Cyperus prophyllatus: An endangered aquatic new species of Cyperus L. (Cyperaceae) with a exceptional spikelet disarticulation pattern among about 950 species, including molecular phylogenetic, anatomical and (micro)morphological data

Cyperus prophyllatus, an endangered new species of Cyperus (Cyperaceae) from an aquatic ecosystem of the Atlantic Forest, Espírito Santo State, southeastern Brazil, is described and illustrated. The spikelet morphology of Cyperus prophyllatus is unique among the c. 950 species of Cyperus in having both a conspicuous spikelet prophyll and a corky rachilla articulation, which remain persistent at the base of the spikelet after disarticulation. Our molecular phylogenetic data support the placement of C. prophyllatus in the C3 Cyperus Grade and more precisely in the clade representing Cyperus sect. Oxycaryum, which also includes C. blepharoleptos and C. gardneri. Anatomical and (micro)morphological analyses corroborate the phylogenetic results, provide a better understanding of ecology and taxonomy, as well as reveal compatibility of structures with survival and dispersion in aquatic environments. A distribution map, table with distinctive characters of allied species, and conservation status are made available.


Introduction
Cyperus L. is the second largest genus in Cyperaceae and the most diverse in tribe Cypereae, comprising about 950 species [1][2][3]. Cyperus

Taxonomy and morphological analysis
The first specimens of the new species were found in the herbaria CVRD and UFP (abbreviations according to Thiers [41]), but it was not possible to describe neither the plant habit nor the rhizome type. Based on data from these first collection, we conducted two field expeditions to Espírito Santo State in September 2018 (no flowering plants were found) and September 2019 (with flowers and fruits available), and therefore it was possible to observe and study the rhizome and plant habit of the new species in its natural habitat. The field expeditions and site access were authorized by Marcio Elias Santos Ferreira, manager of Reserva Natural Vale. In addition, we analyzed the specimen collections deposited in the herbaria of Espírito Santo State (CVRD, MBML,VIES, SAMES) and other states of Brazil (ASE, CEN, EAC, FLOR, HUEFS, IBGE ICN, MAC, MOSS, RB, SP, UB, UFP, and UFRN), besides images of exsiccatae from the Brazilian herbaria available in SpeciesLink [42]. High resolution images of the type specimens of Cyperus species present in several international herbaria (B, C, G, K, P, MO, NY, US) were also examined. The morphological descriptions of the character states followed Radford et al. [43].
Conservation status was assessed based on IUCN Red List criteria [44] with area of occupancy (AOO) and extent of occurrence (EOO) estimated using the Geospatial Conservation Assessment Tool [45]. Distribution map of new species and allied species was made using software QGIS v.3. 16.0 (https://qgis.org).

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.
In addition, new names contained in this work have been submitted to IPNI, from where they will be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix http://ipni.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS.
Total DNA was extracted from 15-20 mg of silica-dried leaf tissue using a modified CTAB (cetyltrimethylammonium bromide) protocol [47]. PCR conditions for amplification and primers followed Reid et al. [46]. Cycle sequencing was performed with the same primers used for amplification and Sanger sequencing was conducted at Jodrell Laboratory of the Royal Botanic Gardens Kew (London, UK). DNA sequences were assembled in Geneious v.7.1.9 [48] per marker and aligned using the MAFFT v.7 [49], with subsequent manual adjustment in PhyDE v.0.9971 [50].
Phylogenetic hypotheses were reconstructed using both Bayesian Inference (BI) and Maximum Likelihood (ML) approaches. We first inferred gene trees for each of the four regions, which were concatenated afterwards since no conflict was found for supported nodes. The best models of nucleotide substitution were determined with PartitionFinder2 [51], using the Akaike Information Criterion (AIC), in this case, each marker was treated as a separate partition. The GTR+G model was determined to be the best model for all partitions. Bayesian Inference was performed using MrBayes v.3.2.7 [52]. Four independent runs of four Monte Carlo Markov chains each were conducted with 20,000,000 generations, sampling every 1000 generations, and 25% burn-in. We assessed convergence and effective sample size (ESS) values using Tracer v.1.7 [53]. ML analyses were performed with RAxML v.8.2.12 [54] using the rapid bootstrap with 1000 replicates, combined with a search of the best-scoring ML tree, and the remaining options set to default. All analysis of BI, ML and PartitionFinder2 were executed in the CIPRES Science Gateway [55]. Outputs of BI and ML were read using Figtree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Micromorphology and Scanning Electron Microscopy (SEM)
The samples were prepared from leaf blade, ligule, spikelet prophyll, glumes, and achenes sourced from existing herbarium specimens and specimens newly collected by us. Deformed and flaccid immature achenes and spikelets were not considered. The samples were mounted onto aluminum metal stubs using carbon double-stick tape and sputter-coated with platinum without pre-treatment. The images were captured using a SEM (Jeol JSM 7001S) under 15 KV at the Electronic Microscope Laboratory of the University of Brasília. The descriptive terminology followed Ellis [56], Haines & Lye [19], Hefler & Longhi-Wagner [57], and Shalabi & Gazer [58].

Anatomy
The leaf samples were obtained from the middle third of the leaf blades collected at field or from the herbarium specimens. At least three leaf blades were analyzed in each accession. The samples were stored in the ethanol aqueous solution 70% (v / v) at 4˚C and after rehydrated in the glycerol aqueous solution 1: 1 (v / v), until the preparation of free-hand sections [59]. The leaf transverse sections were cut in the table microtome (type R. Jung A. G. Heidelberg) available in Laboratório de Anatomia Vegetal in Universidade de Brasília (UnB). The best sections were selected and bleached with sodium hypochlorite aqueous solution 20% (m / v), and after 50% (m / v) for at least five minutes or until the complete discoloration of the tissues [60].
Before the staining, the samples were dehydrated in progressive ethanol concentrations in aqueous solution (50%, 70%, 92,6%, and 100% [v/v]) for fixation of the stain in the butyl acetate. The double staining was performed with safranine aqueous solution 1% (m / v) and alcian blue aqueous solution 1% (m / v). The permanent slides were mounted with colorless glass varnish, according the protocol described by Paiva et al. [61]. The descriptive terminology followed Ellis [62].
The images were acquired with the Leica DM 750 microscope in Laboratório de Criptógamas in Universidade de Brasília. The examination of the images and the evaluation of the tissues and cells were executed with the software Leica Application Suite (version 4.5).

Distribution
At present, C. prophyllatus is known only from the aquatic vegetation of the phytophysiognomy of Seasonal Semideciduous to Evergreen Forest, belonging to the Atlantic Forest in the Reserva Natural Vale, Espírito Santo State, Southeastern Brazil (Fig 3). The geographical distribution of related species C. blepharoleptos and C. gardneri are wider than C. prophyllatus. Whilst C. blepharoleptos can inhabit margins of waterbodies as well as be a floating aquatic macrophyte in tropical and subtropical areas of Africa and America, C. gardneri grows exclusively as a floating aquatic macrophyte in perennial rivers, lakes, or lagoons only in Neotropics (Fig 3).

Conservation status
C. prophyllatus appears to have a restricted distribution, being known only from a few subpopulations from Reserva Natural Vale. This species is estimated to have an extent of occurrence (EOO) and area of occupancy (AOO) of 12 km 2 , and its geographic range is restricted to less than five locations. According to criteria proposed by IUCN [44], C. prophyllatus has ecological parameters that could belong to two categories: Endangered (EN) due to AOO (12 km 2 ) between 10 km 2 and 100 km 2 or Critically Endangered (CR) due to EOO (12 km 2 ) less than 100 km 2 . Nevertheless, the IUCN [44] recommends choosing from the higher risk category for a more precautionary approach to making urgent decisions based on limited information. Although C. prophyllatus occurs in a Protected Area (PA) with population greater than 400 individuals, the surroundings of Reserva Natural Vale are under pressure due to fragmentation caused by urban development and agriculture, like most of the range of the Atlantic Forest [63]. Therefore, C. prophyllatus can be preliminary considered Critically Endangered (CR) B1ab(iii), while more studies are required to expand the botanical collection effort and increase the knowledge about its geographic range.

Phenology
Flowering and fruiting collections were made within July and October.

Etymology
The name of the specific epithet refers to the conspicuous spikelet prophyll that remains attached to the base of the spikelet after disarticulation of the rachilla (Figs 1E, 1F, 2I, 2J and 5E). Moreover, C. prophyllatus has the rachilla articulation with 0.2-0.4 mm long, corky,

PLOS ONE
blepharoleptos, 10-32 in C. gardneri), the spikelet prophyll (Figs 1E, 1F, 2I, 2J and 5E) is conspicuous and remains attached to the base of the spikelet after rachilla disarticulation at maturity (absent in C. blepharoleptos and inconspicuous or not persistent at the base of the spikelet in C. gardneri), the rachilla articulation (Figs 1E, 1F, 2I, 2J and 5E) is corky, tumid, protuberant, semiring to ring-shaped, yellowish to reddish, and persistent at the base of the spikelet prophyll after the disarticulation (rachilla articulation is absent in C. blepharoleptos and absent or when present is flat, not protuberant, and not persistent at the base of the spikelet in C. gardneri), the anther (Fig 2H)

Phylogenetic relationships
Phylogenetic trees resulting from ML and BI analyses of the concatenated dataset recovered congruent topologies. In general, relationships in C 3 Cyperus Grade are strongly supported, whereas relationships are poorly supported in the C 4 Cyperus Clade (Fig 4). Cyperus prophyllatus is resolved in a clade with C. blepharoleptos and C. gardneri that represents Cyperus sect. Oxycaryum (Nees) Larridon of the C 3 Cyperus Grade (Fig 4).

Micromorphology observed in Scanning Electron Microscopy (SEM)
In the abaxial surface of the leaf blade, the intercostal zone has one or two rows of diacytic stomata intercalated with common epidermal cells with papillose wall. The stomata are present only in the abaxial side (Fig 5B), which classify the leaf as hypoestomatic. The costal zone contains two rows of thick-walled epidermal cells, tabular or square-shaped with one or two papillae with base entire or stellate per cell (Fig 5B). The leaf scabrosity is due to the antrorse prickles which are present in leaf margins and in the costal zone of the midrib (Fig 5C).
In the adaxial surface of the leaf blade (Fig 5A), the intercostal zone has common epidermal cells with papillose wall (Fig 5A). The costal zone possesses two rows of thick-walled epidermal cells tabular or square-shaped with one or two papillae with base entire or stellate per cell interrupted by common epidermal cells smooth-walled (Fig 5A). Antrorse prickles are present in costal and intercostal zones (Fig 5A). Ligule has ciliate single-celled hairs abundant on the apex (Fig 5D).
In the spikelet prophyll (Fig 5E), the costal zone contains two rows of thick-walled epidermal cells tabular or square-shaped with one or two papillae with base entire or stellate per cell (Fig 5E). The rows of epidermal cells tabular or square-shaped are in similar position to the ribs (nerves) of the spikelet prophyll (Figs 2I, 2J and 5E). Stomata are scarce and present only next to the two carinas of the spikelet prophyll (Fig 5E). Common epidermal cells have abundant papillae on the cell wall (Fig 5E), which are similar in morphology to the leaf blade. The margins of the spikelet prophyll have abundant ciliate single-celled hairs, mainly on the apex (Fig 5E).
In the glume (Fig 5E-5H), the coastal zone has two or three rows of thick-walled epidermal cells tabular or square-shaped with one or two papillae with base entire or stellate per cell ( Fig  5G). The rows of epidermal cells tabular or square-shaped are in similar position to the ribs (nerves) of the glume (Figs 2I, 2J and 5G). Stomata are abundant next to the regions of the carina and mucron or arista, but they are absent or scarce in other regions of the glume (Fig 5F  and 5H). Common epidermal cells have abundant papillae on the cell wall (Fig 5G), which are similar in distribution and morphology to the leaf blade and spikelet prophyll. The margins of the glume are ciliate with single-celled hairs, except in the region of the mucron or arista, which is glabrous (Fig 5E-5H).
Achenes (Fig 6A-6D) are approximately smooth with slight sinuosities on anticlinal walls on the middle third and most of the surface of the basal and apical third (Fig 6A-6D). The sinuosities at base (Fig 6A and 6C), is more prominent on the anticlinal walls and 1-5 grooves occur with reticulate depressions and protuberant anticlinal walls. The apex (Fig 6A and 6D) also contains sinuosities more prominent next to the style insertion and reticulate depressions have protuberant anticlinal walls. Papillae, hairs and silicified cells are absent on achene surface.

Anatomy
In the transverse section, the radially arranged and elongated mesophyll cells of the chlorophyll parenchyma are absent around the vascular bundles (Fig 7A, 7C and 7D) in all accessions analyzed as well as the Kranz sheath (Fig 7A, 7C and 7D). The colorless parenchyma surrounds the vascular bundles and has thin-walled cells that are larger in the region close to the adaxial side (Fig 7A, 7C and 7D). In lacunosous parenchyma, several braciform cells (Fig 7B) were observed in large air lacunae surrounded by chlorophyll parenchyma (Fig 7A-7D).
The cuticle (Fig 7A and 7C-7E) is thin in the both abaxial and adaxial sides and leaf blade is V-shaped in cross section (Fig 7A). Epidermis is unistratified with common epidermal cells round shaped and greater in the adaxial side (Fig 7A and 7C-7E). The leaf scabrosity is due to the prickles (Figs 5A, 5C and 7E), which are often distributed next to the midrib, bulliform cells, and on the edge of the leaf margin (Figs 5A, 5C and 7E). Prickles are acute, have enlarged base and thickened outer wall (Figs 5A, 5C and 7E). Bulliform cells fill up to half of the mesophyll ( Fig 7A) and occur only in the adaxial side of vascular bundle on the midrib. Fibre caps (Fig 7A, 7C and 7D) have strongly thickened wall and occur in the mesophyll next to adaxial and abaxial epidermis along the entire leaf surface.
The vascular system (Fig 7A, 7C and 7D) comprises collateral vascular bundles from 1 st to 3 rd order elliptical, circular or oval. The vascular bundle sheath (Fig 7A, 7C and 7D) is doubled in all orders, the inner sheath is complete and formed by sclerenchyma, and outer one is often incomplete, sometimes complete and always constituted by parenchyma.
The midrib (Fig 7A) has a V-shaped and a prominent keel and a colorless parenchyma more developed in the adaxial side (Fig 7A). The vascular system from the midrib comprises one of 1 st order vascular bundle and two of 2 nd order ( Fig 7A). The 1st order one is rounded, central, and next to the abaxial side (Fig 7A).
The leaf blade margins can be from acute to rounded (Figs 5C and 7D). The vascular system of the leaf blade margins is composed by one of 2 nd order and one of 3 rd order (Fig 7D). The edge of the leaf margin ( Fig 7D) contains fibres forming a thick cell caps and scabrosity constituted by prickles or hooks, both with base not bulbous (Figs 5C and 7D).

Discussion
The new species Cyperus prophyllatus is unique among the c. 950 species of the genus Cyperus, being recognized by a exceptional spikelet disarticulation pattern that includes a combination of the following characters: spikelets 3-many-glumed; a conspicuous spikelet prophyll that remains attached to the base of the spikelet after rachilla disarticulation at maturity; rachilla articulation 0.2-0.4 mm long, corky, tumid, protuberant, semiring to ring-shaped (Figs 1E, 1F, 2I, 2J and 5E), yellowish to reddish (2I-J), and persistent at the base of the spikelet prophyll after the disarticulation [10][11][12][13]15,16,19,21]. Cyperus sect. Neohemicarpha Bauters and C. sect. Lipocarpha (R.Br.) Bauters also hold species with the spikelet deciduous as a single unity attached to its prophyll at base, but in those sections the spikelet prophyll is inconspicuous and spikelets are reduced to a single glume (sometimes absent) covered by spikelet bract [9,13].
Anatomical data also corroborates the placement of C. prophyllatus in C 3 Cyperus Grade. The absence of the radial chlorophyll parenchyma and Kranz sheath are associated to the eucyperoid anatomy type as well corresponds to the C 3 photosynthetic pathway [3,8,10,11,16,64,65]. The new species shows other anatomical characteristics observed in C 3 Cyperus Grade as a colorless parenchyma surrounding the vascular bundles and large air lacunae immerse in chlorophyll parenchyma in mesophyll tissue [64,66,67]. The air lacunae make survival in aquatic environments possible, since it promotes oxygen flow from leaves to submerged organs [68][69][70]. In allied species C. blepahroleptos, braciform cells also were observed in air lacunae [67]. The braciform cells provide structural support to air lacunae as well partake in the gas flow among distinct tissues of aquatic plants [67,[71][72][73][74][75][76].
Other structures present in C. prophyllatus are recurrent linked to efficiency of sedges from aquatic environments such as achene and rachilla articulation corky, and hollow rhizome. Corky or spongy thickenings in achenes, rachilla internodes or glumes enable the diaspore floatation in species of section Oxycaryum and have been reported in species of other Cyperus sections as C. pectinatus Vahl and C. pedunculatus (R.Br.) J.Kern [10,16]. Hollow internodes in the rhizome have not been reported yet in Cyperus, although hollow culms already have been observed in other genera of Cyperaceae [16,19]. The hollow rhizome keeps large air cavities even when submerged and would allow aquatic species to float in its native environments [77][78][79].
The micromorphology of C. prophyllatus observed by SEM revealed the presence of hypoestomatic leaves and antrorse prickles in the leaf blade margins, which has already been reported in other Cyperus species [64,67,[80][81][82]. Denton [66] recorded to Luzulae Group (informal group), C 3 Cyperus Grade, similar features found in the new species as two rows of thickwalled epidermal cells tabular or square-shaped with one or two papillae with base entire or stellate per cell as well as common epidermal cells with papillose wall. In C. sect. Oxycaryum, similar characteristics as the presence of ciliate single-celled hairs in both spikelet prophyll and glumes, as well achene approximatelly smooth on surface of the middle third had already been observed also in C. blepharoleptos and C. gardneri [10]. Although both C. prophyllatus and C. gardneri have achene trigonous, it is possible to differentiate them by the achene base which in C. prophyllatus is approximately smooth or with 1-5 grooves, as in C. gardneri there is a puncticulate depression area in each side at base [10].
Our ecological data show that C. prophyllatus is an endangered and endemic species from the Atlantic Forest in Southeastern Brazil. It occurs in the Reserva Natural Vale, a private Protected Area (PA) in Espírito Santo State with a high number of endemic plant and animal species [83][84][85][86]. In most PAs in Brazil, the knowledge about the flora is still incomplete or nonexistent [87,88]. Nevertheless, PAs can be threatened in Brazil by reduction or even extinction of species not yet discovered due to the corporate and political lobbying and loss of vegetation cover by anthropogenic interference [36]. Whereas several Protected Areas are threatened in Brazil, botanical and taxonomic studies using integrative approach combining analyses of multiple data sources are fundamental to reinforce and help the continuity and effectiveness of biodiversity conservation.