Eithea lagopaivae, a new critically endangered species in the previously monotypic genus Eithea Ravenna (Amaryllidaceae)

Abstract Eithea lagopaivae Campos-Rocha & Dutilh, sp. nov. is described as the second species of the formerly monotypic genus Eithea. It is characterized by a one flowered inflorescence, completely hollow scape, white or lightly magenta-striated flower that is enclosed by spathe bracts fused for more than the lower fifth of its length. Comments on its range, habitat, phenology, as well as photographs and illustrations are provided. In addition, a distribution map and an identification key for the two species of the genus are presented and anatomical and ecological differences compared. Known by only two small populations exposed to several types of threats and without any guarantee of protection, E. lagopaivae is considered a Critically Endangered (CR) species.


Introduction
Eithea Ravenna (2002) was proposed as a monotypic genus with the transfer of Griffinia blumenavia K.Koch & C.D.Bouché ex Carrière, historically a species of somewhat uncertain classification. It was described from material collected in Santa Catarina state, Brazil, cultivated at the Berlin Botanical Garden and originally placed in the genus Griffinia Ker Gawl. (Carrière 1867) presumably because of the pseudopetiolate, evergreen leaves. Combinations for both Hippeastrum Herb. (Sealy 1937) and Amaryllis L. (Traub 1938) were subsequently proposed. Transfer to Hippeastrum was indicated due to perceived similarities with the flowers and seeds of H. reticulatum Herb. (Sealy 1937) while Traub (1938) proposed the transfer to Amaryllis, in line with his belief that the type specimen of Amaryllis belladonna L. was an American plant (see Goldblatt 1984, for full details of this controversy). Satô (1938) published a chromosome number of 2n = 77 for the species, which would be consistent with x = 11 chromosomes as a basic chromosome number for Hippeastrum (Naranjo andAndrada 1975, Flory 1977). Almost fifty years later, Traub (1983) proposed the restoration of the species in Griffinia based on the work of Arroyo (1982), who reported 2n = 20 chromosomes for the species.
A phylogenetic analysis of nrDNA ITS sequences resolved G. blumenavia as having a closer relationship to the genus Rhodophiala Presl, than with either Griffinia or Hippeastrum (Meerow et al. 2000). This work also reported a chromosome number of 2n = 18 for G. blumenavia, the number found in most Rhodophiala species (Satô 1942, Ficker 1951, Naranjo 1969, Flory 1977. Meerow et al. (2000) considered that these findings would justify the separation of G. blumenavia from Griffinia and Hippeastrum with recognition as a distinct monotypic genus. The authors of the current paper analyzed different individuals of the species and found the main somatic chromosome number of 2n = 18. However, a few cells from some individuals exhibited 19 to 20 chromosomes, perhaps explaining Arroyo (1982) report of 2n = 20. These additional smaller or supernumerary chromosomes are considered B-chromosomes (Jones and Rees 1982, Dutilh 1989, Ising 1990, Ising and Wide-Andersson 1991 due to their erratic behavior. Ravenna (2002) described the species as Eithea blumenavia (K.Koch & C.D.Bouché ex Carrière) Ravenna based on some morphological considerations, not on phylogeny nor chromosome number.
Currently, Eithea is positioned in tribe Hippeastreae, which includes Hippeastrum and Rhodophiala (Meerow et al. 2000, Oliveira 2012, García et al. 2014, 2017. In the same studies, Griffinia, sister of monotypic Worsleya, is included in the tribe Griffinieae Ravenna, a strongly supported clade. Eithea has some unusual morphological attributes for Hippeastreae, such as pseudopetiolate oblanceolate leaves with attenuate base, as well as globose seeds with elaiosome, features also found in Griffinia. However, testa of Eithea seeds contains phytomelanin, a typical trait of Hippeastreae, not found in Griffinia.
At the end of the 1990s, the researcher Celso do Lago Paiva discovered the new species in Piracicaba, São Paulo state, Brazil. Morphological, ecological and anatomical analyses conducted on material of the species over the past ten years have confirmed that it is an undescribed species of Eithea.

Material and methods
The following national and international herbaria with the most important collections of Brazilian Amaryllidaceae, particularly those from the state of São Paulo, were visited: BR, C, ESA, HRCB, IAC, K, MBM, MO, NY, RB, SP, SPF, SPSF, UEC, and US (acronyms according to Thiers 2017). The terminology used for the morphological description follows Radford et al. (1974), Meerow andSnijman (1998), andStearn (2004), with minor modifications. In addition, comments on the conservation status of the taxon are provided as recommended by IUCN (2016). GeoCAT (Bachman et al. 2011) was used to calculate Area of Occurrence (AOO) and Extent of Occurrence (EOO). Climatic data was obtained from Banco de Dados Climáticos do Brasil (EMBRAPA 2003) for the municipalities of Indaial (Santa Catarina) and Piracicaba (São Paulo). These two localities present collections of E. blumenavia and E. lagopaivae respectively. The map was drawn with ArcGIS 10.5 (ESRI).
For the morphological analysis, measurements were made on at least 15 individuals of each species, fresh plants and exsiccates. For the anatomical analysis, slides were prepared with fresh mature leaves of E. blumenavia and E. lagopaivae. At least five fully expanded and mature leaves of each species were fixed in Karnovsky (Karnovsky 1965) for 24 h, dehydrated in ethanol series (10%, 30%, 50% -1 h each) and stored in 70% ethanol. Samples of leaves from both species were selected, encompassing the middle region of the pseudopetiole and the lamina, which was subdivided into midrib region and area between the midrib and margin. Infiltration was performed in plastic resin (Leica Historesin®) according to manufacturer's instructions. Transverse and longitudinal sections of 7 µm thickness were obtained with manual rotary microtome (Leica®) and stained with 0.05% toluidine blue (Sakai 1973) in citrate buffer. Slides were mounted in synthetic resin "Entellan®" (Merck®) and images were obtained with an Olympus DP71 digital camera attached to an Olympus BX51 microscope.
Vouchers of the species and populations were deposited at UEC.
Distribution, habitat and ecology. Eithea lagopaivae is known from only two small populations separated about 50 km, each composed of less than 50 individuals. The type population (Piracicaba) occurs in the understory of an abandoned Eucalyptus plan-tation, next to fragments of deciduous and semideciduous forest, where the Corumbataí river meets the Piracicaba river. The second is located in a small fragment of semideciduous forest, near the junction of the basins of the Piracicaba and Tietê rivers in the municipality of Tietê (Figure 4). Both fragments are located on gravelly soils of litholic limestone origin (Oliveira and Prado 1989). The region presents a well-defined seasonality, with total annual rainfall of 1230 mm and precipitation of 50 mm or less, for six months, during autumn and winter. During spring and summer, rainfall exceeds 100 mm for six months, reaching close to 250 mm in January (EMBRAPA 2003). Ants were observed removing elaiosomes from the seeds of E. lagopaivae in their natural habitat, indicating that these animals might be dispersal agents, as is known for Griffinia.
Phenology. Eithea lagopaivae has been collected in bloom between October and January, and occasional blooming occurs until early March. Fruits have been observed from November.
Conservation status. With estimated AOO of 8 km 2 and EOO of 13.7 km 2 , Eithea lagopaivae can be considered as Critically Endangered [CR, B1ab(iii) + B2ab(iii)], due to the low number of known locations (≤ 5) and decline in quality of habitat (IUCN 2016). In the municipality of Piracicaba, at the end of the year 2016, when the species was again visited, two small scattered groups were encountered ca. 500 meters apart. The smaller of the two groups was in a trash dump on the side of the wooded area, and the second in an area of higher humidity, near a small stream. This fragment, on the edge of the urban sprawl of Piracicaba, is highly disturbed and subject to regular episodes of fire. The population of the Tietê municipality is in a slightly larger fragment of semideciduous forest with an impoverished understory, intense edge effects, with many lianas and invasive exotics. The area is located within a livestock breeding facility.
Etymology. The epithet is a tribute to Celso do Lago Paiva, environmental analyst at ICMBio, who has collected the plant for the first time and has dedicated his life to the study and conservation of the flora of Brazil.
Additional Notes. Eithea lagopaivae and E. blumenavia form a clade with maximal support in all phylogenetic analyses performed by García et al. (2017). Eithea lagopaivae can be distinguished from E. blumenavia by a number of characteristics (Table 1). It is a smaller plant (ca. 12-25 cm), usually with 2-3 leaves, rarely 4, which are deciduous before the onset of winter. Eithea blumenavia however is evergreen, with 2-8 leaves, and up to 50 cm in height, although specimens of extremely reduced size are known, also with several flowers.
Eithea lagopaivae is known from an area originally of deciduous and semideciduous forests with a well-defined dry season. In turn, E. blumenavia is found in wetlands of the Atlantic rainforest, from the south of the state of São Paulo to eastern Santa Catarina (Dutilh 2010, Dutilh andOliveira 2015) (Figure 4), especially in the coastal mountains. The region has some of the highest average annual rainfall (1650 mm) of any area of extra-Amazonian Brazil, distributed throughout the year, but more intensely during the summer, although with an average under 200 mm/month. From April to July, monthly averages are close to 100 mm (EMBRAPA 2003).
Eithea blumenavia is considered an Endangered (EN) species (MMA 2014). Anatomy. The three most obvious anatomical characteristics differentiating the two species of Eithea are: 1. Margins and cortex of the pseudopetiole ( Figure 5A Cross section of pseudopetiole margins of E. lagopaivae are flatter, more laminar ( Figure 5A, arrow) than those of E. blumenavia, which are angular ( Figure 5B, arrow). The pseudopetiole is composed by chlorenchyma, aerenchyma and vascular bundles. In E. lagopaivae, 1-3 aerenchyma lacunae were found below the vascular bundles (Figure 5A), while in E. blumenavia several lacunae above and below the bundles could be observed ( Figure 5B). In the cross section of leaf lamina, epidermal cells were more elongated in E. lagopaivae and polyhedral in E. blumenavia ( Figure 5C-D, respectively). We found periclinal thickening on the outer wall of the epidermal cells of both species as occurs in Hippeastrum puniceum (Lam.) Kuntze (Alves-Araújo et al. 2012). Ornamentation of the external periclinal epidermal cell wall of E. blumenavia ( Figure 5D, arrow) was not found in E. lagopaivae ( Figure 5C). Mesophyll of E. lagopaivae and E. blumenavia is composed of about 6-8 layers of chlorenchyma with arm-palisade cells (also called arm-cells, H-palisade or H-cells) ( Figure 5E-F), which showed their typical morphology in paradermic sections ( Figure 5G-H). Arm-cells were first described by Haberlandt (1880) as a morphological modification of palisade cells and seem to be more common in plants of forest understory, probably increasing photosynthetic capacity (Chatelet et al. 2013). In the midrib region, the lacunae of the aerenchyma were larger and wider in E. lagopaivae than in E. blumenavia ( Figure 5I-J).
A protrusion on the abaxial leaf surface opposite the central vascular bundle was evidenced in both species ( Figure 5I-J, arrows) and the parenchyma cells in this region were regular and rounded. However, adaxial surface of the leaf in E. lagopaivae was flat ( Figure 5I), while in E. blumenavia it was possible to observe two protrusions opposite to the vascular bundles adjacent to the midrib ( Figure 5J, arrowheads). The alternation of aerenchyma with vascular bundles found in Eithea species was described for other species of the family (Arroyo and Cutler 1984, Meerow 1989, Raymúndez et al. 2000,