DOI: 10.5061/dryad.2v6wwq02k
title: Phylogenetic diversity and endemism of the vascular flora of the páramo ecoregion

description: The páramo ecoregion is known for its remarkable biodiversity and
 evolutionary histories shaped by complex biogeographical processes. To
 protect and conserve the ecological diversity and evolutionary lineages in
 this hotspot requires analyzing spatial patterns of phylogenetic diversity
 and endemism. This study uses spatial phylogenetic methods, combining
 georeferenced occurrence data with a genus-level phylogeny of the region’s
 entire vascular flora to investigate the spatial distributions of
 phylogenetic diversity and endemism. In this study, we assembled a
 comprehensive dataset of 361 equal-area grid cells covering the páramo
 biome and compiled occurrence records for 567 vascular plant genera. A
 phylogeny of vascular plants in the páramo was generated by combining
 publicly available genetic sequence data with novel genetic data from
 Colombian herbarium collections. This study examined the vascular plant
 diversity in the páramo ecosystem and identified distinct centers of neo-
 and paleoendemism, as well as a strong pattern of phylogenetic clustering
 across most páramo regions. The phylogenetic beta diversity analysis
 identified several phylogenetically distinct bioregions, characterized by
 both widespread, shared evolutionary histories and localized, distinct
 assemblages. This study provides a scope to prioritize certain areas for
 protection and to further identify and understand biogeographical patterns
 in the páramo ecoregion. The dataset provided here is an alignment of 5
 chloroplast DNA marker sequences aiming to represent all á
 vascular plant genera.
description: # Phylogenetic diversity and endemism of the vascular flora of the páramo
 ecoregion Dataset DOI:
 [10.5061/dryad.2v6wwq02k](https://doi.org/10.5061/dryad.2v6wwq02k) ##
 Description of the data and file structure ***DNA sequence collection and
 generation via herbarium genomics.***\ In order to create a phylogenetic
 tree containing all the taxa found in the páramo region, representative
 sequence data were sourced from the public repository NCBI GenBank
 [(Benson et al., 2018)](https://paperpile.com/c/HQ3xa5/jcoGo). For this,
 the highly conserved chloroplast-genome maturase K gene (*matK*),
 chloroplast NADH dehydrogenase F gene (*ndhF*), RuBisCO large subunit gene
 (*rbcL*), and *trnF(GAA)* gene, as well as the internal transcribed spacer
 of the nuclear ribosomal DNA (*ITS*), were selected. From the initial list
 of 1833 taxa,  available sequences from at least one of the selected
 markers were found for about 1670 genera. These sequences were identified
 and downloaded from NCBI using the Python script Matrix Maker [(Freyman
 & Thornhill, 2016)](https://paperpile.com/c/HQ3xa5/7IrAF). If at least
 one species of a genus had a publicly available sequence for the marker in
 question, the sequence was included in the study to represent its genus.
 To supplement this set of DNA sequences, leaf tissue samples were
 collected from 33 dried herbarium plant specimens in the Herbario Luis
 Siguifredo Espinal Tascón (CUVC) at the University of Valle in Cali,
 Colombia. Genomic DNA was extracted from these leaf tissue samples using
 the Qiagen DNeasy Plant Mini Kit, following the manufacturer’s protocol.
 After DNA extraction, the concentration of the extracted DNA was measured
 using the Qubit 4.0 Fluorometer (Life Technologies) with the dsDNA HS
 Assay Kit. Following extraction, DNA samples were sheared to the desired
 fragment size (400-600 bp) using a Covaris ME220 focused ultrasonicator.
 The quality and size distribution of the DNA extracts were then assessed
 using an Agilent 4200 TapeStation instrument, using HS D1000 ScreenTapes
 and reagents, to ensure they were suitable for the library preparation.
 The sheared genomic DNA extracts were used to construct Illumina
 sequencing libraries following the BEST (blunt-end single-tube) library
 preparation protocol [(Carøe et al.,
 2018)](https://paperpile.com/c/HQ3xa5/KPXnl), which involves three main
 steps: blunt-end repair, adapter ligation, and adapter fill-in. After a
 qPCR analysis to determine the optimal number of cycles, the purified
 libraries were subjected to dual-indexing PCR with 16-30 cycles. Each PCR
 reaction was carried out in a 100-uL volume containing: 7.5 µL of library
 template, 0.8 µL of dNTPs (25 mM), 2 µL each of F and R index primers (10
 µM), 1 µL of AmpliTaq Gold polymerase (5 U/µL), 10 µL of AmpliTaq Gold
 buffer (10X), 10 µL of MgCl2 (25 mM), 2 µL of BSA (20 mg/mL), and 64.7 µL
 of H₂O. The resulting libraries were purified and size-selected using a
 1:1 ratio of purified DNA:SPRI beads, and then assayed and quantified on
 the Agilent 4200 TapeStation prior to equimolar pooling. Sequencing was
 performed by the Novogene UK commercial sequencing service on the NovaSeq
 X sequencing platform (150-bp paired-end chemistry).  The raw sequence
 data for each sample were subjected to *de novo* assembly using
 GetOrganelle 1.7.7.0 [(Jin et al.,
 2020)](https://paperpile.com/c/HQ3xa5/a7IBi). The software GeSeq from the
 toolkit Chlorobox [(Tillich et al.,
 2017)](https://paperpile.com/c/HQ3xa5/T2tQA) was used to annotate genes in
 the resulting chloroplast genome assemblies. The sequences of the
 aforementioned highly conserved genes, along with 1,000-bp flanking
 regions on either side of the gene, were extracted from their respective
 annotation files to include in alignments with the data obtained from
 GenBank. The *de novo* assemblies resulted in circularized genomes for 14
 samples, with base coverages ranging from 67.5X to 1509.8X and an average
 chloroplast genome length of 153,875 bp (Table S1). Uncircularized or
 partial assemblies were obtained for 17 samples, while two samples were
 completely unsuccessful in generating any assembled sequences. For 28
 samples, at least one of the four target chloroplast markers (rbcL, matK,
 ndhF, and trnF) was assembled and annotated. For 23 samples, all four
 markers were annotated and included in the analysis.  ***Sequence
 alignment and phylogeny***.\ An initial sequence alignment for each marker
 was created using MAFFT version 7 [(Katoh & Standley,
 2014)](https://paperpile.com/c/HQ3xa5/eRB8l). The software Geneious Prime
 version 2024.0.5 was used to manually inspect the sequence alignments,
 editing the sequences to remove long strings of entirely ambiguous bases
 (encoded as N), as well as to trim sequence ends, and to refine the
 alignments. In some cases, some low-quality sequences were removed
 entirely from the alignment. The alignments were refined using the
 ClustalW algorithm [(Sofi et al.,
 2022)](https://paperpile.com/c/HQ3xa5/wAVdp) within Geneious Prime, with
 gap penalties set to default values. After alignment, low-quality ends and
 ambiguous regions were trimmed from each sequence to retain only the
 high-quality, core region of the multiple sequence alignment. To identify
 potential issues with alignment and misidentified sequences, a
 single-marker phylogeny was generated for each marker using RAxML v8.2.11
 [(Kozlov et al., 2019)](https://paperpile.com/c/HQ3xa5/CnFPG), and these
 issues were resolved for each of the alignments prior to concatenation of
 the 5 single-marker multiple sequence alignments using a custom Python
 script. The final multi-marker multiple sequence alignment was 13,217 bp
 in length and contained 1696 sequences, including the newly generated
 sequences from 31 herbarium specimens. This alignment was used to infer a
 final maximum-likelihood phylogenetic tree with RAxML, using a GTRGAMMA
 nucleotide substitution model and five data partitions. The resulting
 phylogenetic tree was visualized with iTOL [(Letunic & Bork,
 2024)](https://paperpile.com/c/HQ3xa5/KOgxk). The phylogenetic tree
 comprises 7 classes, 67 orders, 231 families, and 1696 genera. The
 placement of each branch tip was checked for consistency with the
 reference phylogeny from Kew Tree of Life Explorer [(Baker et al.,
 2022)](https://paperpile.com/c/HQ3xa5/a4COE) and confirmed to belong to a
 monophyletic group at the order level. In visualizing and rooting the
 tree, the class Lycopodiopsida was selected as an outgroup based on an
 available phylogeny of vascular plants [(Cole et al.,
 2019)](https://paperpile.com/c/HQ3xa5/h7VeY). ### Files and variables ####
 File: 5markers_partitions.txt **Description:** Indicates the location of
 each of the five concatenated DNA markers (matK, ndhF, rbcL, trnF, ITS)
 within the FASTA alignment
 'Concatenated_tree_Final_Sep06_V4.fasta'. #### File:
 Concatenated_tree_Final_Sep06_V4.fasta **Description:** Multiple sequence
 alignment of 5 sequences for all the genera included in the study. Marker
 sequences that were not available for particular genera are filled instead
 with Ns. #### File: File_S1_final_phylogeny.new **Description:** Phylogeny
 used for all spatial phylogenetics analyses in the tree (in standard
 Newick file format). Tip labels indicate the genus.