A new species of Thinouia (Paullinieae, Sapindaceae) from the Amazon and its phylogenetic placement

Abstract Thinouia is a Neotropical genus of lianas with approximately 12 species and is the only genus in tribe Paullinieae with actinomorphic flowers. During a taxonomic revision of the genus and fieldwork in south-western Amazonia, we found a new species that appears similar to Thinouiatrifoliata (ex Allosanthus) because of its racemiform inflorescence. However, before describing the new species, we had to confirm that Allosanthus was congeneric with Thinouia so we could place the new species in the correct genus. The results of the phylogenetic analysis, based on molecular data (trnL intron and ITS sequences), show that Allosanthus should be included in Thinouia. Thus, the new taxon is described here as Thinouiacazumbensissp. nov. The new species is described, illustrated and phylogenetic trees showing relationships within supertribe Paulliniodae and Thinouia and the congeneric Allosanthus are given.


Introduction
Thinouia is a neotropical genus of lianas that includes around 12 species, of which T. myriantha Planch. & Triana is widely distributed, including records from Mexico, Central America and northern South America (Ferrucci and Somner 2008;Acevedo-Rodríguez et al. 2011). The remaining species are distributed in Brazil, Bolivia, Paraguay and Peru, except for T. tomocarpa Standl. which is restricted to Mexico, Belize and Guatemala. Most Thinouia species occur in rainforest; a few species occur in savannah (BFG 2015).
Thinouia was proposed by Triana and Planchon (1862). It is characterised by the presence of umbelliform and racemiform thyrses, actinomorphic flowers with marginal or bifid petal appendages, an annular disc and schizocarpic fruits that split into three mericarps, each with a distal wing (Ferrucci and Somner 2008;Acevedo-Rodríguez et al. 2017).
Molecular phylogenetic studies show that Thinouia is a monophyletic group in Sapindaceae. In the most recent phylogenetic study, tribe Paullinieae (i.e. Cardiospermum, Lophostigma, Paullinia, Serjania, Thinouia and Urvillea) is a well-supported clade with Thinouia sister to the remaining genera (Acevedo-Rodríguez et al. 2017). In the same work, the monospecific genus Allosanthus (A. trifoliatus Radlk.) was maintained as a synonym of Thinouia, based on morphological characters. The only differentiating character (i.e. a racemiform inflorescence) was not considered worthy of generic recognition (Acevedo-Rodríguez et al. 2011. During a taxonomic revision of the genus and fieldwork in south-western Amazonia, we found a new species of Thinouia that is similar to Thinouia trifoliata (Radlk.) Acev.-Rodr. & Ferrucci because of its racemiform inflorescence. Since we now have high-quality DNA material for the taxa previously assigned to Allosanthus, we re-analysed the placement of Allosanthus within Thinouia and further tested the monophyly of Thinouia s.l., which revealed the correct position of the new species.

Plant material
We collected the new species in Reserva Extrativista do Cazumbá-Iracema in Sena Madureira, Acre, Brazil. The collection was pressed and dried for vouchers, leaves were collected in silica gel for DNA extraction and reproductive structures were fixed in 70% alcohol for morphological analyses, which were performed using a stereomicroscope. The morphological structures were described using the terminology in Radford et al. (1974) and Weberling (1989). The herbarium abbreviations cited in the text follow Thiers (2020, cont. upd.).

Phylogenetic analysis
The phylogenetic analysis included the same taxa and molecular markers of Acevedo-Rodríguez et al. (2017), 93 taxa, plastid marker trnL intron and nuclear ribosomal internal transcribed spacer, ITS. Six samples (Allosanthus sp., Allosanthus trifoliatus, Thinouia mucronata, T. myriantha, T. obliqua and Thinouia sp.), including the new species, were added to the analysis, using the same molecular markers. For these additional taxa, approximately 60 mg of leaf tissue were pulverised with Tissuelyzer (Qiagen, Duesseldorf, Germany) for 3 min at 60 hz. The DNA extraction used the DNA NucleoSpin Plant II kit (Machery-Nagel, GmbH & Co. KG, Dueren, Germany) following the manufacturer's protocol. Primers and the PCR amplification were used, as described in Acevedo-Rodríguez et al. (2017). Products were purified and sequenced by Macrogen (Seoul, South Korea). All sequences, vouchers and GenBank accession numbers are summarised in Appendix I.
The alignments were performed using MAFFT (Katoh et al. 2002) using the default parameters implemented in Geneious 2020.0.5 (Kearse et al. 2012). Poorlyaligned regions were removed and adjusted manually. We used jModelTest 2.0 (Guindon et al. 2010; Darriba et al. 2012) and the Akaike Information Criterion (AIC) to select the best-fit model of nucleotide substitution for each dataset. The GTR+I+G was selected as the best model for the ITS dataset, whereas the GTR+G was selected as the best model for the trnL dataset. Bayesian Inference (BI) analyses were conducted using MrBayes 3.2.2 (Ronquist et al. 2012) in the online CIPRES Science Gateway interface (Miller et al. 2015) with four Markov Chain Monte Carlo (MCMC) runs using a random starting tree and 10 million generations, with a sampling frequency of one every 1000 generations. We used Tracer 1.7 (Rambaut et al. 2018) to check for convergence of the MCMC and to check for stationarity. We discarded 25% of the trees as burn-in.

Phylogenetic results
The ITS dataset included 99 terminals and 876 bp, the trnL dataset included 99 terminals and 727 bp and the combined dataset included 99 terminals and 1604 bp. Phylogenetic trees from the analyses of the combined dataset showed high posterior probability values (PP > 0.8). Only the topology from the combined analysis is described here (Fig. 1). Separate analyses of each locus did not reveal any strong groupings that would indicate incongruences. Supertribe Paulliniodae is strongly supported as monophyletic ( Fig. 1 A, PP = 1.0). The tribe Paullinieae is also strongly supported as monophyletic (PP = 1.0) and the genus Thinouia (including Allosanthus) is recovered as the clade, sister to the remaining genera of the tribe Paullineae (PP = 1.0). Thinouia species are grouped in two main clades that are in a polytomy with the new species Thinouia cazumbensis. The first clade (PP = 1.0) includes Thinouia obliqua, T. mucronata, T. restingae and T. cf. mucronata species. The second one (PP = 0.8) includes Thinouia sp., T. myriantha and T. trifoliata (= Allosanthus trifoliatus Radlk.) (Fig. 1).
Thinouia cazumbensis is differentiated from most species of Thinouia by the thyrses axillary, racemiform ( Fig. 2A, C) and the 5-lobed nectary disc, a character that is unique and for the first time recorded in the genus (Fig. 2E). The lobed nectary disc within Thinouia should be further investigated through morpho-anatomical studies to understand how nectaries evolved within the genus.
Distribution and ecology. Thinouia cazumbensis is known only from the Reserva Extrativista do Cazumbá-Iracema (Fig. 3) where it is an infrequent liana that reaches the canopy of the open rainforest with abundant bamboo (Guadua spp.) (Silveira 2005).
Phenology. Collected in flower and fruit during July. Etymology. The epithet cazumbensis refers to Reserva Extrativista do Cazumbá-Iracema, where the species was collected. In the 1980s, local rubber tappers and extractivists fought against the area becoming a rural settlement and on 19 September 2002 succeeded in getting the area designated as a conservation unit (ICMBio 2007). Situated in the State of Acre between the municipalities of Sena Madureira and Manoel Urbano, the Reserva Extrativista do Cazumbá-Iracema covers an area of 750,794.70 hectares of the Western Amazon Corridor, one of the seven major ecological corridors proposed for Brazil (Ricardo and Lima 2004).
Conservation status. The species is only known from one locality in Acre and is categorised as Data Deficient (DD) according to IUCN (2019). Further field studies are needed to evaluate its conservation status more accurately.

Discussion
The broader relationships that we recovered within supertribe Paulliniodae largely agree with those in Acevedo-Rodríguez et al. (2017). Additionally, with the inclusion of new sequences of Thinouia in this study merged with sequence data from Acevedo-Rodríguez et al. (2017), our results recovered the same clades in tribe Paullinieae, where Thinouia forms a clade that is the earliest diverging lineage. Therefore, our phylogenetic results reinforce including Allosanthus in Thinouia as proposed by Acevedo-Rodriguez et al. (2011), based on morphological characters. The only differentiating morphological character (i.e. the racemiform inflorescence) was not considered worthy of generic recognition by Acevedo-Rodriguez et al. (2011 and the molecular data in the present study corroborate this conclusion. The position of the new species as a member of Thinouia is strongly supported albeit its relationship to other species is not fully resolved, perhaps because of our limited sampling of Thinouia or because only two markers have been sequenced.

Conclusion
Thinouia cazumbensis is supported as a distinct taxon, based on morphological and molecular sequence data. Its position within the genus is still undetermined, highlighting the need for in-depth taxonomic studies on this genus. Ongoing systematics studies, based on molecular and morphological analyses of Thinouia, should provide additional insights into the evolution and biogeographic history of this neotropical genus (H. Medeiros et al. in prep.).