Three new species of Bertolonia (Melastomataceae) from Espírito Santo, Brazil

We describe and illustrate three new species of Bertolonia, all endemic to the state of Espírito Santo, Brazil. Bertolonia duasbocaensis and B. macrocalyx occur close to each other, in the municipalities of Cariacica and Viana. Bertolonia ruschiana has a wider distribution, occurring in the municipalities of Santa Leopoldina, Santa Maria de Jetibá and Santa Teresa. The first two species are classified as critically endangered (CR), and the latter as endangered (EN). We also present an identification key for the species of Bertolonia that occur in Espírito Santo.


MATERIAL & METHODS
The authors have been collecting Bertolonia in Espírito Santo since 2008. Specimens were collected and processed following the usual procedures for botanical specimens (Mori et al., 1989). Morphological descriptions follow Radford et al. (1974), Baumgratz (1983Baumgratz ( -1985 and Baumgratz (1990). The study was based on literature (Baumgratz, 1990;Baumgratz, Amorim & Jardim, 2011;Bacci et al., 2016) and the analysis of specimens at CEPEC, MBM, MBML, NY, RB, UPCB, VIES (acronyms according to Thiers, 2015). Ecological and geographic distribution data were also obtained from herbarium labels. Conservation status assessments were based on range size (criterion B), according to IUCN Standards and Petitions Subcommittee (2014). The Extension of Occurrence (EOO) and Area of Occupancy (AOO) were calculated through the GeoCAT tool (Bachman et al., 2011). Collection permits were issued by the ''Instituto Estadual do Meio Ambiente'' (IEMA/Espírito Santo: 14489/07) for the plants inside ''Reserva Biológica de Duas Bocas'' and by the ''Instituto Chico  for the plants inside the ''Reserva Biológica Augusto Ruschi.'' The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants (ICN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. In addition, new names contained in this work which have been issued with identifiers by IPNI will eventually 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

Diagnosis
Bertolonia duasbocaensis is similar to B. formosa. The new species has petioles and leaves covered only with sessile or short-stalked glands (vs. petioles hirsute and leaves also covered  with unbranched trichomes in B. formosa), smaller flowers (13.3-14.7 mm long vs. 17-22 mm long in B. formosa), inconspicuous and truncate sepals with triangular external teeth (vs. conspicuous, 3.2-3.7 mm long sepals, these rounded and lacking external teeth) and by the petals with the apex covered with sessile or short-stalked glands (vs. petals glabrous).

Distribution and conservation status
Bertolonia duasbocaensis has been collected a few times since 2008, mostly along the same trail (''Pau-Oco'') inside Duas Bocas Biological Reserve, but with one specimen occurring in a private property near the Reserve. The Biological Reserve is a fully protected area, kept by the state government (IEMA/ES). It is located in the municipality of Cariacica, in central Espírito Santo, between 20 • 14 40 S-20 • 18 30 S and 40 • 28 01 W-40 • 32 07 W. Its 2,910 ha are covered with well-preserved Atlantic Rainforest that protects threatened species of both fauna and flora (Novelli, 2010). In the last five years, three new plant species were described from Duas Bocas: Eugenia amorimii Fraga & Giaretta (Giaretta & Fraga, 2014), Leandra magnipetala R. Goldenb. & E. Camargo (Camargo & Goldenberg, 2011), and Ouratea cauliflora Fraga & Saavedra (Fraga & Saavedra, 2014). Specimens of Bertolonia duasbocaensis occur in shaded and moist slopes at 500-600 m elev. They were collected with flowers in January and February and with fruits in January, February and July. According to IUCN Standards and Petitions Subcommittee (2014)

Etymology
The epithet ''duasbocaensis'' refers to the Duas Bocas Biological Reserve, the type locality of the new species.

Remarks
Bertolonia duasbocaensis is characterized by the ovate to elliptic leaves with rounded to slightly cordate bases, margins entire to slightly crenulate and ciliate, 3-5 main veins, and covered only with sessile and short-stalked glands, the widely campanulate hypanthium, truncate sepals, triangular external calyx teeth, apiculate petals with the apex of the adaxial surface covered with sessile and short-stalked glands, and oblong-subulate stamens with a dorsally thickened appendage. The new species closely resembles B. formosa, also endemic of the state of Espírito Santo. For more details on the distinction between the two of them, see the diagnosis above.
Bertolonia hoehneana Brade, B. nymphaeifolia Raddi and B. sanguinea share with the new species the leaves with cordate bases and also the branches and adaxial surface of the leaves covered only with sessile and short-stalked glands. The first is a rare species, endemic to the state of São Paulo (Baumgratz, 1990;Baumgratz, 2016) and can be distinguished by the leaves with a cordate-lobate base (vs. rounded to slightly cordate in B. duasbocaensis), eciliate margins (vs. ciliate margins) and an acute, attenuate, or sometimes acuminate apex (vs. rounded to slightly acute), by the smaller flowers (10-13 mm long vs. 13.5-15 mm long) with the hypanthium covered only with sessile and short-stalked glands (vs. covered also with scattered unbranched trichomes, mostly on its upper half) and sepals widely ovate, lacking external teeth (vs. sepals truncate, with external teeth). Both Bertolonia nymphaeifolia and B. sanguinea are endemic to the state of Rio de Janeiro and differ from the new species by the bigger leaves, with 7-9 and 5-7 acrodromous veins, respectively (vs. 3-5 in B. duasbocaensis), petioles hirsute at the base or seldom on the whole petiole (vs. petioles covered with sessile and stalked glands, the young ones sometimes with caducous, unbranched trichomes), longer flowers, 18-23 mm long in B. sanguinea (vs. shorter ones, 13.3-14.7 mm long in B. duasbocaensis) and by the long subulate stamens with an acute extension at the apex, forming a tube below the apical pore (vs. oblong-subulate stamen, without the extension at the apex). The morphology of the calyx is also different: B. nymphaeifolia has widely ovate, membranaceous sepals with fleshy, rounded external teeth, this forming a concave cavity on the sepals and B. sanguinea has fleshy, widely ovate sepals lacking external teeth (Baumgratz, 1990), while B. duasbocaensis has truncate sepals with a triangular external teeth.
Bertolonia duasbocaensis closely resembles B. macrocalyx; both are endemic of Espírito Santo and occur very close to each other. The differences between both species are listed in the diagnosis of B. macrocalyx (see below).

Diagnosis
Bertolonia macrocalyx is similar to B. duasbocaensis. Both species have similar vegetative morphology, but B. macrocalyx has the calyx with longer (ca. 2 mm long), ovate and ciliate external teeth (vs. shorter, ca. 1 mm long, triangular and eciliate in B. duasbocaensis), short-terete hypanthium (vs. widely campanulate hypanthium), smaller, 7-8.5 × 3-3.5 mm petals with the upper half of the adaxial surface densely covered with sessile and short-stalked glands (vs. bigger 10.8-12 × 4.5-5 mm petals, with only the apex of the adaxial surface sparsely covered with sessile and short-stalked glands).

Description
Herbs ca. 20 cm tall, terrestrial or rupicolous, reptant; adventitious roots branched, growing from several points along the stem, but larger next to its base; stem ca. 3 mm thick, quadrangular and slightly costate, the older portions plagyotropic and aphyllous, the young ones erect and bearing leaves. Branches, leaves, inflorescences and bracts with sparse to dense, sessile and short-stalked (then less than 0.1 mm long) glands. Leaves opposite, occasionally subopposite; petioles 1.5-5.3 cm long, quadrangular, slightly costate, covered with the same trichomes as the branches, also with unbranched trichomes, denser at the apex; blade 10-13.5 × 4.7-6.3 cm, flat, ovate, narrowly elliptic to lanceolate, chartaceous,  base slightly cordate, apex rounded to slightly acute, margins entire to slightly crenulate and ciliate, adaxial surface dark-green, sometimes light green along the primary vein, abaxial surface light-green or lilac, veins three to five, plus a shorter marginal pair that do not reach the leaf apex, basal, main veins at the abaxial surface with pocket domatia at their bases. Thyrsoids 11.3-16.2 cm long, terminal (but pseudo-lateral in older, fruiting specimens), with one pair of paraclades, these cymose, scorpioid, the branches greenish to light-pink; bracteoles ca. 0.5 mm long, linear to narrow-lanceolate, apex aristate and tipped with one glandular trichome ca. 0.5 mm long, both surfaces covered only with sessile glands. Flowers 5-merous, 9.5-11.2 mm long, on pedicels 1.5-2 mm long, light pink, densely covered with sessile and short-stalked glands. Hypanthium light green, 2.5-2.7 × 2 mm, short-terete, 10-costate, ridges pink, the ones between the external teeth ending in one trichome, with the same indument as the pedicel, also with unbranched trichomes, on the ridges at the apex of the hypanthium. Calyx caducous on fruits, tube ca. 0.8 mm long, sepals truncate, external teeth ca.2 mm long, with the abaxial surface pinkish, abaxial light green, covered with the same trichomes as the pedicels and also with unbranched trichomes (0.6-0.8 mm long) on the adaxial surface, ovate, apex rounded, margins entire, ciliate at the apex. Petals white, with light-pink apex, 7-8.5 × 3-3.5 mm, irregularly elliptic, base slightly atenuate, apex acute and apiculate (ca. 0.5 mm long), the apiculus bending outwards (to the abaxial surface of the petal), ending in a long unbranched trichome, margins entire, both surfaces papillose, upper half of the adaxial surface densely covered with sessile and short-stalked glands. Stamens ten, 6-6.5 mm long, isomorphic; filaments 3.5-4 mm long, slightly widened at the base; thecae yellow, 2-2.5 mm long, oblong-subulate, slightly undulate, pore apical, introrse; connective prolonged ca. 1.5 mm below the thecae, with a dorsal calcar, ca. 0.3 mm long. Ovary free, apex glabrous, 3-locular, placentation axillary; style ca. 5 mm long, curved at the apex, glabrous; stigma slightly capitate, papillose. Capsules bertolonidium-type (following Baumgratz, 1983Baumgratz, -1985, 3.5-5 × 4.5-5.3 mm, obtriquetrous; seeds unknown.

Distribution and conservation status
Bertolonia macrocalyx is endemic to the state of Espírito Santo. It had been collected only twice, both in 2008. The species occurs near the limits of the Duas Bocas Biological Reserve, in the municipalities of Cariacica and Viana. The area is covered with wellpreserved Atlantic Rainforest. Bertolonia macrocalyx was collected near a local roadside and in a private property. It occurs in shaded and moist slopes, with flowers in January and with old fruits in January, May and June. According to IUCN Standards and Petitions Subcommittee (2014) criteria B1ab(ii) + B2ab(ii), with EOO (Extent of Occurrence) = 0 km 2 and AOO (Area of Occupancy) = 8.000 km 2 , this species should be classified as ''critically endangered'' (CR).

Etymology
The epithet ''macrocalyx'' refers to the calyx with long external teeth, one of the diagnostic features of the new species.

Remarks
Bertolonia macrocalyx is characterized by the short petioles (1.5-5.3 cm long) covered with sessile and short-stalked glands and also with unbranched trichomes at the apex, leaves with 3-5 main veins, both surfaces covered with sessile and short-stalked glands, hypanthium short-terete, light green with pinkish ridges, calyx with truncate sepals and external teeth light green on the abaxial surface, ovate and ciliate (trichomes mostly at the apex), petals with the upper half densely covered with sessile and short-stalked glands, short-apiculate, with the apiculus ending in an unbranched trichome, and stamens oblong-subulate, with a dorsally thickened connective appendage.
Bertolonia duasbocaensis also has petals with the apex covered with sessile and shortstalked glands, but they are concentrated at the apex of the adaxial surface and are much more sparsely distributed when compared with B. macrocalyx. Another diagnostic feature of B. macrocalyx is the morphology of the calyx: The sepals are truncate (like B. duasbocaensis), but with a long (ca. 2 mm), ovate and ciliate external teeth.

Diagnosis
Bertolonia ruschiana is similar to B. nymphaeifolia. Both have branches, adaxial suface of the leaves, pedicels and hypanthium covered only with sessile and short-stalked glands, white petals with a pinkish apex, hypanthium widely campanulate and a fleshy calyx with widely ovate sepals. The new species differs by the leaves with 5 main acrodromous veins (vs. 7-9 main acrodromous veins in B. nymphaeifolia) and with ciliate margins (vs. eciliate margins), petioles covered with sessile and short-stalked glands, seldom with unbranched trichomes (vs. petioles covered with sessile and short-stalked glands, and also hirsute), shorter flowers (8-9 mm long vs. 12-15 mm long), petals apex covered with sessile and short-stalked glands on the adaxial surface (vs. petals apex glabrous) and ovary apex glabrous (vs. ovary apex covered with glands).

Distribution and conservation status
Bertolonia ruschiana occurs in the central region of the state of Espírito Santo, in three neighboring municipalities: Santa Leopoldina, Santa Maria de Jetibá and Santa Teresa (Fig. 7). Most populations were found in Santa Teresa, within three different conservation units and in several private properties. Two populations with several individuals were found inside the Augusto Ruschi Biological Reserve, a Brazilian Federal Conservation Unit with 3,598 ha (IBAMA-Instituto Brasileiro do Meio Ambiente e Recursos Renováveis, 2000). A few specimens of B. ruschiana were collected in some trails inside the Santa Lúcia Ecological Station and in the São Lourenço Municipal Natural Monument (also called ''Caixa D'água''), kept by the Museu de Biologia Professor Mello Leitão (MBML). The first has 400 ha and the second has 363 ha, both covered with Atlantic Rainforest (Mendes & Padovan, 2000). Specimens of B. ruschiana usually are terrestrial or rupicolous herbs that inhabit moist and shaded slopes or rocks with shallow soil near water, but sometimes they are epiphytes growing on lower portions of tree trunks. It was collected with flowers from January to March and with fruits all year. According to IUCN Standards and Petitions Subcommittee (2014) criteria B1ab(ii) + B2ab(ii), with EOO (Extent of Occurrence) = 298.881 km 2 and AOO (Area of Occupancy) = 68.000 km 2 , this species should be classified as ''endangered'' (EN). It has been collected more than 30 times, inside three conservation units and several private properties. Nevertheless, its habitat is fragmented and the conservation of the species depends on the maintenance of these units.

Etymology
The epithet ''ruschiana'' honors the Brazilian naturalist Augusto Ruschi (1915Ruschi ( -1986, who fought against the deforestation of the Atlantic Forest, mainly in the state of Espírito Santo. In addition, the type locality of the species is in the Augusto Ruschi Biological Reserve.

Remarks
Bertolonia ruschiana is characterized by the adaxial surface of the leaves, pedicels and hypanthium covered only with sessile and short-stalked glands, the leaves with 5 main acrodromous veins and ciliate margins, hypanthium widely campanulate, sepals widely ovate and fleshy, with triangular, slightly concave external teeth. The widely depressed, slightly convex and fleshy petals with the apex of the adaxial surface covered sparsely with sessile and short-stalked glands are also important for the recognition of the new species. Bertolonia ruschiana closely resembles B. nymphaeifolia (for more details, see the diagnosis above).
Several specimens of Bertolonia ruschiana had been determined as Bertolonia sanguinea, due to the similar vegetative morphology. Both species share the big leaves with cordate bases, and both surfaces of the leaf covered only with sessile or short-stalked glands, but B. sanguinea has hirsute petioles and eciliate leaf margins (Baumgratz, 1990). The morphology of the flowers is also different. Bertolonia sanguinea has sepals lacking external teeth (vs. sepals with a triangular, concave external teeth in B. ruschiana), longer flowers (18-23 mm vs. 8-9 mm), glabrous petals (vs. adaxial surface apex covered with sessile and short-stalked glands), longer stamens (18-19 mm long vs. 6-7.5 mm), these with an extension at the apex, forming a tube (vs. stamens without an extension at the apex) (Fig. 7).