Floristic survey of vascular plants of a poorly known area in the Brazilian Atlantic Forest (Flona do Rio Preto, Espírito Santo)

Abstract Background The Atlantic Forest is one of the most threatened biomes in the world. Despite that, this biome still includes many areas that are poorly known floristically, including several protected areas, such as the "Floresta Nacional do Rio Preto" ("Flona do Rio Preto"), located in the Brazilian State of Espírito Santo. This study used a published vascular plant species list for this protected area from the "Catálogo de Plantas das Unidades de Conservação do Brasil" as the basis to synthesise the species richness, endemism, conservation and new species occurrences found in the "Flona do Rio Preto". New information The published list of vascular plants was based on field expeditions conducted between 2018 and 2020 and data obtained from herbarium collections available in online databases. Overall, 722 species were documented for the "Flona do Rio Preto", 711 of which are native to Brazil and 349 are endemic to the Atlantic Forest. In addition, 60 species are geographically disjunct between the Atlantic and the Amazon Forests. Most of the documented species are woody and more than 50% of these are trees. Twenty-three species are threatened (CR, EN and VU), while five are Data Deficient (DD). Thirty-two species are new records for the State of Espírito Santo. Our results expand the knowledge of the flora of the Atlantic Forest and provide support for the development of new conservation policies for this protected area.


Introduction
The Atlantic Forest houses one of the highest levels of species diversity and endemism in Brazil (Werneck et al. 2011). Despite that, the Atlantic Forest has been reduced to small and disconnected forest remnants, with only ca. 28% of its original forest cover remaining, of which less than half are protected (Rezende et al. 2018).
Unique in its occupation history, the State of Espírito Santo is completely inserted within the Atlantic Forest and exhibits, from the coast to the mountains, contrasting vegetation types (Dutra et al. 2015). Garbin et al. (2017) described the vision of many naturalists who passed through Espírito Santo in the 19 and early 20 centuries, providing a nice historical overview for the State. These naturalists highlighted the exuberance and distinction between the vegetation located in the southern and northern portions of the Doce River.
Floristic inventories were produced for the State of Espírito Santo during the last 30 years (e.g. Peixoto and Gentry 1990, Peixoto et al. 2008, Thomaz 2010, Rolim et al. 2016. The most comprehensive checklist of the angiosperms for the Espírito Santo was produced, based on a synthesis of online data (Dutra et al. 2015). These efforts resulted in the publication of several taxonomic and floristic studies for selected plant families in subsequent years (e.g. Luber et al. 2016, Chagas et al. 2017, Moreira et al. 2020a, Moreira et al. 2020b. Despite that, the State includes multiple knowledge gaps and botanical data remain scattered (Garbin et al. 2017). A full understanding of the forest dynamics of Espírito Santo depends on more detailed floristic inventories and vegetation data for the State. Information of this nature is crucial to fill knowledge gaps and establish sound conservation policies.
The "Catálogo de Plantas das Unidades de Conservação do Brasil" (https://catalogo-ucsbrasil.jbrj.gov.br/) was launched online in 2018 to contribute to the knowledge of the Brazilian protected areas. This digital platform provides comprehensive lists of land plants from the Brazilian protected areas, providing information about species correct names, th th conservation status, native/non-native status and digital images. The catalogue currently contains plant lists from 18 conservation units located in different Brazilian phytogeographic domains (e.g. Caatinga, Cerrado, Atlantic Forest and Amazon). The checklist of the vascular plants of the "Parque Nacional do Itatiaia" , Moreira et al. 2020a) and "Parque Nacional do Caparaó" (Moreira et al. 2020b, Carrijo et al. 2021 were the first to be launched, while the checklist of the vascular plants of the "Flona do Rio Preto" was recently added (Carrijo et al. 2021).
The "Flona do Rio Preto" was officially created in 1990 and is located in the northern portion of Espírito Santo (IBGE 2012). This protected area is mainly covered by dense ombrophilous lowland forest (IBGE 2012), despite the vegetation from the northern Espírito Santo having been classified as seasonal evergreen forest (Rolim et al. 2016). Historically, the area has undergone loss of natural vegetation due to anthropogenic disturbances, such as logging, grazing and fires, followed by the cultivation of Eucalyptus and sugar cane (Souza and Resende 1999). Despite these threats, the "Flona do Rio Preto" still offers good quality environments and floristic integrity. However, information about the local flora is restricted to the first forest inventory which focused on the documentation of woody trees and climbers (Souza and Resende 1999). Therefore, the vascular plant richness and composition of this protected area is still underestimated, especially due to a lack of documentation of herbaceous and shrubby species. Here, we summarise the information from a recent inventory (Carrijo et al. 2021) and present information about species richness, endemism, conservation and new species records from the "Flona do Rio Preto."

Sampling methods
Study extent: During this project, we undertook field expeditions to the "Flona do Rio Preto" fortnightly or monthly, from March 2018 to February 2020. Sampling covered all physiognomies ( Fig. 1) and was conducted by walking randomly across trails (Fig. 2). Collected materials were dried following standard methods for plant taxonomy (Peixoto and Maia 2013) and deposited at the Herbarium VIES (acronym from Thiers 2021), where samples were also digitised. Stages of data cleaning performed in R software to obtain a list of vascular plant species of the "Flona do Rio Preto," Brazil. Specimens kept on the list are shown in grey; specimens removed from the list are shown in red.
To obtain a list of species with currently accepted nomenclature, we manually selected all specimens identified to the species level, which led to the following: Search 1: JABOT determined = 5,237, undetermined = 2,174; REFLORA determined = 14,024, undetermined = 4,362; and Splink determined = 16,525, undetermined = 3,586; Search 2: JABOT determined = 10,057, undetermined = 3,079 (Fig. 3). We then selected specimens for which the localities fall within the area covered by the "Flona do Rio Preto" and removed duplicates, based on the catalogue code, collector name and number and the year when the sample was collected (Fig. 3). For the list of determined specimens, we corrected and updated species names using the function get.taxa from the package flora (Carvalho 2017) in R 4.0.2 (R Development Core Team 2019). This function compared the names in our list with those from the Flora do Brasil 2020 (http://floradobrasil.jbrj.gov.br).
After these corrections, we had taxonomists check a preliminary list with 690 determined species (1,558 specimens) and 870 undetermined specimens using images available from online databases (Fig. 3). When a taxonomist modified a plant species name, at least one specimen of that species was updated in the REFLORA database, but not in Splink. Infraspecific taxonomic categories or hybrids were not considered. The final list was published by Carrijo et al. (2021) and is available at (https://catalogo-ucs-brasil.jbrj.gov.br/).

Life forms
We obtained information on life forms from the Flora do Brasil 2020 (http://flora dobrasil.jbrj.gov.br) through the function get_lifeform of the flora package (Carvalho 2017).
Considering that the Flora do Brasil 2020 indicated more than one life form for several species, we conducted searches using three categories: (i) woody species (trees, shrubs and subshrubs, excluding climbers); (ii) herbaceous species (including palms and excluding climbers); and (iii) climbers (woody and/or herbaceous). When species were simultaneously assigned as a "climber" and "shrub" in the database, we chose to classify them as a "shrub" since "scandent shrubs" are often mistakenly treated as "climbers." As far as substrates are concerned, plants were classified as aquatic, epiphytic, hemiepiphytic, hemiparasitic, mycoheterotrophic or terrestrial.

Endemic and threatened species
We classified all species as native or non-native and endemic or non-endemic to Brazil using information from the Flora do Brasil 2020 (http://floradobrasil.jbrj.gov.br). Threat categories were assigned according to the Brazilian National Red List (CNCFlora; http://www.cncflora.jbrj.gov.br/portal). For binomials not included in the Flora do Brasil 2020, their native/non-native or endemic/non-endemic status was obtained from taxonomists and experts in those groups. We considered as non-native all species indicated as cultivated or naturalised within the Flora do Brasil 2020 (http://floradobrasil.jbrj.gov.br). We classified species as endemic to the Atlantic Forest when their distribution was classified as restricted to this phytogeographic domain in the Flora do Brasil 2020 (http://floradobrasil.jbrj.gov.br). We obtained this information using the function get_domains of the flora package (Carvalho 2017).   (Fig. 4). These families comprise 50% (i.e. 363 spp.) of all species found in the area. Forty-two families are represented by a single species. These findings are consistent with floristic inventories conducted in other forests remnants located in the Brazilian States of Espírito Santo and Bahia (Paula 2006, Amorim et al. 2008, Siqueira et al. 2014, Rolim et al. 2016, where the same richest families are generally documented. Despite the high floristic similarity documented in those inventories, only four species of Orchidaceae were found in the "Flona do Rio Preto," contrasting with the 103 spp. of Orchidaceae documented by Rolim et al. (2016). The low number of orchid species documented might be due to a shorter sampling period, incipient sampling efforts for epiphytes, in general, at the "Flona do Rio Preto," differences in climatic conditions or even the historical conservation of the "Flona do Rio Preto."

Life forms
In total, 447 species (61.9%) occurring in the "Flona do Rio Preto" are woody (trees, shrubs and subshrubs, excluding climbers), of which 219 are trees. Herbs (excluding

Endemic and threatened species
The list of the "Flona do Rio Preto" includes 711 native and 11 non-native species to Brazil. Some non-native species were found in the "Flona do Rio Preto," but were not included in the final list because no herbarium records were found, such as Mangifera indica L.  Field sampling in the "Flona do Rio Preto" started in 1953, even before its creation and extended until 2020. The years of 2019 (1,015 specimens), 2018 (257 specimens), 2020 (184 specimens) and 1995 (156 specimens) were the most intensively sampled. Collections conducted between 2018 and 2020 highlight the importance of the project "Rediscovering species threatened with extinction: Basis for management and information access," for increased knowledge about the flora of the "Flona do Rio Preto."

Conclusions and prospects
The list of the vascular plants of the "Flona do Rio Preto" expands the knowledge of the flora of the Atlantic Forest and provides important data for the development of sound conservation policies. Our study documented 253 endemic Atlantic Forest taxa at the "Flona do Rio Preto" and 32 new records of angiosperms for the State of Espírito Santo. The small number of epiphytes found in the studied area suggests that the "Flona do Rio Preto" has a vegetation type that does not match an Ombrophilous Dense Forest, the phytophysiognomy under which it was previously classified. The species list, provided here, contributes important information for an improved vegetation classification.