Genlisea hawkingii (Lentibulariaceae), a new species from Serra da Canastra, Minas Gerais, Brazil

Genlisea hawkingii, which is a new species of Genlisea subgen. Tayloria (Lentibulariaceae) from cerrado in southwest Brazil, is described and illustrated. This species has been found in only one locality thus far, in the Serra da Canastra, which is located in the Delfinópolis municipality in Minas Gerais, Brazil. The new species is morphologically similar to Genlisea violacea and G. flexuosa, but differs from them in having a corolla with a conical and curved spur along with sepals with an acute apex and reproductive organs that only have glandular hairs. Moreover, it is similar to G. uncinata’s curved spur. G. hawkingii is nested within the subgen. Tayloria clade as a sister group to all the other species of this subgenus. Therefore, both morphological and phylogenetic results strongly support G. hawkingii as a new species in the subgen. Tayloria.


Introduction
Genlisea A.St.-Hil. is a mainly Neotropical genus that includes ca. 30 species of carnivorous plants [1,2]. The genus belongs to the family Lentibulariaceae [3] and has recently aroused special interest for its tiny genomes, such as that of Genlisea tuberosa, which is the smallest genome that has ever been found in angiosperms [4]. Genlisea was first split into two sections and separated according to fruit dehiscence: Genlisea sect. Genlisea, which has a circumscissile dehiscence, and G. sect. Tayloria, whose fruits open through longitudinal slits [5]. In more recent times, Fromm-Trinta's Tayloria [5] was raised to the subgeneric rank [6]. Both subgenera are considered to be monophyletic groups according to molecular phylogenetic data [1,7].
The Genlisea subgen. Tayloria includes eight species all endemic to Brazil [1,8,9], and chiefly colonizing the highlands in Minas Gerais. Particularly, most of the species are from Serra do Espinhaço in Minas Gerais [10]. They are mainly annual species, though some are known to be perennials [1]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 During field trips, we discovered Genlisea plants that did not seem to belong to any species that had been previously described. Although detailed comparisons revealed similarities between G. violacea and G. flexuosa, significant differences were also observed.
Here, we describe a new species of Genlisea, endemic to the Serra da Canastra region of the Delfinópolis municipality in Minas Gerais. We also provide a full taxonomic description, comments, photographs and illustrations for the new species as well as a phylogenetic analysis based on the chloroplast DNA regions of Genlisea subgen. Tayloria, in order to assess the phylogenetic position of the new taxon.

Plant material
We collected the specimens in Serra da Canastra in the Delfinópolis municipality, Minas Gerais, Brazil. The individuals were pressed for dry vouchers and then fixed in a FAA70 (Formaldehyde-Glacial Acetic Acid-Ethyl Alcohol 70%) solution for the morphological analyses, which were performed using a stereomicroscope and light microscope. In addition, some specimens were fixed in a mixture of 5% glutaraldehyde with 2.5% formaldehyde in a 0.006-M cacodylate buffer (pH 7.2) and the morphological analyses were performed using a scanning electron microscope (SEM). For the SEM, the fixed material was dehydrated, subjected to critical drying point using liquid CO 2 , and sputter-coated with gold and digital photos of the analyzed material were taken with a Hitachi S-4700 scanning electron microscope (Hitachi, Tokyo, Japan). The morphological traits were measured for all of the collected specimens using a digital caliper and a Leica1 stereomicroscope using the Leica1 IM50 program, calibrated for the magnification used. The comparison between similar species was based on field observations, herbarium specimens, and information that has been gathered from species' protologues and monograph on Genlisea [1]. The morphological terminology and structure of the description follow [1]. The herbarium abbreviations that are cited in the text follow [11].

Molecular analyses
For the phylogenetic analyses, DNA was extracted from floral axes and flowers of the new Genlisea species using a Qiagen1 DNeasy Plant Mini Kit, thus avoiding any contamination from other organisms, such as prey, from the photosynthetic leaves and carnivorous traps. The chloroplast DNA regions were amplified and sequenced from the rps16 region using the primers RPSF 5'-GTGGTAGAAAGCAACGTGCGACTT-3' and RPSR2 5'-TCGGGATCGAACATCAA TTGCAAC-3' [12], which were used in previous Genlisea studies [7] and matK using the primers 3F-KIM 5'-CGTACAGTACTTTTGTGTTTACGAG-3' and 1R-KIM 5'-ACCCA GTCCATCTGGAAATCTTGGTTC-3', which were designed for plant DNA barcoding studies by Kim [13]. Reaction conditions for the rps16 region included denaturation at 95ºC for 5 min followed by 29 cycles of 30 sec at 95ºC, 30 sec at 52ºC and 2 min at 72ºC, followed by a final extension at 72ºC for 5 min. For the matK region, the reaction was performed with denaturation at 94ºC for 1 min followed by 35 cycles of 40 sec at 94ºC, 20 sec at 52ºC and 50 sec at 72ºC, followed by a final extension at 72ºC for 5 min. All amplifications were performed in a PTC-100 (MJ Research) thermal cycler. The sequences were amplified for both strands (forward and reverse) and the consensus sequences were assembled using BioEdit v. 7.0.5 [14].

Phylogenetic analyses
Using the sequences of other Genlisea species, Utricularia gibba and Pinguicula alpina (Table 1), which are available in GenBank (NCBI), the sequences were aligned using the online version of MAFFT v. 7 [15]. The Pinguicula and Utricularia sequences were used as the outgroup. The matrices were trimmed according to the Genlisea amplified sequences and the genes were concatenated into a single matrix. All gaps were treated as missing. Two methods were used for the phylogenetic reconstruction: maximum likelihood (ML) using RAxML v. 8 [16] software and Bayesian inference (BI) using Mr. Bayes v. 3.2.2 [17]. For the BI, 5×10 7 generations were calculated until the standard deviation reached a value below 0.01 using two runs with four chains. In each run, the trees were sampled every 1,000 generations at a sample Table 1. Genbank accession numbers of the taxa that were used in this study. The sequences indicated by an � were newly generated. The species names of G. flexuosa and G. metallica were changed from the Genbank reference according to [1].

Species
Genlisea subgenus matK rps16 region  frequency of 100. The first 25% of the trees that were initially produced were discarded as burn-in. The BI analyses was conducted using the TVM+G model and was calculated using jModeltest v. 2 software [18] following the Akaike information criterion [19]. The ML was run using the GTRGAMMA model and the bootstrap support values were generated with 1,000 pseudo-replicates using a rapid bootstrap algorithm [20] implemented in RAxML. All of the phylogenetic and model test analyses were conducted using the CIPRES Science Gateway online platform [21]. The trees were edited using TreeGraph v. 2 [22].

Nomenclature
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Ethics statement
Because the samples were not collected from a conservation unit, no collection permits were required. Thus, the field studies did not involve endangered or protected species.

Description
Habit small terrestrial annual herb, up to 30 cm tall; lax rosette of ca. 20 leaves. Leaves numerous, spatulate, lamina obovate, green or dark green, with apex rounded, up to 12 mm long and 1.5-3.0 mm wide, glabrous, petiole 4-5(8) mm long, flattened, widened into the lamina. Rhizophylls many, uniform, up to 53 mm long, the largest part consisting of the trap arms; stalk short, up to 2.8 mm long and 0.5 mm wide; vesicle narrowly cylindrical, up to 1.3-3.6 mm long up to 1.3 mm wide; neck 3.5-6.8 mm long and up to 0.7 mm wide; trap arm up to 30 mm in length and width with ca. 24 twists. Inflorescence lax raceme, many-flowered, unbranched, up to 27 cm long and 0.4 mm thick diameter (0.6 mm near the base); scape, bracts, bracteoles and calyx densely covered by stalked glandular capitate hairs 0.15-0.2 mm long. Scapes up to 3, bracts narrowly obovate to linear-triangular, 1.4 mm long and 0.5 mm wide; bracteoles subulate, up to 1.1 mm long and 0.3 mm wide. Flowers 1-7 per inflorescence distally inserted on the branched axis; Pedicels slightly curved at anthesis, 7-14 mm long, 0.4 mm diameter, during fructification pedicels are elongated, up to 18 mm long and curved downwards, and densely covered with glandular capitate hairs. Sepals subequal, lanceolate with acute apex, about 0.7 mm long and 0.6 mm wide, densely covered with glandular capitate hairs. Corolla 7-10 mm long (excluding the spur), pale lavender to lilac, with two yellow ridges forming a round marking at the base of the lower lip, a few individuals have a white blotch in the lower lip, the upper lip often has darker purple streaks along the nerves, margins with glandular capitate hairs; upper lip ovate has one third divided in two lobes, each lobe ca. 1.5 mm wide, with apex cleft; lower lip up to 7 mm long and 10 mm wide, trilobate, lobes subequal, median lobe 3 mm wide, lateral lobes 2.5 mm wide, short with apex obtuse to slightly retuse; spur conical, apex curved, straightening towards the apex, longer than the lower lip, 6 mm long and 1.3 mm in diameter at the base and 0.3 m in diameter at the apex, covered with glandular capitate hairs. Capsule globose, (1.7)2-3 mm, densely covered with only glandular hairs, opening longitudinally bivalvate. Seeds prismatic, 0.20-0.26 mm long and 0.24-0.31 mm wide, 0.14 mm high, testa reticulate with cells polygonal, isodiametric, anticlinal boundaries straight and raised periclinal walls tabular.

Distribution
So far Genlisea hawkingii has only been found in Serra da Canastra, in the Delfinópolis municipality in Minas Gerais, Brazil (Fig 4).

Etymology
The species epithet 'hawkingii' was attributed as homage to the great English theoretical physicist and cosmologist, Stephen William Hawking, who died on March 14, 2018. We were impressed with his life's trajectory and his outstanding discoveries in cosmology. He became a signpost not only for other scientists but for all people.

Conservation status
The conservation status according to the IUCN [23] is Data Deficient (DD). The only known population of this species is currently found in Serra da Canastra near the Delfinópolis municipality (Minas Gerais, Brazil). The population occupied~90 m 2 and around 80 individuals were found. However, more populations may be found as this area has been poorly explored. This location suffers from an anthropogenic impact-cattle, and horses trampling, as it is located inside a farm near the boundaries of the Serra da Canastra National Park.

Ecology and phenology
Genlisea hawkingii is a montane species (1,080-1,140 m) which grows among rocks, on shallow and sandy soils and also been near perennial water bodies such as streams and waterfalls.

Species comparison
According to the subgeneric classification of Genlisea [1,10], G. hawkingii can be placed in the Genlisea subgen. Tayloria due to its bivalvate capsule dehiscence, which is curved downward in its fruit (Figs 1 and 2) and its Neotropical distribution.
Genlisea hawkingii resembles a robust G. violacea or G. flexuosa, except for the flower spur, which resembles G. uncinata (Table 2). However, in its vegetative state, G. hawkingii can be distinguished from its related species by having glabrous and dark green leaves, not green and with a few hairs. Regarding the reproductive organs, G. hawkingii is distinguished from G. violacea and G. flexuosa by the flowers, which have sepals with an acute apex and a long conical spur that is curved upward differently from G. flexuosa and G. violacea whose spurs are cylindrical, straight and are rarely curved downward, and are as long as the upper lip or slightly shorter. All the individuals found (~80 individuals) presented the same long conical and curved upward spur, therefore these characteristics are possibly stable for the new species. In addition, G. hawkingii only has glandular capitate hairs.

Phylogenetic analyses
The combined gene analyses (rps16 + matK) resulted in a matrix of 1,436 bp with 591 bp from the rps16 and 844 bp from the matK fragments.
The phylogenetic position of the new Genlisea hawkingii species, as it is presented in this study was strongly supported to be in G. subgen. Tayloria according to the BI and ML analyses (Fig 5). Moreover, despite having morphological characteristics that are similar to G. flexuosa and G. violacea, it is an early branching species to the other species of G. subgen. Tayloria, and is followed by G. uncinata, which is similar to G. hawkingii in a few morphological characteristics such as the curved spur (Table 2). Variations in flower morphology, including the spur shapes and sizes, can affect the pollination success [24], therefore the curved spur found in Genlisea can be explained as a possible adaptation to different pollinators [7,25]. However, further studies regarding the pollinator fauna are needed to clarify the pollination biology for the species of Genlisea.
Considering that G. hawkingii and G. uncinata present curved spur and for the other species of G. subgen. Tayloria this structure is straight (in G. metallica it can be slightly curved downwards near the apex) [7], two hypotheses can be explored by optimizing the transformations of this character in the tree, and having both accepted with the parsimony approach (ACCTRAN and DELTRAN [26]) since both hypotheses assume the same number of steps (two in this case). Thus, the curved spur can be the effect of parallelism for G. hawkingii and G. uncinata, as a result of two autapomorphies, or this state can be assumed to be a synapomorphy to the G. subgen. Tayloria clade with the posterior reversion to the G. violacea-G. lobata-G. flexuosa-G. metallica clade.
G. hawkingii is shown to be an early-branching lineage considering the G. subgen. Tayloria clade (Fig 5), thus contradicting the assumption that G. uncinata could be a relict lineage exhibiting plesiomorphic states for some characters [7]. For instance, the entire upper lip of G. uncinata can be the result of a reversion of this character, considering that G. hawkingii and other members of G. subgen. Tayloria (except G. uncinata) show a bilobate upper lip [1,10].
Even with the presented hypothesis based on two markers (Fig 5), a more complete analysis with additional species, populations and DNA sequences could bring a robust and   hawkingii, G. flexuosa, G. violacea and G. uncinata. The measurements were taken from collected specimens and literature [1,10]. "-" denotes missing data. comprehensive hypothesis to the phylogeny of G. subgen. Tayloria. For example, the morphological diversity between populations of G. violacea and its paraphyly in phylogenetic  (rps16 + matK). The numbers above and below the branches refer to the BI posterior probability and to the ML bootstrap support, respectively. The numbers beside the species names are references to the different specimens/populations and follow the numbers in Table 1.
https://doi.org/10.1371/journal.pone.0226337.g005 hypotheses (Fig 5) [7] suggest that it represents a complex of different species and, therefore, further studies based on morphological and molecular data are necessary to address this issue. Therefore, a phylogenomic approach based on plastidial [27] and mitochondrial genomes is under construction (Silva et al., in preparation) and possibly will result in a more robust phylogeny for the group.