An updated checklist reveals strong incongruities with previous studies: insights after revisiting a regional orchid list

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Introduction
Updated species lists are important tools for taxonomy, and they are also fundamental for biogeographical analysis and conservation planning (Clark et al. 2011;Söderström et al. 2007;Konrat et al. 2010).Detailed checklists also have application in areas such as in vitro propagation, since they ensure solid botanical information to develop structured conservation programs of endangered species (Silva & Acharya 2014).
Nevertheless, many biologically important regions lack updated species lists.As an example, the knowledge on species composition, richness, and endemism in Brazil is strongly biased especially in favor of the spatial distribution of the most important universities and research centers in the country (Oliveira et al. 2016).The Neotropics alone harbors about 37 % of the world's seed plant species (Antonelli & Sanmartín 2011), and specifically among the Angiosperms, the monocot family Orchidaceae (APG IV 2016) is one of the most diverse in the region (Chase et al. 2015;Dressler 2005) as well as in Brazil (BFG 2015;2018).In this country Orchidaceae are especially rich in the Amazon basin and Atlantic Forest (BFG 2015;2018), which were pointed out by Oliveira et al. (2016) as the Brazilian ecosystems with the lowest and highest collection densities, respectively.
Although among the poorest studied areas of Brazil botanically speaking (BFG 2015;2018), the northeastern state of Maranhão has great potential to have high levels of biodiversity due to its geographic position between the Amazon (30 %) and Cerrado (69 %) domains, and its variety of ecosystems and habitats (Abreu 1949;Froés 1953;Ribeiro 1971).However, before this study only about 200 records of Orchidaceae from Maranhão (excluding duplicates) were available in Brazilian herbaria, whereas smaller states (in territory) of northern and northeastern Brazil have many more orchid records (Amapá: ca.500, Roraima: ca.1000, Rondônia: ca.700, Ceará: ca.800, Paraíba: ca.700, Pernambuco: ca.2,000, Alagoas: ca.1,200, Sergipe: ca.700, based on Specieslink and Reflora, available at splink.org.br and reflora.jbrj.gov.br,respectively), indicating a strong Wallacean shortfall.It is also important to highlight that the majority of the orchid records from the state (ca.55 %) are deposited in the herbaria of other states (mainly CEN and MG, acronyms according to Thiers 2021).
The first known records of Orchidaceae from the state of Maranhão are presented by Cogniaux (1893Cogniaux ( -1896Cogniaux ( , 1898Cogniaux ( -1902Cogniaux ( , 1904Cogniaux ( -1906)), who listed five species, later by Pabst & Dungs (1975;1977), who cited ten species, which demonstrate the lack of collection efforts in the area for almost a century.The first more comprehensive list of species was provided by Silva et al. (1999), who included 103 orchid species in their checklist.However, for several taxa no voucher was cited, and some of the cited vouchers correspond to specimens collected in the neighboring state of Pará (available at MG); thus, the data are not accurate.
Currently, according to the Flora do Brasil 2020 (2020), 105 species and 41 genera are recorded in Maranhão.These data are mainly based on the list by Silva et al. (1999) and information from some recently published studies (Ferreira et al. 2017).This number of species is fairly low if compared to smaller but better studied states of northeastern Brazil such as Pernambuco (ca. 190 species, BFG 2015, 2018), whose territory corresponds to a third of that of Maranhão, but which has been studied in recent years (Pessoa & Alves 2012;2014;2015).
Thus, the aim of this study is to provide an updated checklist of the orchid species from the botanically poorly studied state of Maranhão, based on a review of herbaria and new field expeditions.A revised list will be important not only for taxonomic but also for conservation purposes, since Orchidaceae can be used as a model group for optimization of the conservation of plants in general (Swarts & Dixon 2009).Furthermore, it will clarify our knowledge on the composition of the orchid community in this Amazon border region that may have served as a corridor for species exchange with Atlantic Forest during the Pleistocene (Maciel et al. 2017;Pessoa et al. 2018;2021).In addition to the list of species, this study presents maps of spatial distribution of collection records and species richness in the state, and for each species information is provided on life form and ecological domain of occurrence.

Study area
The state of Maranhão is located in the Northeast region of Brazil, has a total area of 333,365.6 km 2 , and is delimited to the west by the North region of Brazil (IMESC 2008;IBGE 2014).Its territory includes parts of the Amazon (30 %), Cerrado (69 %) and Caatinga (1 %) (Fig. 1A) domains, therefore having markedly transitional vegetation (Abreu 1949;Froés 1953;Ribeiro 1971;Muniz 2004;Almeida & Vieira 2010;Celentano et al. 2017;Silva et al. 2017;Silva-Moraes et al. 2019).Its coastal northern portion has low altitudes that do not exceed 60 m elev., the western Amazonian portion can reach 300 m elev., while the highest points of the state are concentrated in its Center-South portion with altitudes up to 600 m in areas of Cerrado (Silva-Moraes et al. 2019).The climate is Tropical Savanna, dry in the summer along the coast (As sensu Köppen 1948), while dry in the winter toward the interior of the state (Aw sensu Köppen 1948), the annual mean temperature is 26-27 °C, and precipitation varies from 700-1700 mm in the southern half of the state to 1700-2900 mm in the northern half (Montes et al. 1997;Maranhão 2011).

Data collection
Our initial database was constructed based on the lists provided by Silva et al. (1999) andBFG (2015;2018) and new records published more recently (Ferreira et al. 2017;2019;Rodrigues et al. 2019;Saraiva et al. 2020;Oliveira et al. 2021;Gomes et al. 2021;Silva et al. in press).Subsequently, the main Brazilian herbaria and local herbaria were visited (CEN, HABIT, HB, HUEFS, INPA, IAN, MAR, MG, RB, SLUI, SP, SPF, UB, acronyms according to Thiers 2021, continuous adapted) in order to find specimens collected in the state to serve as vouchers of the taxa reported in the literature.Information available on the on-line platforms SpeciesLink and Reflora was also used for herbaria not visited (only if images were available).The occurrence in ecological domains and the geographic distribution in Brazil of each species were consulted in the Flora do Brasil 2020 (2020).
Fieldwork was carried out from July 2012 to February 2020, covering areas not yet collected in the state.The samples were submitted to the usual taxonomic procedures (Mori et al. 1989) and then deposited at HABIT, MAR, or SLUI, with duplicates sent to RB.The identification of the material collected and the re-identification of previously collected specimens in the herbaria analyzed were based upon the most important references on Orchidaceae taxonomy for species in northern and northeastern Brazil (Dunsterville & Garay 1959;1961;1965;1966;1972;1976;Pabst & Dungs 1975;1977;Carnevali et al. 2003;Toscano-de-Brito & Crib 2005) and analysis of type specimens when available on-line or in the herbaria visited.
A database of geographical coordinates from field collections and herbarium specimens was prepared to analyze the spatial distribution of collection records and species richness in the state.Specimens with no georeferenced data had their localities determined using online gazetteers (Google Earth).Kernel density maps (heat maps) were produced with the software QGIS v. 2.18.12 (QGIS Development Team 2020), using a radius of 30,000 m and the WGS84 datum.

Results and discussion
The herbaria review and field expeditions recovered a total of 51 genera and 119 species of Orchidaceae with occurrences confirmed to , of which 77 species (64.7 %) are epiphytic, 38 species (31.9 %) are terrestrial, three species (2.5 %) are hemi-epiphytic, and one species (0.8 %) is myco-heterotrophic (Tab.1).Epiphytic species occurred mainly (about 75 %) in environments in western Maranhão in the Amazon Forest, whereas the terrestrial species more often grow (about 84 %) in areas of Cerrado.Our results corroborate those described by Dressler (1993), who reported that in wet tropical forest at least 2/3 of the orchid species are expected to be epiphytic and also Dressler (1993), Batista & Bianchetti (2003) and Ferreira et al. (2010), who highlight the higher percentage of terrestrial species in open environments such as the Cerrado.
The most representative genera in Maranhão are Habenaria (17 spp.), Catasetum (14 spp.) and Epidendrum (10 spp.), while 35 other genera are represented by a single species in the state.Representatives of the subtribes Laeliinae (21 spp.), Oncidiinae (17 spp.), and Habenariinae (17 spp.) correspond to about 46 % of the species.Representatives of Laeliinae make up a large part of the orchid flora in the Neotropics (Van den Berg 2000) and have been pointed out as the most representative orchid subtribe in other studies in northeastern Brazil (Pessoa & Alves 2015;Monteiro et al. 2012).Habenaria has been found to be the most species-rich in areas of Cerrado, such as in the Federal District (Batista & Bianchetti 2003), and Catasetum is especially diverse in the transition zone between Amazon and Cerrado, as pointed out by Petini-Benelli (2012) for the Brazilian state of Mato Grosso.
Among the species, six (5 %) are endemic to Maranhão, all representatives of the genus Catasetum, and 27 (22.7 %) are endemic to Brazil.Although connections between the Amazon and Atlantic Forest along the coast have been hypothesized to have happened during the Pleistocene (Maciel et al. 2017), among the orchids of Maranhão, only nine species (7.6 %) have disjunct distributions between the Amazon domain and the Atlantic Forest of northeastern Brazil.The results show that most of the orchid species that grow in the state are widespread in South America (Govaerts et al. 2020).Most of the species (87, 73.7 %) grow in two or more Brazilian domains, pointing out the transitional characteristics of Maranhão, 27 species (22.9 %) are known only to the Amazon domain, while only six species (5 %) are exclusive to the Cerrado (Tab.1).
Considering the Northeast region of Brazil, 27 species listed here are found only in Maranhão (BFG 2015;2018), including its six endemic species and others restricted to the Amazon domain, which in the Northeast region is limited to Maranhão.However, this area, a part of the Belém Endemism Center (Almeida & Vieira 2010), is constantly being degraded by human activities and social conflicts, a situation that puts the orchid flora increasingly at risk (Almeida & Vieira 2010;Silva-Moraes et al. 2019;Silva-Junior et al. 2020).According to the National Flora Conservation Center (CNCFlora 2020), four species are under some degree of threat: Cycnoches pentadactylon, Cyrtopodium poecilum and Notylia microchila are endangered (EN), while Cattleya nobilior is near threatened (NT).
Our checklist includes 30 species that are not listed in Flora do Brasil 2020 (2020) to the state, however many of them have been recorded in previous publications (Silva et al. 1999;Ferreira et al. 2017;2019;Rodrigues et al. 2019;Saraiva et al. 2020;Oliveira et al. 2021;Gomes et al. 2021;Silva et al. in press).Thus, excluding these, a total of six species are cited here for the first time to Maranhão, all also being new records for northeastern Brazil (Tab.1).All specimens of these six species were found as unidentified or misidentified in the herbaria visited.We also provide the first record of Cycnoches and Paphinia to northeastern Brazil.
Although representing only a small fraction of change in terms of the number of species when comparing the lists provided by Silva et al. (1999) and Flora do Brasil 2020 (2020), 103 and 105, respectively, vs. 119 in this study, we detected strong incongruity among the data.After an update of the names listed by Silva et al. (1999) following the current widely accepted systematics of the family, we found that of the 103 taxa, 25 species lack vouchers from the state and were not collected in our expeditions; thus, these taxa were not confirmed to Maranhão.For the Flora do Brasil 2020 (2020) list we found 20 species that were also not confirmed for the same reasons.The excluded species from Silva et al. (1999) and Flora do Brasil 2020 (2020) are not entirely the same, while 10 species excluded here are common to both lists, 14 are only in the former list and 10 are only in the latter.A list with the 34 excluded taxa is provided in Tab.2; most of these species do occur in the neighboring state of Pará.
The new number of confirmed orchid species in Maranhão is still low, however according to Flora do Brasil 2020 (2020) it is higher than the majority of states in northeastern Brazil, except for Pernambuco (188 spp.) and Bahia (526 spp.), while it is equivalent of the species richness recorded for Acre (114 spp.) and Amapá (130 spp.) in the North region.The geographical position of the state in a transition zone, from a drier area along the border with the state of Piauí to a moister area along the border with Pará (Fig. 1A), can explain the wide distribution of the species, and low number of endemic species, since ecotones are characterized by the "edge-effect" (Dawson 2001).The central and southern portions of the state are still poorly collected (Fig. 1B), and a better collection effort in these regions may result in the discovery of more species from the Cerrado not listed here.It is also a reflection of the strongly biased botanical gathering in Brazil, especially due to the difficulties of access and infrastructure for researchers in some areas (Oliveira et al. 2016), as also reported by Siqueira et al. (2014).

An updated checklist reveals strong incongruities with previous studies:
insights after revisiting a regional orchid list

Species
From the four hotspots of collection effort in the state (Fig. 1C), three coincide with university campuses (municipalities of Caxias, Imperatriz and São Luís).Only the one in the northwest region (in the vicinity of the municipality of Candido Mendes) is the result of a previous study (Silva et al. in press).This area is also the richest in orchid species in the state (Fig. 1C), but also the most exposed to threats such as change in land use/deforestation and altered fire regime (Almeida & Vieira 2010;Silva-Moraes et al. 2019;Silva-Junior et al. 2020).

Figure 1 .
Figure 1. A. State of Maranhão and its three phytogeographic domains.Green = Amazon, Orange = Cerrado, Yellow = Caatinga.B. Distribution of the records of Orchidaceae.C. Species richness of Orchidaceae.

Table 2 .
List of species cited in the literature to Maranhão but excluded here due to the lack of voucher specimens.