# Chromosome Number and Genome Size Evolution in _Brasolia_ and _Sobralia_ (Sobralieaee, Orchidaceae)
## 4 Materials and Methods
### Phylogenetic Analysis  
For phylogenetic reconstruction we sampled 57 individuals from 53 species distributed in four genera: 11 _Brasolia_, four _Ellanthus_, 34 _Sobralia_, and four _Neotia_ species (outgroup) (Table S1). We sampled species favoring those with available material for cytogenetic data (chromosome number and genome size), but that also represent all groups detected in previous studies [4, 5]. Genomic DNA extraction was performed in two ways, both AX Sherlock kit (A&A Biotechnology, Gdansk, Poland) and CTAB 2\(\times\) protocol [41] excluding \(\beta\)-mercaptoethanol were used. For both protocols, a fragment of plant tissue (\(\sim\)30 mg) and a ceramic sphere were placed in FastPrep tubes and the samples were homogenized. The final pellet was resuspended in 50 \(\mu\)L TE buffer. Two sequences were amplified: nrITS and _mat_K. The nrITS sequences were amplified using two different pairs of primers: 101F and 102R [42] and 17 SE and 26 SE [43], while, for the _mat_K gene, the primers 19F [44] and 1326R [45] were used for all samples. The PCRs, for both of the markers, were performed in a total volume of 25 \(\mu\)L, containing 1 \(\mu\)L temple DNA (\(\sim\)10-100 ng), 0.5 \(\mu\)L of 10 \(\mu\)M of each primer, 1.0 \(\mu\)L dNTP mix (0.2 mM of each dNTP), 2.5 \(\mu\)L 10\(\times\) Hybrid buffer contains 15 mM MgCl\({}_{2}\), 0.5 \(\mu\)L Hybrid DNA Polymerase (2U/\(\mu\)L) and water. The reaction parameters for the nrITS were: 94 \({}^{\circ}\)C, 4 min; 30\(\times\) (94 \({}^{\circ}\)C, 45 s; 52 \({}^{\circ}\)C, 45 s; 72 \({}^{\circ}\)C, 1 min); 72 \({}^{\circ}\)C, 7 min. For the _mat_K fragment, we set the following parameters: 95 \({}^{\circ}\)C, 3 min; 33\(\times\) (94 \({}^{\circ}\)C, 45 s; 52 \({}^{\circ}\)C, 45 s; 72 \({}^{\circ}\)C, 2 min 30 s); 72 \({}^{\circ}\)C, 7 min. The products of PCR reaction were cleanup using Wizard SV Gel and a PCR Clean Up System (Promega, Madison, WI, USA), following the manufacturer's protocol. Purified PCR products were sequenced by Macrogen (Seoul, South Korea), using the same primers mentioned above. DNA sequence chromatograms were examined/edited in FinchTV software ([[https://finchtv.software.informer.com/1.4/accessed](https://finchtv.software.informer.com/1.4/accessed)]([https://finchtv.software.informer.com/1.4/accessed](https://finchtv.software.informer.com/1.4/accessed)) on 12 October 2021). The phylogenetic reconstruction was based on the two obtained matrices, from nrITS and _mat_K, aligned in MAFFT v. 7.453 software using progressive alignment and iterative refinement methods [46]. The aligned sequences were trimmed using BioEdit v. 7.2 [47] and concatenated using Mesquite v 3.61 [48]. The generated matrix was analyzed under Maximum Likelihood (ML) and Bayesian Inference (BI). The ML analysis was performed using IQ-TREE2 [49], searching for the best-scoring ML tree under the following nucleotide substitution models: TNe+G4 to nrITS and K3 Pu + F + G4 to matK, both chosen by ModelFinder applying the Bayesian Information Criterion (BIC) [50]. We used 100 bootstrapping to assess branch supports. The BI was performed using MrBayes v. 3.2.7a [51] with GTR+I+G as the selected evolutive model for both partitions. Four independent runs, one hot and three cold chains, were started from different random trees to ensure that individual runs had converged to the same result. We used 20 million generations per chain with a sampling frequency of 2000. Split frequencies below 0.01 were used to check for convergence and the effective sample size (ESS) for each run was checked in the Tracer v. 1.7.1 [52]. In this case, 25% of trees were excluded as burn-in. Saved trees were summarized in a majority rule consensus tree created with nodal confidence assessed by posterior probabilities (PP), which were strongly supported when equal to or higher than 0.95 [53, 54].