Original Flora of Acanthaceae of Iguaçu National Park, Paraná, Brazil

Species of Acanthaceae are predominantly associated with conserved forest environments such as Iguaçu National Park (PARNA Iguaçu), which is composed of Semideciduous Seasonal Forest and Araucaria forest. The aim of this work was to perform a floristic study of Acanthaceae of PARNA Iguaçu, with botanical illustrations, an identification key and descriptions of the species. Collections were carried out monthly from August 2015 to July 2016, in the areas of Céu Azul, Capanema and Foz do Iguaçu. The individuals collected were deposited in the UNOP herbarium and the specimens present in the herbaria EVB, HCF, MBM and UNOP, as well as in the virtual herbaria Reflora and SpeciesLink , were analyzed. A total of 13 native species were recorded from Atlantic Forest, distributed in seven genera. Justicia was the most representative, with five species. In all, 12 new records were made for PARNA Iguaçu, of which eight are new records for Semideciduous Seasonal Forest. Of the species found, three are threatened with extinction, one of which is considered vulnerable and two are categorized as endangered, which reinforces the role of PARNA Iguaçu in in-situ conservation in the state of Paraná.


Introduction
Lamiales is a monophyletic order and Acanthaceae is one of its most diverse family in Brazil with 39 genres and 453 species (BFG 2018).
Acanthoideae is the subfamily with the largest number of species, approximately 4,000. It is characterized by having loculicidal capsules with retinacula, which are lignified and developed funiculi, hook shaped that support the seeds, and when the capsules open the elastic movement of the wall launches the seeds a certain distance (Witztum & Schulgasser 1995). It is composed of a basal clade, the tribe Acantheae, which is sister to the other tribes, whose morphological synapomorphy is the presence of cystoliths and includes Ruelliae and Justiciae (McDade et al. 2008).
The distribution of Acanthaceae is mainly tropical, with about 4,000 species gathered in 220 genera (Stevens 2012). In Flora do Brasil (2020), 39 genera and 453 species are identified and for Paraná, 17 genera and 54 species.
They range from herbs to shrubs, lianas and rarely trees. They have simple, generally opposite leaves, flowers solitary or in inflorescences, bracts that are often colorful and showy (Kameyama 1995(Kameyama , 2006Judd et al. 2009). Flowers in Acanthaceae usually have two or four didynamous stamens and sometimes, staminodes, bilocular ovary with two syncarpous carpels with axial placentation and a maximum of two rows of anatropous ovules in each carpel. The fruit is mostly a loculicidal capsule Generally, the flowers are colorful, and the inflorescences are axillary or terminal, in spikes of solitary flower or cymes, sometimes grouped in inflorescences of superior orders condensed in glomeruli or simple capitula (Ezcurra 1999).
Despite the difficulty in using vegetative characters for the identification of genera and species in Acanthaceae, a character that can help in the identification is the presence of cystoliths, which can be found in the genera Barleria, Ruellia, and Justicia, and are more easily observed in material already herborized with the aid of a stereomicroscope (McDade et al. 2000).
In spite of a previous checklist of the Acanthaceae of Paraná (Kameyama 2014), there are few studies of this family in Paraná. There is only one recent taxonomic study for Ruelliinae Nees, which is one of the largest subtribes of Acanthaceae, but this study only addresses the genera Dyschoriste Nees, Hygrophyla R.Br. and Ruellia L. (Silva 2011). For PARNA Iguaçu, one of the largest conservation units in the state, only Justicia brasiliana Roth is reported in the management plan (ICMBio 2018).
In view of the above, this work aimed to perform a floristic study of the Acanthaceae of PARNA Iguaçu, presenting morphological descriptions and phenology of the species, an identification key, and illustrations of the diagnostic characteristics to contribute to the knowledge of the family for the state.

Study area
Iguaçu National Park (PARNA Iguaçu) is a conservation unit located in the state of Paraná, between the coordinates 25º05' to 25º41'S and 53º40' to 54º38'W, with 185,262.5 hectares of extension and a perimeter of 420 km (ICMBio 2018).
The climate of PARNA Iguaçu is classified as subtropical, with a hot summer, no dry season and little frost. The average temperature of the hottest month is above 22 °C, and that of the coldest month is below 18 °C (Alvares et al. 2013;IAPAR 2010). In the PARNA Iguaçu area there is variation in altitude, the lowest part is the southern portion, with altitudes up to 200 m along the Iguaçu River and predominance of the Semideciduous Seasonal Forest (SSF), which covers most of the park. In the North the altitudes reach up to 600 m and is characterized as a transition area of Araucaria Forest (AF) and SSF (ICMBio 2018).

Collection and identification
Trails were followed in the three different areas of PARNA Iguaçu. In area 1, six trails were visited, totaling 17 km. In area 2, three trails were visited, about 64 km. These were walked or accessed with the use of a motorboat, and along the Iguaçu Riverbank trail, the plants sighted were collected. In area 3, eight trails were traveled, totaling 16 km (Fig. 1).
Monthly collections were performed from August 2015 to July 2016, using the walking method described by Filgueiras et al. (1994) and detailed by Walter & Guarino (2006). Whenever possible, at least three fertile branches were collected from each individual, which were prepared using herborization techniques (Bridson & Forman 2010) for incorporation into the UNOP herbarium and duplicates sent to MBM and SP, acronyms according to Thiers (continuously updated). The collected specimens were photographed, described in the field and part of these samples were fixed in 70% alcohol for morphological studies in the laboratory.
Identification was performed through specific literature (Barroso 1991;Kameyama 1995;Ezcurra 1993Ezcurra , 2002Wasshausen & Wood 2004;Kameyama 2006) and comparison with samples at herbaria such as MBM and HCF (Thiers, continuously updated). The spelling of the scientific names and their authors follow The International Plant Names Index (IPNI 2016) and BFG 2018).
For the morphological descriptions, the vegetative and reproductive characteristics of the collected materials were analyzed with the aid of stereomicroscopy and the terminology was based on Weberling (1981) and Radford et al. (1974), in addition to the qualitative and quantitative characteristics observed in the herbarium samples of EVB, HCF, MBM and UNOP.
F o r t h e b o t a n i c a l i l l u s t r a t i o n s , diagnostic characters were selected, present in the identification key and important for the identification of the species, which were illustrated in line drawing or in photographs of living plants Data on geographical distribution were verified in the specialized literature, while comments on flowering, fruiting and area of occurrence in PARNA Iguaçu were based on the samples collected and materials examined.
Information on the conservation status of each of the species found in PARNA Iguaçu was obtained from the National Center for Conservation of Flora (CNCFlora 2016). L.) and Justicieae (Justicia L., Pachystachys Nees and Poikilacanthus Lindau) (McDade et al. 2008).
Justicia was the most representative genus, with six species, followed by Ruellia with three. The other genera were represented by only one species each.
Identification key to the species of Acanthaceae of PARNA Iguaçu

2004.
Figs. 2a; 3a Subshrubs, erect, up to 1 m tall. Branches cylindrical, never dilated in the internodes, pubescent, cystoliths absent. Leaves petiolate, petiole 2-4 cm long, blade narrowly elliptical to oblanceolate, 9-18 × 2-5.5 cm, pubescent on both surfaces, cystoliths absent, apex acute to acuminate, base attenuate, margin entire to slightly sinuous, pubescent. Inflorescences paniculate, terminal with spikes 1.5-12.5 cm long, sometimes with supernumerary spikes, inflorescence axes pubescent; bracts and bracteoles narrowly elliptical   This species can be found in Argentina, Bolivia and Brazil, being exclusively tropical (Profice & Andreata 2011). In Brazil, it occurs in the North, Central-West, Southeast and South regions, in areas of Riparian or Gallery Forest, Upland Forest, Ombrophilous Forest and Restinga (BFG 2018). According to the CNCFlora (2016), this species is characterized as least concern. In PARNA Iguaçu it occurs in the three areas, including SSF and AF, along almost all trails and was found with flowers from February to October and fruits in the month of July.
Aphelandra longiflora is mainly recognized by the spike or panicula with congest flowers, short 2-labiate, reddish orange corolla with a yellow throat and middle region. Herbs erect, up to 1.5 m tall. Branches quadrangular, never dilated in the internodes, glabrescent, cystoliths present. Leaves sessile, blade narrowly elliptical to lanceolate, 4-14 × 1-3 cm, adaxial surface glabrescent and abaxial surface pubescent along veins, cystoliths present, apex acuminate, base attenuate, margin entire, glabrescent. Inflorescences cymose, verticillate, axillary, inflorescence axes pubescent; bracts and bracteoles lanceolate, 13-14 mm long, pubescent. Flowers sessile; calyx 5-lobed, lobes narrowly lanceolate to linear, 5-8 mm long, glabrescent; corolla white to lilac, throat with lilac spots, glabrous, bilabiate, straight, 1-1.1 cm long, tube 0.4-0.5 cm long, upper lip 3-4 mm long, bilobate, oblong, lower lip 3-4 mm long, trilobate, lobes 1-2 mm long, lateral lobes 1 mm wide, central lobe 1 mm wide, ovate; stamens 4, exserted, trichomes base glabrous, anthers dithecous, thecae inserted at the same height at the connective, divergent, base sagittate; nectariferous disk absent; ovary cylindrical, about 1mm long, glabrous. Fruits capsules, 0.7-1 cm long. Seeds 5, elliptical. Hygrophila costata can be found from Mexico to Argentina (Wasshausen 1995) and in Brazil occurs in the North, Northeast, Central-West, Southeast and South, in Riparian Forest and Ombrophilous Forest (BFG 2018). In PARNA Iguaçu it can be found in the three areas, Céu Azul, Capanema and Foz, in SSF and AF, in more humid places. This species blooms and bears fruit practically all year round. This species can be recognized for having verticillate cymes and corolla from white to lilac, generally found in humid areas, near watercourses. Roth, Sp. Pl. Nov. 17. 1821. Figs. 2b; 3b Subshrubs, erect or decumbent, up to 2 m tall. Branches subquadrangular to cylindrical, dilated in the internodes, visibly contracted above the internodes, glabrescent with a longitudinal band of trichomes, cystoliths obscure to conspicuous.   It can be found in Brazil, Paraguay and Argentina (Ezcurra 2002). In Brazil it occurs in the Central-West, Southeast and South and is found in Riparian Forest and Ombrophilous Forest (BFG 2018). It occurs in several physiognomies of the Atlantic Forest and in several conservation units, with great abundance in southern Brazil and, therefore, characterized as of little concern (CNCFlora 2016). It occurs in the three areas of PARNA Iguaçu, in SSF and AF along practically all trails and was found from February to November with flowers and fruits.

Justicia brasiliana
Justicia brasiliana is recognized for having a distinctly bilabiate, red corolla and subsessile spikes.  This species can be found in Argentina, Paraguay, Colombia, Ecuador and Brazil (Ezcurra 2002;Profice 2013). In Brazil, it is registered in the Southeast and South, occurring in the Semideciduous and Ombrophilous Seasonal Forests (BFG 2018). This species was not evaluated for conservation status. In PARNA Iguaçu, Justicia carnea only occurs in Foz do Iguaçu (SSF), on the Poço Preto trail. In the study area it blooms mainly from October to March, but it was also collected with flowers in June.
Justicia comata is recognized for having a panicle of verticilate spikes. Leaves, petiole up to 0.8 cm long, blade elliptical to ovate, 2.8-5.6 × 1.3-2.7 cm, glabrescent on both surfaces mainly on the central vein, cystoliths present, apex acuminate, base attenuate, decurrent along the petiole, margin entire to slightly sinuous, glabrescent. Inflorescences paniculate, composed of secundiflorous spikes, some flowers opposite, axillary; inflorescence axes pubescent with a longitudinal band of trichomes; bracts and bracteoles narrowly elliptical to linear 3-4 mm compr, long, glabrescent. Flowers sessile; calyx 4-lobed, lobes lanceolate, 5-6 mm long, pubescent; corolla whitish externally, purple with white lines internally, bilabiate, straight, 1.0-1.2 cm long, tube 0.5-0.7 cm long, upper lip 0.4-0.5 cm long, bilobate, narrowly elliptical, lower lip 0.4-0.5 cm long, trilobate, lobes 4.0-5.0 mm long, lateral lobes about 2 mm wide, central lobe 2-3 mm wide, rounded; stamens 2, extended to the tip of the lips, trichomes present at the base, anthers dithecous, inferior thecae sterile, reduced to a slight thickening on the filament; nectariferous disk present; ovary cylindrical, about 1 mm long, glabrescent. Fruits clavate capsules, 0.4-0.5 cm long. Seeds 4. It is distributed in South America, exclusively in Brazil, Argentina and Paraguay (Ezcurra 2002). In the national territory it is found only in the South and Southeast, in Ombrophilous Forest (BFG 2018). The National Center for the Conservation of Flora (2016) characterizes this species as of little concern, but in PARNA Iguaçu it was found only on the Cataratas Trail (SSF), with flowering in July.
Justicia lythroides is recognized for having an externally whitish and internally purple corolla with white spots and cystoliths in the stems. Figs. 2d-e; 3e Subshrubs, erect, up to 1.6 m tall. Branches, cylindrical to subquadrangular, never dilated in the internodes, pubescent, cystoliths absent. Leaves, petiole 0.5-1.0 cm long, blade ovate to rhombic, 4.5-14 × 2.5-6 cm long, glabrescent on both surfaces, cystoliths present, apex acute, base attenuate, margin entire, glabrescent. Inflorescences spicate, sessile, axillary and terminal; inflorescence axes pubescent; bracts and bracteoles elliptical, 7-11 mm long, thickly pubescent. Flowers, calyx 5-lobed, lobes lanceolate to linear, 4-6 mm long, pubescent; corolla purple, throat with a white straight line and lateral spots, bilabiate, straight, 3.2-4 cm long, 2.5-3 cm long, upper lip 6-11 mm long, bilobate, narrowly elliptical, lower lip 5-13 mm long, trilobate, lobes 5-6 mm long, lateral lobes 2-4 mm wide, central lobe 2-4 mm wide, oblong; stamens 2, exserted, without trichomes at the base, anthers dithecous, thecae inserted at different heights in the connective, divergent; nectariferous disk present; ovary slightly pyriform, 2-3 mm long, glabrescent. Fruits not observed. This species can be found in Peru, South and Central-West Brazil, Bolivia, Paraguay and Argentina (Ezcurra 2002). In Brazil it only occurs in Mato Grosso and Paraná, in the domains of Cerrado and Deciduous Seasonal Forest (BFG 2018). According to the National Center for the Conservation of Flora (2016), the species is considered vulnerable, possibly due to the low efficiency of the pollinator, expressed by the small amount of pollen recorded in hummingbirds, and may show stress due to the incidence of threats. However, in PARNA Iguaçu this species occurs only in SSF, in the areas of Foz do Iguaçu and Capanema and can be found with flowers in the months of May and June.
Justicia ramulosa is recognized by the pubescent to densely pubescent bracts and corolla with purple coloration, throat with a straight line and white lateral spots. It was described as Chaetothylax vestitus Rizzini, for ParNa Iguaçu by Rizzini (1952), and it was mistakenly considered by Ezcurra (2019) as synonymous with Justicia goudotii V.A.W. Graham, but should therefore be treated as synonymous with Justicia ramulosa, as already discussed by Profice (2010). In addition Justicia araucariensis Profice, a new name for Chaetothylax vestitus, should also be considered synonymous with Justicia ramulosa.
Pachystachys dubiosa can be differentiated from the other species in the area by the linear bracts and an unusual long and narrow corolla tube.
It occurs in South America, Brazil, Paraguay, Uruguay and Argentina (Ezcurra 1993). In the national territory it occurs in the Central-West, Southeast and South in Cerrado, Semideciduous Seasonal Forest, Ombrophilous Forest and Araucaria Forest (BFG 2018). This species was not evaluated for conservation status. In PARNA Iguaçu, Ruellia angustiflora occurs in the three areas in SSF and AF and blooms practically all year round, being found with fruit in the months of June, August, September and October.
Ruellia angustiflora can be recognized by its subsessile cymes and the red or magenta, tubular corolla. It is differentiated from Ruellia brevifolia by the straight corolla tube (vs. ventricose).
Ruellia brevifolia has a tropical and subtropical distribution in South America, being found in Colombia, Ecuador, Peru, Brazil, Paraguay and Argentina (Ezcurra 1993). It can be found in the North, Central-West, Southeast and South, in areas of Cerrado and Ombrophilous Forest (BFG 2018). This species has not been evaluated for conservation status. However, in PARNA Iguaçu, this species was only found in SSF, in the area of Foz do Iguaçu. It blooms from October to August, with fruits in the months of March, June and July. This species is recognized by its nonbilabiate, red and especially its ventricose corolla.
Herbs, erect, up to 0.45 m tall. Branches subquadrangular to cylindrical, sometimes dilated in the internodes, glabrescent with a horizontal band of trichomes near the nodes, cystoliths inconspicuous. Leaves, petiole up to 0.5 cm long, blade elliptical to rhombic, 3.5-4.6 × 1.7-2.4 cm, glabrescent on both surfaces and pubescent along the veins, cystoliths present, apex acuminate, base attenuate, decurrent along the petiole, margin slightly sinuous, pubescent. Inflorescences spicate with leaf-like bracts, axillary, opposite; inflorescence axes pubescent; bracts and bracteoles elliptical to ovate, 1.1-2 cm long, pubescent. Flowers, calyx 5-lobed, lobes with four segments joined in two smaller pairs, 2-4 mm long and a larger one 7 mm long, lobes lanceolate, pubescent; corolla lilac to blue externally, without differentiation along the expanded and unexpanded portion, infundibuliform, straight, 2.8-3 cm long, tube 1-1.5 cm long, lobes 3-4 mm long; stamens 4, included, trichomes present at the base, anthers dithecous, thecae inserted at the same heights in the connective, divergent only at the base, base sagittate; nectariferous disk present; ovary pyriform, about 3 mm long, glabrescent. Fruits ovate capsules, about 8 mm long. Seeds 4, suborbicular. This species occurs in Mexico, Bolivia, Paraguay, Brazil and Argentina (Ezcurra 1993). In Brazil, it can be found in the Central-West, Southeast and South, in areas of Cerrado and Ombrophilous Forest (BFG 2018). According to CNCFlora (2016), this species is widely distributed in Brazil and can be found in forest areas, being considered of little concern. However, in PARNA Iguaçu it was found only in SSF, in the area of Foz do Iguaçu, with flowers and fruits in August.
Ruellia erythropus is recognized by its spicate inflorescences, with leafy bracts and purple corolla, and the capsule which placenta and the retinaculum are released from the capsule wall (Ezcurra 1993).
Herbs, subscandent, stem up 1.6 m tall. Branches, subquandrangular, never dilated in the internodes, pubescent, cystoliths absent. Leaves, petiole 1-2 cm long, blade elliptical to ovate, 2.5-6 × 1.5-3.5 cm, glabrescent on both surfaces and pubescent along the veins, cystoliths absent, apex rounded, base attenuate, margin entire to slightly sinuous, pubescent. Inflorescences spicate, pedunculate, terminal; inflorescence axes pubescent; bracts and bracteoles elliptical to obovate, 10-11 mm long pubescent. Flowers, calyx 5-lobed, lobes lanceolate, 4-5 mm long, pubescent; corolla lilac to bright pink, without differentiation along the throat, glabrous, infundibuliform, straight, 8-9 mm long, tube 3-4 mm long, lobes 3-4 mm long; stamens 4, included, trichomes present along the base, anthers monothecous, base not sagittate; nectariferous disk present; ovaries cylindrical, about 1 mm long, glabrous. Fruits obovoid capsules, 0.7-0.8 cm long. Seeds 4, ellipsoid. It occurs in Ecuador, Peru, Brazil, Paraguay and Argentina, sporadically in Bolivia and the Andes (Wasshausen & Wood 2004). In Brazil, on the other hand, it can be found in the Southeast and South, only in Ombrophilous Forest (BFG 2018). This species was not evaluated for conservation status. However, in PARNA Iguaçu, this species was found only in SSF, in the area of Foz do Iguaçu. Stenandrium mandioccanum blooms from August to February and in June and was found with fruits in February and October. This species is recognized by its obovate bracts and infundibuliform corolla, up to 1 cm long, lilac to pink in coloration, without differentiation in the corolla and glabrous.
In addition to these 13 species, the occurrence of three exotic species of the Atlantic Forest was verified: Odontonema tubaeforme (Bertol.) Kuntze, a species originating in Central America, Thunbergia grandiflora var. laurifolia (Lindl.) Benoist, originating in India and Sanchezia oblonga Ruiz & Páv., native of Brazil, but with natural occurrence only in Acre (Lorenzi 2013). These were not included in the taxonomic treatment, since their records are exclusively in anthropized areas in PARNA Iguaçu, such as near the Park School and Cataratas Hotel, and the family is widely known for its ornamental potential.
Among the species found, two are threatened with extinction, one is considered vulnerable (Justicia ramulosa) and Pachystachys dubiosa is categorized as in endangered. For the latter, there is only one record in the state of Paraná within PARNA Iguaçu, in the areas of Céu Azul and Foz do Iguaçu, reinforcing the need to preserve the Park for the conservation of these species in the study area and in the state of Paraná.
PARNA Iguaçu presents a high richness of native species of Acanthaceae when compared to other studies such as those of Kameyama (2014), which identified 52 species, and BFG 2018), which identified 54 species for the entire state of Paraná. Marchioretto et al. (2015), in turn, found 34 species for the entire state of Rio Grande do Sul.
In a survey conducted throughout southern Brazil, Argentina, Chile, Paraguay, and Uruguay, 124 species were found (Zuloaga & Belgrano 2015), of which the Acanthaceae species in PARNA Iguaçu corresponds to almost 10%, despite being a much smaller area.
In more local studies, the number of species is even lower as reported by Braz et al. 2002 with six species for a small reserve in Minas Gerais. While in the flora of Ilha do Cardoso, 13 species were found (Kameyama 2015) and in Serra do Rodriguésia 72: e00762019. 2021 Cipó, 13 species were also found (Kameyama 1995).
In addition, some of the species found are considered endangered or restricted in occurrence, which demonstrates the importance of conservation of this area for the maintenance of biodiversity in interior plant formations and the need for studies with non-arboreal taxa.