Found and lost again: Rediscovery of Mollinedia myriantha (Monimiaceae) after 123 years and perspectives for conservation of the family in Brazil

Mollinedia myriantha Perkins (Monimiaceae) is a tropical tree described by Perkins (1900) based on a collection of the French botanist Auguste F. M. Glaziou collected in 1892 in the mountainous region of Rio de Janeiro state, Brazil. The species was rediscovered in 2015 after 123 years, from Glaziou's report at the type locality, however, in subsequent fieldwork (2018 – 2019) the sole rediscovered individual was found dead. In this study, we report this rediscovery and we present the risk assessment including the Probability of extinction — P(E), also we provide a lectotypification, along with morphological description, geographic distribution map and conservation comments for this species. We also discuss the conservation status of Monimiaceae in Brazil.


Introduction
Global biodiversity in the Anthropocene is decreasing very fast (Butchart et al. 2010;Pena Rodrigues & Lira 2019). The rate of animal and plant species extinction has increased worldwide in the last centuries, deforestation and fire being a global concern for the conservation of biodiversity (Ceballos et al. 2015;Humphreys et al. 2019;Mortara et al. 2020). The IUCN Red List of Threatened Species is regarded as the most effective tool to identify which species and ecosystems are facing high extinction risk and which are the main drivers of species' population declines, similar to a "barometer of life" (Stuart et al. 2010;IUCN 2022a). A recent study estimates that 39.4% of all vascular plant species are threatened with extinction in the world, but only 10.5% had been assessed up to 2020 (Nic Lughadha et al. 2020). Rare species are a key component of plant biodiversity yet most of them are poorly known because they are hard to find and study. When we lack basic information about any given species, it is challenging to understand its threat status and propose evidence-based conservation actions. Consequently, many rare species that have their extinction risk assessed are categorised as Data Deficient because there is not enough information to understand their conservation status (Jaric et al. 2016;Nori & Loyola 2015).
Analysing a global dataset of presumed extinct plant species (1,234 species excluding synonyms), Humphreys et al. (2019) concluded that 571 species have become extinct in the last centuries, while 431 species were erroneously declared extinct and therefore rediscovered. Recently, many species have been rediscovered, mainly due to the extensive fieldwork aimed at finding them (Almeida et al. 2018;Bochorny et al. 2017;Pellegrini & Almeida 2016;Guedes et al. 2019;Marcusso et al. 2019;Guimarães et al. 2020;Mello Rolim & Oliveira 2020). Species that were erroneously declared extinct are often known from only a few or even a single historical locality, which results from their inherent rarity or lack of adequate sampling effort, a phenomenon known in conservation biology as 'Romeo error' or 'Lazarus species' (Collar 1998;Ryan & Baker 2016;Lírio et al. 2018a). In those cases, where possibly extinct species have been collected only once, intensive targeted search at the type locality may be the best way to find new individuals, if they still exist, and improve knowledge of the species and its threat status.
Monimiaceae is a pantropical family of flowering plants, with 28 genera and about 200 species, occurring mainly in humid and well-preserved forests (Renner et al. 2010;Lírio et al. 2020a). The genus Mollinedia Ruiz & Pav. (Ruiz & Pavon 1798), has about 60 species and occurs from south Mexico to the south of South America, Brazil being the richest country, with 46 species, and the Atlantic rainforest the richest domain, with 31 species, 28 of which are endemic to this domain (Lírio et al. 2020a). The Atlantic forest has a vegetation cover estimated from 11% to 28% and it is known to be the home of 15,499 species of Angiosperms, of which 54% are considered endemic to this biome (Ribeiro et al. 2009;Rezende et al. 2018;Flora do Brasil 2020). 67% of Monimiaceae species listed for Brazil occur in the Atlantic forest and 60% are endemic do this biome (Lírio et al. 2020a).
Thirteen species of Neotropical Monimiaceae are restricted to a single locality (Peixoto & Pereira-Moura 2008;Peixoto et al. 2009;Guimarães et al. 2014;Lírio et al. 2018aLírio et al. ,b, 2020bLírio et al. , 2021. So far, 34 out of the 46 species of Monimiaceae known as native to Brazil have been assessed on national level, and 11 of them have been considered as threatened with extinction, (CNC-Flora 2012;Ministério do Meio Ambiente 2014;Martinelli et al. 2018) however, among the 34 species, 7 species were not globally assessed, because they also occur outside the Brazilian territory, resulting in 27 species on the IUCN Red List website (IUCN 2022a). As an example, Mollinedia stenophylla Perkins (1900: 669) has been recently rediscovered within its type locality, 122 years after the last collection (Lírio et al. 2018a). It is found only in the Environmental Protection Area (APA) of Macaé de Cima, Nova Friburgo, in the state of Rio de Janeiro, and based on the newly available information, it has been suggested that the species should be reassessed as Critically Endangered, but presently it is only assessed as Endangered on the IUCN Red List website (Lírio et al. 2018a;IUCN 2022a).
Studies also conducted in APA of Macaé de Cima in 2015 by our research group aiming to collect data on threatened species of Monimiaceae revealed the presence of only one individual of Mollinedia myriantha Perkins (1900: 655), a species last collected in 1892 by the French botanist Auguste F. M. Glaziou. After fieldwork conducted in 2018 -2019 to find new individuals and collect more data about the species, we observed that the only known individual was dead and no new one was found. Here we present an updated morphological description, a geographic distribution map, photographs, lectotypification, extinction risk assessment followed by the probability of extinction P(E) for M. myriantha, and discuss the possible reasons for the loss of this individual. In addition, we present the perspectives for conservation of Monimiaceae in Brazil.

Materials and Methods
The APA of Macaé de Cima covers 350,370 km 2 of mountain landscapes within the upper part of the Macaé river watershed (Map 1). It is located in the Central Corridor of the Serra do Mar mountain range, which includes some of the largest and best preserved vegetation remnants of the Brazilian Atlantic forest, however, it is composed of many private proprieties and (Ribeiro et al. 2009). Mean annual temperature is 18°C and annual precipitation is 2,128mm (Lima & Guedes-Bruni 1997). Altitude ranges from 200 to 1,700 m above sea level and the predominant vegetation is montane rainforest in various stages of vegetal succession . The local flora is very rich, with 964 recorded species of vascular plants (INEA 2014).
Branches of the individual containing flowers were collected in 2015 in the APA Macaé de Cima (Map 1) and deposited in the herbarium RB (acronym follows Thiers 2022). The fieldwork consisted of six expeditions, three in 2015, each one of three days focused on general Monimiaceae species; one in 2016 for six days in five locations of the APA Macaé de Cima focused on general threatened species; and two expeditions in 2018 and 2019, each of four days and focused on M. myriantha and M. stenophylla. The localities where the fieldwork was carried out are shown on Map 2. We also analysed specimens from the herbaria C, F, GH, K, NY, P and RB to examine the types and to search for more specimens. The herbarium records were used (and georeferenced when necessary) to calculate the Extent of Occurrence (EOO) using the area of the minimum convex polygon and the Area of Occupancy (AOO) using a grid of 4 km 2 cells (IUCN 2022b). The analyses were performed using the geospatial conservation assessment tool GeoCat (Bachman et al. 2011) for extinction risk assessment. To assess the conservation status of the species, we followed the Categories and Criteria of IUCN Red List of Threatened Species (hereafter, IUCN Red List) (IUCN 2012;2022b). The probability of extinction followed the new methods for determining when to list species as Extinct (EX) and Critically Endangered (Possibly Extinct) (CR(PE)) on the IUCN Red List (SSC 2019; IUCN 2022b). We followed the instructions in conjunction with the Red List Guidelines (section 11), the data entry template EX_data.xlsx, and the R script RecordsSurveysModel.R (RStudio 2022.02.2+485 "Prairie Trillium" Release (8acbd38b0d4ca3c86c570cf4112a8180c48cc6fb, 2022-04-19) for Windows. The methods were based on two different and complementary models, and a framework for combining their results: the Threats Model ) which estimates the probability that the species is extinct (P(E)) based on qualitative and, where available, quantitative information about the severity, duration and scope of threats and their interaction with the life history traits that determine the species' susceptibility to these threats; the Records and Surveys Model (Thompson et al. 2017) as an iterative model to estimate the probability that the species is extinct, based on a time series of records of the species, and the timing, comprehensiveness and adequacy of any targeted surveys designed to detect the species after the last known record; and the Framework (Akçakaya et al. 2017) which combines the results of the two models to set thresholds of P(E) for classifying species as extinct, possibly extinct, and extant, based on the potential costs and benefits of classifying extinct and extant species.

Results
Mollinedia myriantha was found in an anthropised area of Atlantic forest (a private property called Sítio do Cedro), next to the roads and residences and without undergrowth (Map 1, Fig. 1A, B). The collection was made in 2015 during fieldwork carried out as part of a taxonomic revision of Monimiaceae and also the Brazil Flora Online project (Lírio et al. 2020a). In 2018 and 2019 we carried out more fieldwork to recollect the species and to search for more individuals, however, the only known individual was found dead (Fig. 1C) and no other new individuals have been found. The cause for its death is unknown, however we suspect pests or diseases, because other species of the genus were found dead on the edge of fragments, and marks on the trunk and branches that seem to be infections (Lirio pers. obs.). Despite this setback, the present study led us to an update of the species taxonomy and description because some characteristics were unknown in the protologue, such as the habit, flower colour and bracts and bracteole shape and length, which could help in the rediscovery of new individuals. Furthermore, we were able to confirm and better describe the species habitat and to assess its risk of extinction. Perkins (1900: 655). Type: Brazil, Rio de Janeiro, Nova Friburgo, Alto Macaé, 16 Feb. 1892, Glaziou 19859 (lectotype P00080080! designated here; isolectotypes C10015256!, C10015257!, F0064235F!, GH00041013!, K000587887!, MO3122710, P00080078!, P00080079!).

Mollinedia myriantha
Tree up to 8 m, 40 cm DAP, bark and stems corky, dioecious, cylindrical branches, young branches whitetomentose, then glabrous as they grow. Leaves opposite, 8.5 -10 × 3 -4 cm, elliptical, obovate or oblong, apex long acuminate to acute, base cuneate, entire, rigidchartaceous, discolorous, brownish-dark on the adaxial surface and brownish-light on the abaxial surface when dry, glabrous on the adaxial surface and white-strigose on the abaxial surface, except in the veins where the trichomes are dense and longer, five to six secondary veins, barely apparent on the adaxial surface and protruding on the abaxial surface, white-hirtellous, petiole 0.9 -1 cm, canaliculate, white-puberulous. Staminate inflorescences in triflorous cymes organised in long thyrses up to 24 flowers, axillary or terminal, trichomes white-tomentose, rachis (0.2 -0.4) 2.2 -5 cm, peduncle 0.3 -0.9 cm, pedicel 0.4 -0.6 cm. Staminate flowers greenish-yellow, 3 -3.5 × 4 -7 mm, bracts 3.5 -4 mm, oblong, apex acute, bracteole 1.5 -2 mm, ovate, apex acute, receptacle flat, tepals about 3/4 of the length of the flower, ovate, external with rounded apex, internal with truncated apex and denticulate margin, stamens 22 -23, ovate, non-confluent locule at the apex. Pistillate flowers and fruits remain unknown. Fig. 2. DISTRIBUTION AND HABITAT. The species is endemic to the Atlantic montane forest, only found in Macaé de Cima, an Environmental Protection Area located in Nova Friburgo municipality, Rio de Janeiro state. The patch where we found the specimen was a disturbed secondary forest at 850 m elevation, without understory and a canopy of about 10 m of sparse trees. We found the individual in the superior left side of the fragment of forest. The site where the individual was collected was bordered to the north by a dirt road, to the south by residences and banana crops, to the east by a second dirt road, and to the west by residences (Map 1B). The region is also impacted by invasive species, small farmlands and leisure residences.

EXTINCTION RISK ASSESSMENT AND PROBABILITY OF EXTINCTION P(E).
Mollinedia myriantha is a dioecious species and has a very restricted distribution, known only from the type collection made in 1892 (staminate flowers) and from a recent collection made in 2015 in a private area (also staminate flowers), on the border of a small disturbed secondary forest fragment located within the region of the type locality (Macaé de Cima), at 850 m elevation. Its estimated Extent of Occurrence (EOO) and Area of Occupancy (AOO) based on records is 4 km 2 , falling within the threshold of the Critically Endangered category under criterion B. There is only one known location for this species and the major threats are habitat loss and habitat degradation due to edge effects caused by fragmentation, expansion of roads, residential areas related to tourism and recreational activities, agriculture and invasive species. A continuing decline of area of occupancy, extent/area/quality of habitat, number of locations and mature individuals is inferred based on the threats mentioned, and also considering that since the species' rediscovery in 2015, the single individual recorded alive was later noticed as dead in 2018. Therefore, this species is assessed here as Critically Endangered as it meets the criterion B2ab (ii, iii, iv, v). The Probability that Mollinedia myriantha is extinct P(E) in 2022, when this analysis was carried out using The Threats Model ranges between 0.6 (PE_min) to 0.94 (PE_Max) with a best estimate (PE_best) equal 0.76. The Records and Survey Model resulted in a PE_min = 0.379, PE_max= 0.789 and a PE_best equal 0.588. The average of Probability of extinction -P(E) -from both models for Mollinedia myriantha is 0.674. The threshold for tagging a species as Critically Endangered -Possibly Extinct -CR(PE) is 0.5 and the threshold for assessing species as Extinct is 0.9 (see Supplementary material, ESM1). Therefore, based on the results of analysis of Probability of extinction P(E) using the Extinction assessment tools, we rated this species here as CR(PE). In this case, monitoring and further targeted fieldwork to complete the documentation since the findings from previous fieldwork carried out in 2015, 2018 and 2019 is needed, including documentation of threats across its entire range, as well as information on species biology and population trends. As the cause of death for this individual is unknown, new surveys must be provided including searches for this species in potential habitats (considering the type of forest and altitudinal range within its native distribution) in different months and seasons. Financial support and resources are urgently needed to provide the targeted searches and according to the IUCN Standards and Petitions Committee (IUCN 2022b), once the status of taxa is assigned with a 'Possibly Extinct' tag, its status should preferably be reviewed at five-year intervals. TAXONOMIC COMMENTS. The species resembles Mollinedia gilgiana Perkins (1900: 656)  34 species were evaluated following the IUCN Red List assessment at national or global level (1998 -2021), however, eight of them were made in 1998 using different criteria to what are used now, therefore, in need of update (Table S1, ESM1). Eleven (32%) out of the 34 species assessed are threatened, among these, 4 (12%) are considered Critically Endangered (CR), 5 (14.7%) are Endangered (EN) and 2 (5.9%) are Vulnerable (VU) to extinction, besides 4 (11.8%) are Near Threatened (NT), 1 (2.9%) Data Deficient (DD) and 23 (68%) Least Concern (LC). Among the assessed species, only two of them don't occur in the Atlantic forest, Mollinedia killipii J.F.Macbr. (Macbride 1934: 351) and M. lanceolata Ruiz & Pav., and both assessed as LC. The major threats are deforestation, fragmentation and fire in the Atlantic forest (Table S1, ESM1). Twelve of the 46 species still need a formal assessment. The results of the assessments are summarised in the Supplementary material (ESM1, S1).

Discussion
Over recent decades, there have been many efforts to catalogue the flora of Macaé de Cima. In 1996, a book about its flora with a chapter dedicated to Monimiaceae was published treating the species of the family occurring in the area, with taxonomic descriptions, notes and illustrations which treated 16 species, 10 of them being assessed as threatened (Peixoto & Pereira-Moura 1996, Table S1, ESM1). Also, researchers from the Rio de Janeiro Botanical Garden sampled more than 4,500 trees in two 1-ha plots in the area from 1989 to 1993 (Lima & Guedes-Bruni et al. 1997). Despite this substantial sampling effort, neither Mollinedia myriantha nor M. stenophylla were found. After intense fieldwork to find species of Monimiaceae, our team managed to discover new individuals of M. myriantha and M. stenophylla from 2015 to 2019.
Mollinedia stenophylla was found with 49 mature individuals, between staminate and pistillate, and many young individuals, which indicates that the species is reproducing and growing in its habitat. It is a dioecious shrub with blackish-purple fruits, occurring only in old-growth forests on the Macaé river bank (Lírio et al. 2018a). M. myriantha, a species first collected by Auguste F. M. Glaziou at 'Alto Macaé' (1892) (#19859, deposited in the herbaria A, K and P) and described by Janet R. Perkins (1900) was rediscovered in the same expedition with a single individual. This rediscovery happened 123 years after the previous collection (E. J. Lírio et al. 1196, deposited in RB). The species was located next to roads and residences, out of a fragment of forest (Map 1). The tree was about 8 m tall and had villose leaves, with trichomes concentrated along the rib. Even though the species is dioecious, only one staminate individual was found. It  Lírio et al. 1196).
photos: elton john de lírio. ◂ had long masculine inflorescences, and flowers with a flat receptacle. On the other hand, the discovery of a single individual of M. myriantha in a disturbed forest edge and its subsequent standing death suggests that this species may occur preferentially in undisturbed forests. Indeed, there is evidence that Monimiaceae species are strongly associated with old-growth forests in Macaé de Cima. For example, in a 1-ha plot placed in a 30-year-old secondary forest, only a single individual of M. salicifolia Perkins (1900: 659) was sampled , while in an old-growth forest plot of the same size, 136 individuals from seven species of Monimiaceae were sampled .
Mollinedia myriantha was found in the Environmental Protection Area of Macaé de Cima, Nova Friburgo, an extremely important area for conservation, because of the existence of endemic and threatened species (Diniz et al. 2016). An Environmental Protection Area aims to reconcile the conservation of biodiversity with a certain degree of human occupation and a sustainable use of natural resources. The main economic activities in the APA of Macaé de Cima include tourism, cattle raising and small-scale agriculture, and these occupy about 40% of the total area (INEA 2014). Among the 16 species of Monimiaceae found there (Peixoto & Pereira-Moura 1996), seven are endemic to the area, and two had not been recorded since the 19th century (Lírio et al. 2018b).
Integrated studies consisting of field expeditions, taxonomy and conservation status still bring new scientific knowledge of the flora in Brazil. As previously mentioned, field expeditions can change the conservation status of species while taxonomic studies are fundamental for the knowledge of conservation status. Investments in the assessment of the risk of extinction for the species not included in the IUCN Red List are necessary, as well as for the 12 species that have never been assessed at National or global level, and the reassessment of 13 species evaluated more than 10 years ago. These assessments are important to demonstrate the conservation status of the Monimiaceae family in Brazil, to attract attention to the species threatened with extinction and the priority areas for the conservation of this taxonomic group.
Brazil is the richest country of the world in flora, with 49,993 known species (native, cultivated and naturalised) (Flora do Brasil 2020) and with approximately 18% of its flora assessed at National level (Peixoto et al. 2013;Guimarães et al. 2014;Lírio et al. 2018b;Martinelli et al. 2018, and unedited data in partnership with BGCI, forthcoming). The country has carried out conservation actions including listings of threatened species at global level and conservation planning to prevent the extinction of species; however, this work is being compromised due to the reduction of investment in research and conservation of the environment in recent years Escobar 2019). Additionally, the Brazilian megadiverse forests are decreasing from habitat conversion/alteration for cattle ranching, fires, mining, and agricultural expansion Mortara et al. 2020). Projects like this, focused on the conservation of Monimiaceae bring significant contributions to the knowledge of the native, endemic and rare flora of the country. It also highlights the importance of investment in field expeditions for mapping the global flora and also collaborating with national and international goals such as target 12 of the Convention on Biological Diversity and goals 1, 2, 7 and 16 of the Global Strategy for Plant Conservation (CBD 2020; Sharrock 2020).
This work shows the importance of targeted fieldwork and herbaria visits for the conservation of rare and endangered plant species and the urgent need of conservation actions and monitoring for those species, especially in megadiverse countries such as Brazil. Rare plant species are difficult to find in the wild and without monitoring them, the same outcome that we find here is likely to be repeated. We expect that by doing intense fieldwork focused on this species we have a chance to find other individuals and propose conservation actions such as ex situ conservation, seedling production and reintroduction into the wild. Regarding the perspectives of conservation of the family Monimiaceae in Brazil, we found here that 30% of the assessed species are threatened with extinction, this is less than that predicted for all plants (39.4%), however, it probably reflects the need for an update for some species assessed more than a decade ago and also because some Not Evaluated species are probably threatened. Urgent actions are needed to assess and reassess those, propose actions to conserve them and contribute to avoiding biodiversity loss in the most plant-diverse country.