that provides few characters and convergence that hides the evolutionary differences amongst the species (Taylor and Hickey 1992). In this context, the most useful characters for species identification are the proportion of the sporangium wall covered by the velum, the sporangial wall colouration, habitat, habit, leaf colour and, especially, the size and ornamentation of the mega-and microspores (Pfeiffer 1922;Taylor and Hickey 2004;Troia and Greuter 2014). The cytology of Isoetes (based on n = 11) is also particularly important in the taxonomy and correlates strongly with the megaspore size (Kott and Britton 1980;Taylor et al. 1993;Troia 2001;Pereira et al. 2015).
Amongst the main contributions for the taxonomy of Isoetes in South America are the studies of Fuchs-Eckert (1982;. However, although the author presented a compilation of species (Fuchs-Eckert 1982;, a large number of these species were never validly published and a comprehensive taxonomic study for these species is still lacking. Efforts to understand the taxonomy of Isoetes prompted us to consult herbarium collections where we noted specimens of an apparently undescribed species from the coastal mountains of southeastern Brazil. Although detected earlier and informally named by Fuchs-Eckert (1982), it was never validly published. Accordingly, in this study, we provide a formal description of the species and a key to identify this new species and similar taxa.

Material and methods
Fieldwork was carried out by Ernst Heinrich Georg Ule (1854-1915), a German botanist and botanical explorer of the Amazonas and the Brazilian highlands (Stafleu and Cowan 1986), who collected the materials in March 1894 in Serra do Itatiaia, southeastern Brazil. We also tried three times to locate the new species in the field. Specimens from the following herbaria (acronyms following Thiers 2016) were analysed: BHCB, HBR, ICN, PACA, RB, UPCB and VIC (Brazil); NY and US (USA); B, M, HBG, P and K (Europe). Type specimens of all species of Isoetes analysed in this study were consulted. The species identification was based on the characters of the leaves and the macro-and microsculpture of the spores.
Scanning electron microscope (SEM) images of the spores were made by transferring the spores to aluminium stubs coated with a carbon adhesive. The stubs were then coated with gold-palladium-alloy in a sputter-coater for 180 s, and then digitally imaged using a Zeiss SIGMA VP. A minimum of 10 megaspores and 20 microspores per species were measured. We used the widely accepted terminology proposed by Punt et al. (2007) for the description of the spores and pollen.
The chromosome numbers of the species here examined were taken from Pereira et al. (2015).
Distribution and habitat. Isoetes nana is known only from the type locality at Serra de Itatiaia, Rio de Janeiro ( Figure 2). According to the label on herbarium sheets, the population was found at elevations of about 2300 m and recorded as aquatic, growing in small ponds on rocky outcrops. Comments. The two collections (Ule 98, kept in G and HBG and Ule s.n. kept in P) made by Ule at Serra de Itatiaia (Rio de Janeiro, Brazil) in March 1894 are probably a single collection. Fuchs-Eckert labelled the collection Ule 98 (G) as Isoetes nana and also cited this name in a published paper (Fuchs-Eckert 1982: 255). Hickey (1985) also pointed out the unique characters of the megaspore of I. nana in his doctoral thesis  (Hickey 1985: 142). However, neither Fuchs-Eckert nor Hickey have diagnosed and validly published this new taxon. Isoetes nana is known only from these two collections. Although we tried three times to re-collect Isoetes nana, without success (all our attempts were with bad weather, including heavy rain), no additional collections have been made until now. The lack of recent collections of this species may probably be due to two main reasons: first, because of its rarity; second, because it has been overlooked (as many Isoetes species) by botanists during fieldwork.
In the coastal mountains in southern Brazil, three other species with non-reticulate megaspore occur that are similar to this new species (Figure 2; Table 1-2): I. weberi U.   Tables 1 and 2. Furthermore, although the microsculpture of the megaspore seems to be a reliable source of taxonomic characters and it has been widely used to separate species in Selaginella (Schulz et al. 2013;Bauer et al. 2016), the megaspore microsculpture in Isoetes is rarely studied and used in the taxonomy (but see Macluf et al. 2003;Troia et al. 2012;Schafran et al. 2016). We observed that the microsculpture of the proximal-distal surfaces of the megaspores of I. nana consists of anastomosed bars, whose terminal ends are joined forming bacillae or more rarely echinulae ( Figure 7C). Amongst the analysed species, only I. maxima shows a similar microsculpture pattern ( Figure 7L), while all remaining species present terminal ends of anastomosed bars joined forming echinulae (or more rarely bacillae in I. quiririensis) ( Figures 4C, F, I and 7F, I). These data show that megaspore microsculpture may provide useful characters not only to identify I. nana, but also for the taxonomy of Brazilian Isoetes.
Since there is a correlation between spore sizes and ploidy level (Kott and Britton 1980;Troia 2001;Pereira et al. 2015) and I. nana presents mega-and microspore sizes that are similar to those of the diploids I. quiririensis and I. maxima (Table 2), we hypothesise that I. nana is also diploid.
Conservation status. Since I. nana is currently known from a single (not recently confirmed) locality, it may deserve special attention concerning its conservation status. Key to taxa of Isoetes from Serra do Itatiaia, Rio de Janeiro, Brazil and non-reticulate megaspore species from the coastal mountains of southeastern South America