Taxonomic revision of Mcvaughia W.R.Anderson (Malpighiaceae): notes on vegetative and reproductive anatomy and the description of a new species

Abstract A taxonomic revision of Mcvaughia is presented, including the description of a new species from the state of Piauí, Brazil, and notes on wood, secondary phloem, leaf, and floral morpho-anatomy. We present a key to the species, full morphological descriptions, a distribution map, and notes on distribution, ecology, etymology, and conservation status for each species.

Introduction updated). The indumentum terminology follows Anderson (1981), structure shapes follow Radford et al. (1974), the inflorescence terminology and morphology follows Weberling (1965Weberling ( , 1989, and fruit terminology follows Spjut (1994) and Anderson (1981). Wood and bark anatomical descriptions follow the recommendations of the IAWA Committee for hardwoods and barks (IAWA Committee 1989, Angyalossy et al. 2016. The conservation status was proposed following the recommendations of IUCN Red List Categories and Criteria, Version 3.1 (IUCN 2012). GeoCAT (Bachman et al. 2011) was used for calculating the Extent of Occurrence (EOO) and the Area of Occurrence (AOO). Maps were elaborated using ArcGIS 9.3 software (ESRI 2010), and geographical coordinates were obtained from herbaria specimens and the literature (Anderson 1979;Amorim and Almeida 2015).

Anatomy
Fresh samples of leaves, inflorescence, and stems were fixed in the field with FAA (formaldehyde, acetic acid and 50% ethyl alcohol; 1:1:18, by volume) for 48h (Johansen 1940) and vouchers deposited at HUEFS, and VIC herbaria (acronyms according to Thiers 2018, continuously updated). Additional herborized specimens were sampled for leaf and floral anatomy:  Smith and Smith (1942), dehydrated in an ethanol series and stored in 70% ethanol. Leaves and petals were also submitted to clearing following Vasco, Thadeo, Conover, Daly (2014, modified), to dissociation techniques (Franklin 1945), and mounted in glycerin jelly (Johansen 1940). Samples stored in ethanol were then embedded in methacrylate resin (Historesin Leica; Leica Microsystems, Heidelberg, Germany) and sectioned using an automatic rotary microtome (Leica RM2265, or USA Leica RM2155, Deerfield, USA). Longitudinal and cross-sections were stained with toluidine blue at pH 4.7 (O'Brien and McCully 1981) and mounted in resin (Permount, Fisher Scientific, NJ, USA).
For wood and bark anatomy, Mcvaughia sergipana stems [specimen Amorim 8393 (HUEFS)] were boiled in water and glycerin for a month to soften its extremely stiff wood (Angyalossy et al. 2016), being subsequently embedded in polyethylene glycol 1500 (PEG 1500;Rupp 1964), and sectioned in a sliding microtome with a permanent hard steel knife type "C" (Barbosa et al. 2018) with the aid of a Styrofoam resin (Barbosa et al. 2010). The sections were double stained in Safrablau (Bukatsch 1972 modified by Kraus and Arduin 1997) and mounted in Canada balsam. Imaging was performed using a light microscope (AX70TRF; Olympus Optical, Tokyo, Japan) equipped with a digital camera (AxioCam HRc; Zeiss, Göttingen, Germany). Anatomical analyses of leaves and flowers were conducted at the Plant Anatomy Laboratory -UFV and wood and bark anatomy at the Plant Anatomy Laboratory of the Smithsonian Natural History Museum (SI-NMNH).

SEM analysis
Micromorphological data were obtained using a scanning electron microscope (SEM) at the Center for Microscopy and Microanalysis, Universidade Federal de Viçosa. Fixed samples were dehydrated in an ethanol series, submitted to the critical point technique (CPD 020; Bal-Tec, Balzers, Liechtenstein), sputter coated with gold (Bozzola and Russell 1992), and observed and photographed using a Leo 1430VP SEM (Zeiss, Cambridge, United Kingdom). The anatomical patterns of secretory structure were described as sessile when the secretory epidermis covered all the projected area; subsessile when the secretory epidermis is surrounded by non-secretory epidermis; and stalked when a short stalk is present. cincinni alternate to subopposite, 1-14-flowered; bracts persistent; bracteoles persistent, one of them 1-glandular, the other eglandular, gland green in bud turning yellow in anthesis. Flowers zygomorphic; floral buds slightly flattened at middle; pedicel stout, straight in bud. Sepals leaving petals exposed in pre-anthesis, all 2-glandular. Petals bright to golden yellow, glabrous, the anterior two remaining cupped one inside the other; lateral petals with the margin erose; posterior petal bearing several marginal glands. Stamens 7-8, staminodes 2-3 (stamens opposite the posterior-lateral sepals and the posterior petal); filaments glabrous, those opposite the posterior-lateral petals slightly curved towards the apex; connectives inconspicuous; anthers horseshoeshaped, glabrous, outer locules confluent at apex, reduced to antherodes in staminodes. Ovary 3-carpellate, 3-locular, 2 locules ± anterior, apparently collapsed lacking ovules, 1 locule almost posterior, fertile, 1-ovulate; styles 3, slender, truncate to uncinate at apex; stigma minute, lateral. Drupes rugose, twisted, asymmetric, 1-locular, proximal chamber thick-walled, 1-seeded, distal chamber thin-walled, filled with a viscous secretion (allowing the fruit to float and to be dispersed by water).

Mcvaughia
Etymology. The epithet pays tribute to Dr. Rogers McVaugh (*1909-2009, an American botanist, expert in the Mexican flora. Distribution. Mcvaughia is restricted to sandy soils within sedimentary basins of Northeastern Brazil, with different species being endemic to each sedimentary basin: Mcvaughia bahiana -Tucano basin, M. piauhiensis -Parnaiba basin, and M. sergipana -Sergipe-Alagoas basin (Amorim and Almeida 2015). Sedimentary basins represent conspicuous phytogeographic zones within the Caatinga domain, with a distinct biota from other areas of Caatinga over crystalline shield, holding endemism records for some angiosperm families (Cardoso and Queiroz 2007;Almeida et al. 2018;Silva and Souza 2018).
Biogeography. Mcvaughia is the sister-group of Burdachia and Glandonia, comprising a lineage (Mcvaughia clade) of early diverging Malpighiaceae with waterdispersed fruits, most commonly found growing along wetland floodplains and upland forest throughout the Amazon Basin (Anderson 1981). Davis et al. (2014) estimated that the ancestor of this clade might have arisen around 38.0-33.9 Myr, and its extant lineages diversified from 25 to 15 Myr. The description of M. piauhiensis corroborates the hypothesis of Amorim and Almeida (2015) that extant lineages of Mcvaughia have probably experienced recent radiations along the caatingas and restingas of Northeastern Brazil. This inference is based on the fact that all three Mcvaughia species occur along current or past courses of the São Francisco river (SFR): M. piauhiensis occurs to the west, and M. bahiana and M. sergipana occur to the east near the SFR's delta (Fig. 11). During the upper Pliocene, the SFR flowed to the equatorial Atlantic Ocean via the Piauí or Canindé Rivers, in the Parnaiba river basin, state of Piauí (King 1956;Nascimento et al. 2013). However, the rising of the Grande and Ibiapaba Ridges in northwestern Ceará state during the Pliocene/Pleistocene boundary would have abruptly interrupted the SFR's course (King 1956;Mabesoone 1994;Nascimento et al. 2013;Almeida et al. 2018). By this time, the ancestor of Mcvaughia might have already been established in this area, and its populations might have been isolated from each other by the formation of several lakes in northwestern Bahia, due to the interruption of the river flow (King 1956;Mabesoone 1994;Nascimento et al. 2013). It was not until the Mindel glaciation (ca. 450.000 years ago) that the SFR found its way to the eastern Atlantic Ocean, bordering the states of Alagoas and Sergipe (King 1956;Mabesoone 1994;Nascimento et al. 2013), thus, paving the way for recent vicariant cladogenesis events within these populations.
Secondary phloem anatomy of Mcvaughia sergipana. Growth rings are indistinct, phloem non-stratified ( Fig. 2A-C). Conducting phloem represents a narrow band of 11-12 cells away from the cambium. Nonconducting phloem is marked by the collapse of the sieve tubes, a more significant dilatation of the axial and ray parenchyma ( Fig. 2A-B), and belated sclerosis of some scattered ray cells. Sieve tubes are diffuse (Fig. 2C), solitary or in radial multiples of 2 cells (Fig. 2C), sieve plates are simple, slightly inclined, 441 ± 71 μm 2 in area (24 ± 2 μm in diameter). Sieve tube elements length is 243 ± 45 μm. Slime plug always evident at the sieve plates. One to two companion cells per sieve tube element, as seen in transverse section. When two companion cells are present, one lies on each side of the sieve tube. Dilatation due to cell expansion and division is evident in the rays, axial parenchyma, and the cortex ( Fig. 2A-B). Axial phloem parenchyma constitutes the ground tissue ( Fig   initially green (Fig. 4A) turning yellow during anthesis. The secretory surface of the bracteole gland may vary from flattened ( Fig. 4D) in M. sergipana to convex in the other two Mcvaughia species (Fig. 4E-F). The bracteole glands show an anatomical arrangement similar to that of the leaf glands, with palisade-like secretory epidermis and a vascularized secretory parenchyma (Fig. 4G). The calyces are particularly zygomorphic, due to the lateral displacement of glands in the anterior sepal ( Fig. 4H-I).
Conservation status. Mcvaughia bahiana shows an extent of occurrence of 2,527 km 2 , and an area of occupancy of 16.000 km 2 within the northeastern state of Bahia, Brazil. Its restricted distribution associated with an accelerated habitat degradation categorizes it as Endangered (EN). Mcvaughia bahiana is the only species in the genus not protected within the limits of a conservation unit.
Etymology. The epithet refers to the distribution of M. bahiana, which is restricted to the state of Bahia, Brazil.
Conservation status. Mcvaughia piauhiensis is known only from a single population within the limits of the Serra das Confusões National Park in state of Piauí, Brazil. Until additional fieldwork can be done in seasonally dry forests from Piauí, this species is best categorized as data deficient (DD).
Etymology. The epithet refers to the distribution of M. piauhiensis, which is restricted to the state of Piauí, Brazil.

Mcvaughia sergipana
Conservation status. Mcvaughia sergipana shows an extent of occurrence of 49.735 km 2 and an area of occupancy of 12.000 km 2 . Its restricted distribution and accelerated degradation of habitat categorizes it as Critically Endangered (CR). Even though the populations of M. sergipana are scattered within two municipalities in the state of Sergipe, Brazil, some of them are located within the limits of Santa Isabel Biological Reserve.
Etymology. The epithet refers to the distribution of M. sergipana, which is restricted to the state of Sergipe, Brazil.
Anatomical notes. This species has an unusual distribution pattern of leaf glands (Fig. 3C), in which all conspicuous glands are scattered throughout the leaf blade and visible to the naked eye. The basilaminar glands are generally positioned in pairs, varying from 2-8 glands. Several laminar glands are distally scattered throughout the blade, and one pair is positioned subjacent to the apical leaf tooth (Fig. 3G). The outline of the anticlinal epidermal cell walls is straight on both adaxial and abaxial surfaces (Fig. 3N, P). Leaf glands are yellow, and bracteole glands are green in secretory stage (Fig. 4A), turning yellow at blooming. The basilaminar and laminar leaf glands were anatomically identified as sessile (Fig. 3F, K), while the bracteole gland was recognized as subsessile (Fig. 4D) and the sepal gland as short-stalked (Fig. 4I). The bracteole gland shows a flattened secretory surface. Another important character that distinguishes M. sergipana is the distribution pattern of glands on the posterior petal. About five marginal petal glands are present on the proximal portion of the posterior petal in other Mcvaughia species, but only in M. sergipana do these glands extend the entire length of the petal margin (Fig. 4K, M-N). The proximal petal glands are stalked, in contrast to the small glands distributed distally that are sessile ( Fig. 4R-T).

Discussion on the anatomical characters
Mcvaughia can be characterized by very hard woods, narrow vessels in a radial arrangement, scanty axial parenchyma, heterocellular mixed rays, and large prismatic crystals in ray cells. The bark can be characterized by scattered fiber-sclereids, axial parenchyma in lines filled with druse crystals, intercalating with sieve tubes. Mcvaughia has various features similar to other Malpighiaceae, such as the narrow vessels in radial arrangement, simple perforation plates and simple sieve plates, minute vestured pits, parenchyma strands of 2-4 cells, heterocellular rays, and prismatic crystals in wood and druse crystals in the bark (Solereder 1908, Metcalfe and Chalk 1950, Cabanillas et al. 2017. It is unique within the family because of its very scanty axial parenchyma, making it similar mainly to Byrsonima, which can also have a shrub habit (Solereder1908, Metcalfe and Chalk 1950). However, Byrsonima has shorter radial vessel chains, the rays are wider and heterocellular with body procumbent and square to upright marginal cells (Sonsin et al. 2014), as opposed to Mcvaughia, which has heterocellular mixed rays. Byrsonima also has abundant septate fibers, which are absent in Mcvaughia. From the two genera sister to Mcvaughia, namely Burdachia and Glandonia, only a few aspects are described in Solereder (1908), and Metcalfe and Chalk (1950) and these genera seem to diverge from Mcvaughia in having simple pits in the vessel-ray parenchyma interface, and Burdachia is described as having abundant paratracheal confluent parenchyma. The presence of heterocellular mixed rays is also unusual in this genus, since these types of rays are more common in lianas than in shrubs or trees , Cabanillas et al. 2017 In the phloem, Mcvaughia is unique for the low abundance of sclerenchyma, which is generally quite abundant, at least in the nonconducting phloem of Malpighiaceae , Cabanillas et al. 2017.
Glandonia species also show leaf glands varying from two to four basilaminar and a few to several laminar glands, which can be conspicuous or inconspicuous to the naked eye (Guesdon et al. 2018). Stalked glands have been recorded in the literature for Banisteriopsis (Araújo and Meira 2016;Nery et al. 2017), and Stigmaphyllon (Almeida and Mamede 2016); as well, sessile glands are known in Diplopterys pubipetala (Possobom et al. 2010), and sessile to immersed glands in Amorimia (Mello et al. in press.) and Glandonia species (Guesdon et al. 2018). Dorsiventral mesophyll with a single layer of palisade parenchyma and paracytic stomata are quite similar in all the three genera and commonly found in Malpighiaceae species, as reported by Araújo et al. (2010), Almeida et al. (2017), and Guesdon et al. (2018). The bracteole color observed in the field, could be used to distinguish the Mcvaughioid genera, being typically white in Glandonia (Guesdon et al. 2018), pink in Burdachia (Guesdon et al. unpubl. data) and green turning yellow in all Mcvaughia species. The secretory surface variation of the bracteoles glands in Mcvaughia provides diagnostic characters, as in species of Glandonia species (Guesdon et al. 2018). The short-stalked sepal glands recorded in species of Mcvaughia are also recorded in Glandonia (Guesdon and Meira unpubl. data), while subsessile sepal glands were reported in Banisteriopsis (Araújo and Meira 2016), and Diplopterys pubipetala (Possobom et al. 2015). The stalked and sessile petal glands found in Mcvaughia were also observed in Burdachia (Guesdon et al. unpubl. data), being only previously reported in D. pubipetala (Possobom et al. 2015). Leaf and bracteole glands have been described as true nectaries (Possobon et al. 2010;Meira 2016, Nery et al. 2017;Guesdon et al. 2018). The sepal and petal glands have been identified as elaiophores on the sepals and as osmophores on the petals (Possobom et al. 2015;Araújo and Meira 2016;Guesdon and Meira unpubl. data). The anatomical similarity observed among all these glands support their homology, as suggested by molecular phylogenies (Castro et al. 2001;Davis and Anderson 2010).
The glands of the posterior petal provide promising floral characters for taxonomic studies in Malpighiaceae (Guesdon pers. com.). Previous studies in Mcvaughia (Anderson 1979;Amorim and Almeida 2015) have mentioned the presence of glands only at the proximal region of the posterior petal, and details about number, shape, and size are imprecise. Anatomic studies helped to elucidate the distribution of those structures, revealing not only the stalked glands on the proximal portion of the posterior petal, but also the sessile glands distally distributed exclusively in Mcvaughia sergipana. Additionally, the presence of glands on the lateral petals of Mcvaughia bahiana mentioned by Anderson (1979) was not confirmed in this study. However, this might be a result of intraspecific variation. The petal glands have also been taxonomically useful to distinguish species of Glandonia (Guesdon et al. 2018).

Conclusions
The results presented in this study are the second step towards a complete taxonomic revision of the Mcvaughioid clade using several additional methods in biosystematics, initiated by Guesdon et al. (2018). Additionally, this is the first record of scanty sclerenchyma in the secondary phloem in Malpighiaceae, and of a shrub with heterocellular mixed rays, long radial rows of narrow vessels and scanty axial parenchyma in the wood. The macro and micromorphological data presented here are promising for future taxonomic and phylogenetic studies focusing on understanding the morphological evolution in the Mcvaughioid clade, and in Malpighiaceae, as well.