Vivaria calvasensis—A new genus and species of Araceae (Araceae: Aroidea: Spathicarpeae) from southern Ecuador

We describe a new genus with a new species belonging to Araceae, from southern Ecuador. Vivaria calvasensis gen. et sp. nov. inhabits semi-arid inter- Andean mountains at altitudes ranging between 1100–1300 m a.s.l. The species belongs to the tribe Spathicarpae, which in Ecuador is represented by two other genera, Incarum and Croatellia, both typical for humid environments such as montane forests. This new genus is clearly supported by molecular evidence based on the matK gene, and morphological traits that separate it from the closely-related genera included in this tribe. The analyzed material was collected during several field campaigns carried out during four years in two populations from Loja province (Calvas and Macará), southern Ecuador, near the border with Peru.

Both Incarum pavonii and Croatellia integrifolia occur in highly-humid montane forests, while Vivaria calvasensis gen. et sp. nov. was collected in dry inter-Andean valleys at medium altitudes, where seasonality is marked and human intervention is very intense, with short cycle crops and extensive grazing. Vivaria calvasensis gen. et sp. nov. grows in semi-arid a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 environments, where individuals spend most of the year in a vegetative state with only one leaf, or dormant, a feature which explains why they often go unnoticed [20].
The use of matK for the determination of this new genus and species was based on Goncalvez et al. [16], who reconstructed the phylogeny of the whole tribe based on this gene. It was also more recently used to determine the new monotypic genus Lorenzia [21].

Phylogenetic analysis
Two DNA sequences from matK and two sequences from trnL-F were obtained. However, these sequences were respectively identical. Thus, only one sequence of each gene for one sample of the collected plant was used in the phylogenetic analysis together with the 35 sequences per gene obtained from Gonçalves et al. [16].
The topology of the concatenated phylogenetic tree (Fig 1) was similar to the one obtained by Gonçalves et al. [16]. The matK and trnL-F region of our material clustered within the Spathicarpeae tribe in a well-supported clade containing Gorgonidium and Incarum (Fig 1). Our sequence was located sister to Incarum, and this relationship was well supported by MP and BPP analysis (Fig 1). In any case, our sequence was clearly different from Incarum pavonii (1.7%) and from Gorgonidium (G. intermedium, 1.6%), which was the genus closest to Incarum (Table 1).

Comparisons with similar species
Vivaria is a monotypic genus, closely related to Incarum and Gorgonidium. We refer to 35 of 46 characters previously used by Goncalves [16] to build the taxonomy of the tribe Spathicarpeae which we adopted to taxonomically place Vivaria within this tribe. Of the 35 characters used, 22 are morphological; in character 8 "basic leaf pattern", we include a new description adding the state "pinnatipartite", which is exclusive to Vivaria gen nov. assigning the number 3 in the corresponding character. In character 34 we also include a new category of the "seedling leaf" state by adding the "pinnate" state and assigning it the number 2. The other 13 characters used correspond to characteristics of male flowers that reinforce the separation of Vivaria gen nov. of the most closely related genera in the clade. A comparison of phenotypic traits between V. calvasensis and the four most similar species is given in Table 2.
Vivaria is proposed as a new genus of Araceae (Aroidea, Spathicarpeae), and molecularly it is most closely related to two genera-Gorgonidium and Incarum-with which it forms a single clade. Below we detail the similar morphological characters which are common for the species in the clade, as well as the traits that distinguish Vivaria from its related species: 10. in Vivaria calvasensis the fruit color is white or pale green, while in Gorgonidium and Incarum it is purple or blackish-purple; 11. the base of the leaves in Vivaria is pinnatipartite, and the leaves of the seedlings are pinnatipartite (Fig 14), while in Incarum and Gorgonidium they are simple to cordate.

Conservation status.
The new species has been encountered mainly in areas affected by extensive crop cultivation and livestock grazing (cattle and goats), as well as in moderately conserved areas. Being a seasonal species (the aerial part of the plant grows and shows itself in the rainy season), it is not well known by the local population, so it has no known uses or a vernacular name.
The currently known distribution range of V. calvasensis is small (less than 20 km 2 ). An additional population might be present in a neighboring canton (Macará), although it has not been confirmed until now; even so, the distribution range would not increase substantially. The species has not been encountered in any national reserve or otherwise protected area, which would represent a risk for the conservation of the species. In the Loja province, we encountered no distributional records neither in the herbarium or GBIF database for species of the genera Incarum or Gorgonidium, which are closely related to Vivaria calvasensis. This suggests that the Spathicarpeae tribe of the Araceae family is rare in the study area. According to the criteria issued by the IUCN in 2012 [22], a taxon is considered vulnerable (Vu) and in danger of extinction when its populations face or show any of the following criteria: i) a marked decrease in the individuals of its populations, a criterion that we did not evaluate for V. calvasensis or ii) the estimated area of occupancy is less than 2000 km 2 and the habitat is severely fragmented or it is known that it does not exist in more than 10 localities, this last criterion is the one we use to evaluate the taxon and place it within the vulnerable category (Vu).

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Vivaria calvense a new genus and species of Araceae

Discussion
The Spathicarpeae tribe (Araceae) was composed of 8 genera in 1997 [23]. Goncalves [19] reviewed the genus Asterostigma, specifically, A. pavonii and A. integrifolium, from which it derived two new monospecific genera, Incarum and Croatiella, respectively. This author [16] also reviewed the phylogeny of the whole tribe, including some species of the genus Dieffenbachia, and some species of the genus Bognera. Our results support the addition of a new genus, Vivaria, composed of one species.
Morphologically, Vivaria can be distinguished from Incarum by having pinnatipartite lamina, compared to the entire lamina that are characteristic to the latter. Both Vivaria and Gorgonidium have pinnate leaves, but the number of lobes differs between these two genera. In Gorgonidium, we can usually find between 5-7 lobes per pinna, while in Vivaria the number of lobes is greater 5 (7) -9. Structurally, the three genera have a solitary leaf and a floral peduncle, but in Vivaria the petiole reaches between 0.75-1 m, while in the two other genera, the petiole is smaller, being 0.21-0.45 m in Incarum [15], 0.06-0.1 m in G. beckianun and 0.36-0.4 m in G. striatum. The description of Vivaria calvasensis enriches the so-called "Andean" clade of the Spathicarpeae tribe [21], in which Gorgonidium, Incarum and Spathantheum would be the most closely related genera to Vivaria.
The current distribution of the Spathicarpeae tribe includes areas of Ecuador climatically different from the current distribution of Vivaria. The other closely related species distributed in Ecuador, I. pavonii, is restricted to humid areas. Our findings expand not only the distribution range of Spathicarpeae in Latin America, but, more importantly, bring evidence supporting the replication of an adaptation pattern seen in other species of the clade which inhabit arid and semi-arid areas. In these species, individuals remain dormant most of the year, when humidity is low, and become active during the rainy season, when their leaves and reproductive organs grow.
The occurrence of V. would coincide with the northern limit of the so-called Pleistocene dry arc, i.e. a large area in the South American continent, considered as the geographical distribution zone of certain species typical for forests and dry scrub distributed in the inter Andean dry valleys of Ecuador and Peru [24].
Finally, Cusimano et al. [17] consider that the evolution of Araceae is a complex process, that has resulted in diverse forms of life, ranging from the aquatic Pistia, Jasarum, the strictly rheophytic schistoglottids, to land-based geophytes (Vivaria calvasensis) and epiphytes of various kinds.

Ethics statement
All the botanical collections included in this publication, were carried out under the project "FLORISTIC PROSPECTING AND STUDY OF CRYPTIC SPECIES IN THE DRY SCRUB OF SOUTHERN ECUADOR", carried out between 2012-2015, which was financed entirely by the Universidad Tecnica Particular of Loja (UTPL). AP was supported by the PROMETEO program of the National Secretariat of Science and Technology (SENECYT for its acronym in Spanish) public organization funded by the Ecuadorian Government.

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None of the sampled areas were located inside protected areas, and no specific permit was required for these locations. The field studies did not involve endangered or protected species.

Specimen collection and study site
Field work was carried out between May 2012 and May 2015 visiting the dry inter-Andean valleys of southern Ecuador (Fig 15). During a botany trip financed by the HUTPL herbarium  Table 2. Comparison matrix of phenotypic characters between Vivaria calvasensis and the most closely related species based on the most important morphological characters within the tribe (Table 3) carried out in the Ecuadorian province of Loja, an infertile sample of a possible Araceae was collected, which was initially considered as belonging to the genus Gorgonidium, but with doubts about to the corresponding species. This led to a two-year search for fertile individuals of the species. The processed and labeled specimens (Type and paratypes) have been deposited in the Herbarium of the Universidad Tecnica Particular de Loja (HUTPL).

Morphology
For the description of qualitative and quantitative morphological and floral characters we use some of those cited by Goncalvez et al. [16] in the study of the phylogeny of the Spathicarpae tribe (Table 3). We compare some of the characters of Vivaria gen. et sp. nov. with the same characters of the most closely-related species, in order to obtain a diagnosis that allows us to identify and differentiate the new genus from the rest of the genera in the tribe. Some important measurements were made directly on the collected specimens, and photographs were taken both in the field and in Herbarium.

Molecular analysis
DNA was extracted from two samples of fresh leaf segments using a Plant Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. We amplified two genes, i.e., the plastid gene matK and the trnL intron with the trnL-F intergenic spacer (called simply trnL-F). The gene matK was amplified using the primer combination 371-F [25] and Trnk-2R [26]. The gene trnL was amplified using the primer combination Trnlf-C and Trnlf-F [27]. PCR conditions were as follows: initial denaturizing at 98˚C for 30 s; 30 cycles, each cycle consisting of one step of denaturizing at 98˚C for 10 s; annealing, at 60 ºC for 20 s and extension at 72˚C for 30 s; and final extension at 72˚C for 10 min. A control including PCR mix without DNA template was included in each PCR. Success of the PCR amplifications were tested in 0.7% agarose stained with GelRed. PCR products were purified using the QIAquick protocol (Qiagen). PCR products were sequenced bi-directionally by ABI 3730xl in Macrogen using the same primers as for PCR amplification.

Phylogenetic analysis
Sequences were edited and consensus were generated using CodonCode Aligner Ver.5.0.2. BLAST [28] was used against the NCBI nucleotide database (GenBank; http://www.ncbi.nlm. nih.gov/) to check their similarity with published sequences. Phylogenetic analyses were performed according to Gonçalves et al. [16]. For that, the 35 sequences of each gene (matK and trnL-F) belonging to tribe Spathicarpeae studied by Gonçalves et al. [16] were downloaded from GenBank. Matrices containing sequences belonging to each gene were aligned separately

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using Mafft Ver. 6.620b [29] under the G-INS-i option. After that, we concatenated both alignments in a new matrix using MEGA11 software [30]. Then we performed a Bayesian approach based on Markov chain Monte Carlo (B/MCMC) and a maximum parsimony (MP) analysis. The B/MCMC analyses were conducted using the MrBayes Ver. 3.1 program [31]. We used the most complex substitution model available (GTR+I+G), including two runs each involving four incrementally heated Markov chains over 4 000 000 generations and using random starting trees [32][33][34]. Trees were sampled every 100 generations resulting in 40 000 trees from which the last 24 000 were used to compute a 50% majority-rule consensus tree, enabling the use of Bayesian Posterior Probabilities (BPP) as node support. Stationarity of the process and effective sample size (ESS) values were checked visually with the software Tracer 1.5 [35]. MP analysis for each alignment was calculated on MEGA11 software [36]. Heuristic searches with 1000 random taxon addition replicates were conducted with the tree-bisectionreconnection (TBR) method with search level 1 [37]. Clade support was inferred from bootstrapping [35] performed based on 1000 pseudoreplicates with the same settings as for the heuristic search. Only clades that received bootstrap support of greater than or equal to 70% in the MP or posterior probabilities of greater than or equal to 0.90 in the MrBayes analysis were considered to be well supported [37]. Phylogenetic trees were drawn using FigTree Ver. 1.4.3. (http://tree.bio.ed.ac.uk/software/figtree/).

Nomenclature
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In addition, new names contained in this work have been submitted to IPNI, from where they will be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix http://ipni.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS, RiUTPL, Redalyc, DOAJ. Writing -review & editing: Omar Cabrera.