Pachygenium laurense (Orchidaceae, Spiranthinae), a new orchid species from Argentina—morphological evidence and phylogenetic reconstruction

Background Pachygenium embraces a group of terrestrial species formerly placed in Pelexia sensu lato. The genus currently comprises some 60 species, most of which are known from the southern parts of Brazil and Paraguay, with few species distributed in the Andean countries—only four species have been recorded from Argentina so far. In Jujuy Province, Argentina a new species of Pachygenium was found during our fieldwork. The aim of this article was to provide morphological and molecular evidence for its membership in this genus. Methods Materials from specimens were collected in the field and examined by classical taxonomic and molecular biological techniques, e.g., PCR and sequencing DNA. Phylogenetic reconstruction was performed by maximum-likelihood and Bayesian inference. Results Pachygenium laurense from Argentina is described and illustrated based on morphological evidence and its taxonomic position was confirmed by phylogenetic analyses. A new combination for Pachygenium gutturosa is also proposed. A key for identification is provided for the Pachygenium species occurring in Argentina. Conclusion Pachygenium laurense is the fifth species of the genus recorded from Argentina.


INTRODUCTION
The name Pachygenium was established by Schlechter (1920), who distinguished it as one of the five sections of the genus Pelexia Poit. ex Lindley (Orchidaceae, Spiranthinae). Based mainly on the spur structure, Schlechter recognized, aside from Pachygenium, the following sections: Cogniauxiocharis, P otosia, Eupelexia, and Centropelexia. The genus Pelexia, in its broad circumscription, is difficult to define in the terms of morphology. As a result, the taxonomic status of some of the aforementioned sections has changed. Cogniauxiocharis was incorporated in Pteroglossa Schltr. (Garay, 1982), and Potosia was obtained data were compared with original diagnoses and illustrations of representatives of Pelexia and Pachygenium from Argentina and adjacent countries. We examined over 1000 specimens of these genera stored in AAU, AMES, AMO, B, BA, BIGU, BM, C, COAH, COL, E, F, G, GOET, K, L, M, MO, P, S, SEL, SP, U, US, W, WRSL, WU, and Z.
Information on the occurrence and habitats of the new species was verified during field studies conducted in Argentina in 2020 and 2021. All field experiments complied with provincial, state, and national laws. The Dirección Provincial de Biodiversidad de Jujuy-Secretaría de Gestión Ambiental (Jujuy, Argentina) provided permission to conduct the research under permit number 171/2015-DPB.
''The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants (ICN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. In addition, new names contained in this work which have been issued with identifiers by IPNI will eventually 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: PeerJ, PubMed Central SCIE, and CLOCKSS''.

Molecular analyses
For phylogenetic reconstruction we applied 84 sequences of the ITS region, 90 of the trnL-trnF marker, and 75 of the matK gene representing taxa from seven genera: Cyclopogon, Veyretia, Glohisarcon, Sarcoglottis, Pelexia, Brachystele, and Pachygenium. In selecting the samples for ours analyses, we relied on the results obtained by Salazar et al. (2018). To determine the phylogenetic relations for the new taxon, we used species that grouped together with Pachygenium and Pelexia. Salazar et al. (2018) showed that representatives of Cyclopogon and Veyretia appear as a sister group, therefore we also used these as samples. Cyclopogon obliquus was selected to root the trees.
Most of the sequences used in this article were downloaded from GenBank (http: //www. ncbi.nlm.nih.gov/). A list of the taxa with their accession numbers is included in Appendix 1. Samples (leaf fragments) of Pachygenium laurense used for molecular studies were collected in the field by CM. The exact localities with numbers for names and collector are presented in Table 1. GenBank accession numbers for the sequences for these samples are listed in Table 2. DNA Isolation. Total genomic DNA was extracted from 20-100 mg of dried leaves (Chase & Hills, 1991) using the DNA Sherlock AX Kit (A&A Biotechnology, Poland) following the manufacture's protocol. The pellets of DNA were suspended in 50 µl of TE buffer. Amplification and sequencing. The PCRs and sequencing reactions were performed for three markers, two plastid (mat K and trnL-trnF) and one nuclear (ITS1+5.8S+ITS2). In both, the same pairs of primers for each marker were used: for the ITS region, 101F and 102R primers (Douzery et al., 1999) while for the plastid region trnL-F containing the trnL intron and trnL-trnF intergenic spacer using primers trnL-c and trnL-f as described by  Taberlet et al. (1991), and for the part of the mat K gene, primers 19F (Molvray, Kores & Chase, 2000) and 1326R (Cuénoud et al., 2002).
The ML/Thorough bootstrap workflow analysis was performed using RAxML-HPC2 (Stamatakis, 2014) by searching for the best-scoring ML tree under the previously calculated nucleotide substitution model. The branch support values (BS) were calculated with RaxML by halting bootstrapping automatically under the autoMRE criterion.
Bayesian inference was performed using MrBayes v. 3.2.7a (Ronquist et al., 2012). Two independent runs of four Markov-chain Monte Carlo (MCMC) chains (one heated and three cold) were started from different random trees to ensure that individual runs had converged to the same result. We used 2 million generations for ITS and mat K matrices, and 3 million for the trnL-trnF dataset per run with sampling every 100 generations. Convergence was assessed using the average standard deviation of split frequencies below 0.01 and the effective sample size (ESS) was checked in the Tracer v. 1.7.1 (Rambaut et al., 2018). Thereafter we discarded the initial 25% of the sampled generations of each chain as burn-in. Saved trees were summarized in a majority rule consensus tree and were edited with FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) and Inkscape (https://inkscape.org/release/inkscape-1.0.2/). The nodal confidence was assessed by posterior probabilities (PP), which were considered strongly supported when equal to or higher than 0.95 (Huelsenbeck & Ronquist, 2001).

RESULTS
The results of the maximum-likelihood and Bayesian inference gave similar results. Thus, we present and discuss the 50% majority-rule consensus tree only. The clades that have low support for bootstrap values in the trees of the ML analyses also have low posterior probability values in trees resulting from Bayesian inference. The topology of trees obtained for the mat K and the trnL-trnF regions are similar. Therefore, we decided to perform combined analyses for these markers. We also made a combined analysis for all used fragments of DNA. In this article we present the trees from the ITS region ( Fig. 1) and combined matrices (Figs. 2-3). All other obtained trees are available from the corresponding author. Table 3 lists the statistical data.

ITS matrix
In the ITS tree representatives of Cyclopogon and Veyretia were placed at its base ( Fig. 1). One of species of Cyclopogon (C. obliquus) served as an outgroup. The genus Veyretia is represented by three species in our analyses and all of them are included in one strongly supported group at the base of the tree. The examined species of Cyclopogon did not form a coherent clade and were recovered in a polytomy with clade I (Fig. 1). The main clade I of this tree embraces representatives of Glottisarcon, Sarcoglottis, Pelexia, Brachystele, and Pachygenium. All these taxa appear to be closely related as evidenced by strong support (PP = 1, BS = 100). In clade I we can recognize three smaller groups (clades A-C, Fig. 1). The

Combined matrices (matK + trnL-trnF and ITS + plastid markers)
The trees obtained for the combined matrices showed similar results as for the nuclear marker. Again, Cyclopogon obliquus is an outgroup, whereas species of Veyretia and the other sampled species of Cyclopogon were placed at the base of this phylogram. The main group of this tree (clade I,  embraces representatives of Glottisarcon, Sarcoglottis, Pelexia, Brachystele, and Pachygenium. Clade I was strongly supported both by posterior probability and bootstrap value (PP = 1, BS = 100) and it is divided into two smaller groups. The first, clade A, (Figs. 2-3)    The

DISCUSSION
The new species described in this article is an example of an unusual combination of various characters. While in flowering, Pachygenium laurense brings to mind Porphyrostachys parviflora (C. Schweinf.) Garay from Peru, especially with its leafless stem and densely many-flowered inflorescences. The ecallose, shortly clawed lip resembles that of Eltroplectris Raf., but the relatively slender elongate gynostemium appears to be more similar to that of Pelexia s.s. The narrow leaf blade gradually transiting into the petiole, and the saccate spur suggests a relationship with Pachygenium.
Our analyses of molecular markers have been able to help to clarify the taxonomic position of this odd species. The phylogenetic trees obtained for the single markers (i.e., ITS, mat K, and trnL-trnF) (Fig. 1) and the combined matrices  reveal that this taxon is accommodated in the Pachygenium clade. There are several characters, however, that are unique for this species and unknown for other species of Pachygenium as described to date. There are some species of Pachygenium growing in drier areas which can have either well-developed leaves or leaves withering in flowering. At the best of our knowledge, P. laurense appears to be the only species of the genus in South America which is totally leafless at anthesis. Interestingly, Pelexia gutturosa Rchb.f. from Mesoamerica, has very similar leafless habit and general flower architecture. Furthermore, P. gutturosa also grouped with the Pachygenium clade in our analysis (clade 4, Fig. 1 and clade 3 Figs. 2-3). Therefore, we decided to transfer this species to the genus Pachygenium as well.
The other unique features of the new species are: very characteristic arrangements of the flowers in the inflorescence, ecallose lip, undivided into epichile and hypochile and narrow gynostemium. In our opinion these features afford the plants to be assigned status of species and we describe it below.