A REMARKABLE NEW GYNODIOECIOUS SPECIES OF OCOTEA (LAURACEAE) FROM THE BRAZILIAN ATLANTIC RAIN FOREST, AND ITS PHYLOGENETIC PLACEMENT IN THE OCOTEA COMPLEX

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Introduction
Over the past 30 years, the Phanerogamic Flora of São Paulo State project has been dedicated to inventorying the flora of São Paulo, the most populated state in Brazil and paradoxically the home of some of the most pristine and biologically diverse Atlantic Forest remnants (Wanderley et al., 2011). The Flora project did not, however, detect all existing taxa, particularly those in the Lauraceae, and since its publication , several new species have been described. These have been based on new collections or on previously collected herbarium specimens that had not been recognised as distinct new taxa (Moraes, 2007;Baitello & Brotto, 2016;Baitello et al., 2017).
Although the number of specimens in São Paulo's herbaria has increased considerably in recent years, partly due to new floristic surveys in under-collected areas, the processing and identification of these collections by specialists has been much slower. The issue of new species being unrecognised in herbaria, sometimes for over a hundred years (Arzolla et al., 2009), is found not only among the world's greatest plant collections but also in local, regional and small Brazilian collections.
2 New Ocotea from São Paulo, Brazil The new species presented here was first recognised as distinct in 2016. However, only now has all the necessary evidence been obtained to describe and place it accurately within the Ocotea group, despite its disporangiate stamens and apparent gynodioecy. Rohwer (1986a) published a synopsis of Ocotea Aubl., proposing its subdivision into 29 smaller informal groups based on shared morphological features. Among them, the Ocotea minarum group, to which the species described here is phylogenetically related, corresponds to Nees von Esenbeck's (1833Esenbeck's ( , 1836 concept of the genus Gymnobalanus Nees & Mart.

Ocotea minarum group
A particular characteristic of the fruits in this group is that the berry is not surrounded by a cupule and is seated almost completely free on a more-or-less conical, swollen pedicel, covered with lenticels (which occasionally expand into a rather shallow cupule). Additionally, Rohwer (1986a) pointed out that it was very likely that all members of that group would be gynodioecious or cryptically dioecious, because there are clearly pistillate and apparently hermaphroditic flowers (functionally male?) in different individuals. The latter type of flowers have roundish-trapeziform to almost rectangular anthers, filaments that are usually much shorter than the anthers and mostly ± hairy, staminodes lacking, a receptacle that is very small and shallow, an ovary that is also relatively small but always with a clearly well-developed ovule. By contrast, pistillate flowers have anthers that are smaller and sterile, the ovary is larger than the staminodes, and the style is usually only about as long as the ovary.
In  Mez and O. vaccinioides (Meisn.) Mez, are recorded. Ten taxa are recorded for the Andean region, the Guiana Shield, Central America, and the Caribbean (Rohwer, 1986a). More recently, several taxa have been described as belonging to the group or at least as being morphologically close to it (e.g. due to having ellipsoid or ovoid-ellipsoid fruits sitting free on a small plate-like cupule, gradually narrowed into an often lenticellate pedicel, and/or hermaphroditic and pistillate flowers). These taxa include Ocotea badia van Rohwer, 1986a). Based on results from a RADSeq phylogenomic study, a phylogenetic classification system for the Ocotea complex has recently been proposed, in which the new clade Pluriocotea is described (Penagos Zuluaga et al., 2021). Pluriocotea is further phylogenetically divided into (i) a clade containing representatives of the Ocotea minarum group (sensu Rohwer, 1986a;van der Werff, 2017)

Reproductive traits
The above-mentioned clades and associated taxa are not just phylogenetically closely related but also morphologically similar, particularly in terms of reproductive traits. Although information on breeding systems is mostly lacking, there are a few documented studies for Ocotea species in which evolution of dioecy via gynodioecy has been suggested. Ocotea cuprea (Meisn.) Mez, O. minarum and O. oblonga include individuals with clearly pistillate flowers and others with apparently hermaphroditic yet probably functionally male flowers (Rohwer, 1986b). However, Penagos Zuluaga et al. (2021) have not identified any evolutionary shifts that could point to gynodioecy as an intermediate step on the path from hermaphroditism to dioecism. According to Penagos Zuluaga et al. (2020), "sexual dimorphism is common in Lauraceae, where dioecism has evolved independently multiple times (Rohwer, 1986b;Chanderbali et al., 2001) often led by subtle morphological differentiation between sexes".
Identifying the correct breeding system from herbarium specimens is challenging, mostly due to the presence of only rudimentary structures in unisexual flowers and the difficulty of determining ovule and pollen fertility (Penagos Zuluaga et al., 2020). In Lauraceae, floral dimorphism is not evident between the sexes, but in a few species, the male inflorescence tends to produce more flowers. Additionally, some Lauraceae lineages may contain hermaphroditic, dioecious and gynodioecious species.
In species described as dioecious, pistillate flowers produce staminodes that resemble regular stamens, but the staminodes are usually smaller, borne on a shorter filament and with a flattened, sterile anther. In turn, staminate flowers usually show a pistillode that in some cases is morphologically indistinct from a fertile gynoecium, thus preventing verification of ovule fertility, determination of whether a flower is male or hermaphrodite, and whether the species is dioecious or gynodioecious. Consequently, species usually described from a few fertile specimens with evidently pistillate flowers have been stated as being dioecious, but in some of these, pollen-producing flowers were later described as having a well-developed pistillode (Penagos Zuluaga et al., 2021). Conversely, in the absence of pistillate flowers, plants with pollen-producing flowers have been described as hermaphroditic, overlooking the existence of female trees in the same species (Penagos Zuluaga et al., 2021). Thus, Penagos Zuluaga et al. (2021) have considered that the Ocotea complex is currently known to include only four known gynodioecious species: O. infrafoveolata, O. lenitae, O. oblonga (Penagos Zuluaga et al., 2020) and O. tenera (Gibson & Wheelwright, 1996;Gibson & Diggle, 1997, 1998. Molecular phylogenies have suggested that evolutionary changes in the number of anther cells have occurred multiple times in Lauraceae (Penagos Zuluaga et al., 2021) and support those who have questioned the generic value of two-or four-locellate anthers (e.g. van der Werff, 1984;Burger, 1988;Rohwer et al., 1991;Chanderbali, 2004;Rohde et al., 2017).
Given the morphologically atypical nature of the newly discovered species among the traditionally defined Lauraceae genera, as well as the broad polyphyly within the Ocotea complex involving new generic recircumscriptions (Trofimov et al., 2019;Trofimov & Rohwer, 2020), we used an integrative approach of morphology and phylogenetic analysis of nuclear and plastid DNA sequence data to reveal the evolutionary relationships and describe the new species.  Thiers, continuously updated) were also examined. Photographs of floral parts were obtained using a stereomicroscope (Leica M80) equipped with a camera (Leica IC80 HD; Leica, Wetzlar, Germany), using the software LAS (Leica Application Suite, version 4.3.0, Interactive Measurement module). The new species was compared with collections of Ocotea daphnifolia, because the two taxa are very similar, and with other species of the Ocotea minarum group. The terminology used in the descriptions is based on that used by Moraes et al. (2019).
Preliminary conservation status assessments follow the IUCN Red List Categories and Criteria, version 3.1 (IUCN, 2012) and accompanying guidelines (IUCN Standards and Petitions Committee, 2022). Rapid assessments were performed using the conservation assessment tool GeoCAT (Bachman et al., 2011), with estimations of extent of occurrence (EOO) and area of occupancy (AOO) based on a standard cell size of 2 km 2 .

Molecular data and phylogenetic analysis
We used the DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) to isolate high-quality total genomic DNA of four specimens of the new species and three other specimens of the putatively closely related species Ocotea daphnifolia. To amplify and sequence the psbA-trnH intergenic spacer, we used the forward primer psbA and the reverse primer trnH (Kress et al., 2005), and PCR conditions that consisted of a 4 min denaturing step at 94°C followed by 40 cycles of 1 min at 94°C (denaturation), 30 s at 51-55°C (annealing), 1 min at 72°C (extension), and a further extension for 7 min at 72°C. For the nuclear ribosomal ITS/5.8S region, we used the amplification primers 17SE and 26SE (Sun et al., 1994) and the sequencing primers SSF and LSR (Kollipara et al., 1997), in which the run program involved a 3 min denaturing step at 94°C followed by 28-30 cycles of 1 min at 94°C (denaturation), 1 min at 50-52°C (annealing), 2 min and 30 s at 72°C (extension), and a further extension for 7 min at 72°C. DNA extraction, amplification, and PEG-based purification of the PCR products (Paithankar & Prasad, 1991) were carried out at the Laboratório de Sistemática Molecular de Plantas of the Universidade Estadual de Feira de Santana in Bahia, Brazil. Sequencing reactions in both directions used BigDye Terminator kit version 3.1 (Applied Biosystems/ Life Technologies Corporation, Carlsbad, CA, USA) and were analysed on a ABI3730XL sequencer (Applied Biosystems Inc.) at the sequencing facility of the Rede de Plataformas Tecnológicas in FIOCRUZ-Bahia, Brazil.
Forward and reverse reads were inspected and assembled in contigs, using CodonCode Aligner version 9.0.2 (CodonCode Corp., MA, USA). Each of the 14 newly generated sequences of ITS/5.8S and psbA-trnH were aligned manually using AliView version 1.26 6 New Ocotea from São Paulo, Brazil (Larsson, 2014) into taxonomically more densely sampled individual alignments with focus on the entire Ocotea complex. Our sampling of 230 species was taken from sequences in GenBank (https://www.ncbi.nlm.nih.gov/genbank/), as retrieved with a custom R script using functions from ape package (Paradis & Schliep, 2019). The sampling covers not only morphological and taxonomic diversity but also the amplitude of the geographical range of the Ocotea complex (Trofimov et al., 2019;Trofimov & Rohwer, 2020). GenBank accession numbers and voucher information are shown after the taxon names in the original molecular datasets and the resulting phylogenetic trees (Supplementary files 1 and 2).
Individual DNA alignments were combined into a concatenated dataset using the R package catGenes (https://github.com/domingoscardoso/catGenes; Cardoso et al., 2020;Cardoso et al., unpublished data). The custom R script uses catGenes functions to automatically build the concatenated dataset by maximising the inclusion of taxa that are incomplete or with missing data (Wiens, 2003(Wiens, , 2006. In our concatenated dataset, the taxa lacked only 0.8% (ITS) and 1.2% (psbA-trnH) of sequence data.
Phylogenetic relationships were estimated through Bayesian inference (Lewis, 2001), using the software MrBayes 3.2.6 (Ronquist et al., 2012). The AIC (Akaike information criterion) model selection statistics, as implemented in the program jModelTest2 (Darriba et al., 2012), were used to select the most complex GTR+I+G evolutionary model of nucleotide substitution. Because the psbA-trnH intergenic spacer is largely known for inversions associated with palindromic sequences (Simpson et al., 2006;Whitlock et al., 2010;Cardoso et al., 2013;Trofimov et al., 2019), we excluded the sites 108-117 and 325-336 from the analysis.
The Bayesian analysis involved two separate runs of a Metropolis-coupled Markov Chain Monte Carlo permutation of parameters, in which eight simultaneous chains were initiated with a random tree for 20 million generations through the phylogenetic tree space, sampling one tree at each 10,000th generation. A 50% majority-rule consensus tree from the collection of the post-burn-in trees was generated, in which clade frequencies or posterior probabilities (PPs) represent support measures (Huelsenbeck et al., 2001).
Visualisation and editing of the phylogenetic trees for graphical presentation were carried out using FigTree version 1.4.4 (Rambaut, 2012) and Adobe Illustrator version 25.2 (Adobe, San Jose, CA, USA). All the original molecular datasets, and the associated MrBayes command blocks, are accessible as online supplemental data (see Supplementary files 1 and 2).

Phylogenetic relationships
The Bayesian combined phylogenetic analyses of nuclear ITS/5.8S and plastid psbA-trnH sequence data (Figure 1; Supplementary file 3) essentially recovered the same strongly 7 supported main clades across the Ocotea complex, including the broad polyphyly of the genera Endlicheria Nees, Licaria Aubl. and Ocotea, as previously revealed in comprehensively sampled phylogenies of the group (Trofimov et al., 2019;Trofimov & Rohwer, 2020;Penagos Zuluaga et al., 2021). Both analyses of individual ITS/5.8S sequences and of combined data concur in resolving all multiple accessions of the newly discovered species as phylogenetically close to Ocotea daphnifolia and O. domatiata within the O. minarum-O. smithiana group (see Figure 1; Supplementary files 3 and 4).
Despite the new species being morphologically distinct from both Ocotea daphnifolia and O. domatiata (see discussion in the Species description section), it is not readily differentiated genetically within the ITS and psbA-trnH alignments, which suggests recent speciation. This pattern of non-coalescence resulting in non-monophyletic species in our molecular phylogenies (see Figure 1; Supplementary file 4) is more likely due to incomplete lineage sorting, as largely observed in plant clades inhabiting tropical rain forest biomes (Pennington & Lavin, 2016).

Species description
Distribution. Ocotea bilocellata is currently known from only five municipalities on the north coast of the Brazilian state of São Paulo (a region encompassing the Paraíba river valley and the north coast itself, from Caraguatatuba to Ubatuba), within the Serra do Mar State Park, close to the border with Rio de Janeiro (Figure 7).
Habitat and ecology. It is found as medium-sized trees in the understorey of montane ombrophilous forests of the Atlantic Forest domain, at an altitudinal range of 800 to 1150 m a.s.l. Flowering from October to February, and fruiting in April, July, and September to November.
Etymology. The specific epithet refers to the 2-celled stamens.
Proposed IUCN conservation category. São Paulo is the most populous state in Brazil, with more than 46 million people inhabiting an area of 248.219.481 km 2 (IBGE: https://www. ibge.gov.br/en/cities-and-states/sp.html). The new species Ocotea bilocellata occurs in well-protected areas of the Serra do Mar State Park, in three of its Conservation Nuclei, namely Cunha-Indaiá, Picinguaba and Santa Virgínia, and also in the Boracéia Biological Station. Although its oldest collection dates back to 1941, there are 22 specimens from six locations (sensu IUCN). Ocotea bilocellata has an estimated EOO of 1640.24 km 2 and minimal AOO of 32 km 2 . Both estimates fall within the limits for Endangered (EN) status under criteria B1 (EOO < 5000 km 2 ) and B2 (< 500 km 2 ), but the number of locations is greater than five and falls within the Vulnerable (VU) category. However, its population is not severely fragmented, and there is no continuing decline in the number of mature individuals, which mean that it does not qualify it for criterion B. Moreover, because there are no plausible threats and the species is within well-protected areas, it meets category Least Concern (LC) (IUCN, 2012;IUCN Standards and Petitions Committee, 2022).

Discussion
From the dissection of pistillate and hermaphroditic flowers from the same population, we conclude that the new species is gynodioecious. The hermaphrodite flowers showed the same disporangiate pattern as the pistillate flowers, with well-formed and delineated pollen sacs occupying almost the entire length of the anther and bearing pollen grains with well-developed pistils without any sign of reduction (their stigmas at the same level as the apex of anthers). Their dimensions are also similar to those found in pistillate flowers: pistil 1.2-1.6 mm long (hermaphrodite) vs 1.3-1.6 mm long (pistillate); ovary globose vs ellipsoid, 0.7-0.9 × 0.5-0.9 mm vs 0.8-1 × 0.5-0.7 mm; style slender vs 'stout', slightly shorter than ovary vs shorter than ovary, 0.43-0.74 mm long (mean, 0.59 mm) vs 0.39-0.68 mm long (mean, 0.56 mm); and ovule well developed, 0.4-0.6 × 0.3-0.4 mm vs 0.5-0.7 × 0.2-0.3 mm. Therefore, they are morphologically indicative of hermaphroditism, but its gynodioecy is based on dried herbarium collections, without verification of ovule fertility. We found neither hermaphroditic individuals with flowers and fruits nor fertile stamens attached to the 'cupules' of young fruits. The reduction of the pistil in those cases is not obvious, and this can cause doubt as to whether the flower is uni-or bisexual. Compared with Ocotea oblonga (Penagos Zuluaga et al., 2020), it is possible that O. bilocellata similarly presents a populational hermaphrodite-biased sex ratio and that more fruits are produced by female individuals than by hermaphrodites. Based on keys to the genera of Lauraceae (e.g. Mez, 1889;Kostermans, 1957;Rohwer, 1986a;van der Werff, 1991;Rohwer, 1993), the new species described here does not fit unequivocally in any of the currently circumscribed disporangiate genera. Those keys employ floral characters such as the number of stamens and of anther cells per stamen, presence and shape of staminodes of fourth whorl, sexual condition of the flowers, and tepal heteromorphism, which are often variable within a genus (van der Werff, 1991). Specimens of this new species with pistillate flowers key out to Endlicheria, whose species are dioecious, with unisexual flowers and, frequently, with a rudimentary pistillode in the staminate flower (Chanderbali, 2004). By contrast, specimens with hermaphrodite flowers key out to Beilschmiedia Nees, whose Neotropical species have leaves that can be opposite and with a coarse venation pattern or alternate with a fine venation pattern; staminodes in the fourth whorl that are conspicuous and cordate to triangular in outline; receptacles that are invariably shallow; and fruits lacking cupules and that are freely inserted on their pedicel (Nishida, 1999;Nishida & Christophel, 1999). Moreover, it also keys out to the newly circumscribed Aiouea Aubl., which now encompasses 2-celled and 4-celled anther species (Rohde et al., 2017). These authors have shown that the Neotropical 'Cinnamomum' species form an evolutionary lineage with South American Aiouea rather than with the Paleotropical Cinnamomum species.
As pointed out by van der Werff (1988), the circumscription of the Neotropical genera of Lauraceae attaches much importance to the number of anther cells, which is an artificial character that obscures true relationships. This is supported by Rohwer et al. (1991), who found flowers with 4-celled and 2-celled anthers in a single inflorescence. Ocotea bilocellata can be placed together with several Neotropical species that have the staminal configuration of one genus, yet they can be recognised as belonging to another genus based on all other characters (examples in Rohwer et al., 1991). Despite its 2-celled anthers, which are previously unknown within Ocotea, the phylogenetic placement of Ocotea bilocellata in the O. minarum-O. smithiana group (see Figure 1; Supplementary files 3 and 4) is also supported by its gynodioecious breeding system (unknown for disporangiate genera), with clearly pistillate and hermaphroditic flowers, and the absence of staminodes of the fourth whorl.
Although we are here mainly concerned with the populations of Ocotea daphnifolia in the state of São Paulo, it is worth noting that individuals from known populations from Rio de Janeiro have leaves morphologically similar to those of São Paulo and Paraná, the latter being narrower (see Quinet, 2006;Giannerini et al., 2007;Brotto et al., 2013). Collections from the states of Espírito Santo and Bahia may have much larger leaves but show the other characteristics of the species consistently, including flowers without staminodes of the fourth whorl (Quinet, 2008;Barbosa et al., 2012;Ribeiro, 2019). Flowers with staminodes of 18 New Ocotea from São Paulo, Brazil the fourth whorl have been found exceptionally in collections from Paraná studied by Brotto et al. (2013).
Supplementary file 4. Bayesian majority-rule consensus tree of the Ocotea complex (Lauraceae), derived from the analysis of nuclear ribosomal ITS sequence data (269 terminals by 918 aligned sites), showing the placement of the morphologically enigmatic new species O. bilocellata within the O. minarum group (sensu Trofimov et al., 2019). Posterior probabilities from 0.5 to 1.0 are shown as colour gradient from red to black as well as values on the branches. GenBank accession numbers and vouchers follow the terminal taxon labels.