DOI: 10.5061/dryad.f7m0cfz5d
title: Phylogenomics of Boraginaceae using lineage-specific and angiosperms353 loci

description: During the past 20 years, the phylogenetics of Boraginaceae has taken
 shape using plastid DNA regions and the nuclear ribosomal internal
 transcribed spacer (ITS), but these regions only represent a limited
 understanding of the evolutionary history of the family. Using
 hybridization-enrichment sequencing, 531 nuclear regions from
 lineage-specific and Angiosperms353 loci were sequenced and aligned for 49
 species from across Boraginaceae. Additionally, the Angiosperms353 loci
 were incorporated with a broader dataset of the same loci from 115
 accessions of Boraginales and relatives. Based on multiple phylogenetic
 approaches and datasets, the resolved phylogenies of Boraginaceae were
 quite similar to our current understanding, yet multiple taxa were
 recognized in different positions. These included: 1) Echiochiloideae as
 sister to Cynoglossoideae instead of to the rest of the entire family, 2)
 Moritzinae as nested within Boragininae, and 3) Lasiocaryeae and
 Trichodesmeae not resolved as sisters. These different positions
 recovered, via different methods, using hundreds of nuclear loci suggest
 that incomplete lineage sorting, hybridization, and heterotachy may have
 occurred during the early origin of the family. In analyses of
 Boraginales, Namaceae was resolved as non-monophyletic, providing evidence
 that a broader Hydrophyllaceae may again be appropriate, and Lennoaceae
 was nested in Ehretiaceae. While both sets of loci allowed for a
 well-resolved and well-supported phylogeny to be reconstructed, the
 lineage-specific loci recovered some of the more intriguing phylogenetic
 relationships in part because these loci appear to be less conserved than
 those from Angiosperms353. The two sets of loci provide an interesting
 complement for understanding patterns of evolution within the family and
 order.
description: Six datasets were constructed for phylogenetic analyses of
 Boraginaceae: 1) 353-42 (Angiosperms353 loci with all 49 species, which
 includes 42 loci), 2) 353-305 (Angiosperms353 loci with at least 27
 species, which includes 305 loci), 3) Borage-149 (Borage-specific loci
 with all 49 species, which includes 149 loci), 4) Borage-226
 (Borage-specific loci with at least 27 species, which includes 226 loci),
 5) B353-191 (Borage-specific and Angiosperms353 loci with all 49 species,
 which includes 191 loci), and 6) B353-531 (Borage-specific and
 Angiosperms353 loci with at least 27 species, which includes 531 loci).
 These six datasets represent two approaches for phylogenetic analyses. One
 was to maximize the completeness of the matrix by using only loci that
 were present for all 49 species, and the other was to maximize the number
 of characters for the matrix by including loci that had sufficient
 representation among the 49 species. The former resulted in representation
 for each locus for each species, limiting missing data, and the latter
 resulted in many more loci and characters included, but with a greater
 amount of missing data, with 27 species used as cut-offs to balance the
 incorporation of sequence data with increasing missing data. Furthermore,
 multiple distinct phylogenetic analyses were conducted and results were
 compared to explore potential variation in reconstructed trees, to examine
 the influence of methodological issues in phylogenetics, and to recognize
 areas of the tree or taxa that were more variable or stable depending on
 analysis and dataset.   Individual loci, plastid
 genomes (with only one inverted repeat), mitochondrial genomes, and
 nuclear ribosomal cistrons were each aligned using the global pair option
 with MAFFT (Katoh and Standley 2013). Aligned sequence data was
 subsequently analyzed using Maximum Parsimony (MP) and Maximum Likelihood
 (ML) methods. With MP analyses in TNT (Goloboff and Catalano 2016), the
 following search was conducted for each locus: 1,000 parsimony ratchets
 (Nixon 1999), with 10% upweighting and 10% down weighting, 1,000 drift
 iterations, 100 rounds of tree fusing, and sectorial searches (Goloboff
 1999), and this analysis was undertaken 10 times to find the optimal tree.
 A strict consensus tree was reconstructed, and 1,000 bootstrap and
 jackknife replicates were conducted. For ML analyses with RAxML-NG (Kozlov
 et al. 2019), ModelTest-NG (Darriba et al. 2020) was used to identify the
 optimal substitution model according to the Bayesian Information Criterion
 (BIC), for each locus, among 11 substitution schemes and with nucleotide
 frequencies and rate heterogeneity taken into account. RAxML-NG was run to
 recover the optimal tree, and 1,000 bootstrap replicates were conducted,
 with Felsenstein bootstrap support and Transfer bootstrap expectation
 calculated (Lemoine et al. 2018; Lutteropp et al. 2020). IQ-TREE (Minh et
 al. 2020b) was also employed for ML analyses.  For these analyses,
 ModelFinder (Kalyaanamoorthy et al. 2017) was used to identify the most
 appropriate model as recognized by BIC, and this was followed by searching
 for the optimal tree and 1,000 ultrafast bootstrap replicates (UFBoot)
 (Hoang et al. 2018) with hill-climbing nearest neighbor interchange to
 reduce potential severe model violations (bnni option).  
 Multispecies coalescent (MSC) analyses were conducted with ASTER
 and ASTRAL v5.6.3 (Zhang et al. 2018; Zhang et al. 2020), and the 353-42,
 353-305, Borage-149, Borage-226, B353-191, and B353-531 sets of MP and ML
 gene trees were analyzed.  Additionally, ML trees with Felsenstein
 bootstrap support values were used as input into weighted ASTRAL with
 ASTER (Liu and Warnow 2023), using 100 rounds of searching and resampling,
 to explore another approach for reconstructing species trees from gene
 trees.  Along with phylogenetic analyses of individual
 loci, sequence data were concatenated into the six aforementioned datasets
 (353-42 [44,364 bp], 353-305 [259,895 bp], Borage-149 [196,063 bp],
 Borage-226 [274,651 bp], B353-191 [240,427 bp], B353-531 [535,758 bp]).
 Each concatenated dataset was analyzed using MP and ML, with parameters in
 TNT and IQ-TREE mentioned previously, with one difference for ML analyses.
 With ML, the data were analyzed as both unpartitioned and partitioned by
 locus, with the optimal partition determined by analyses in ModelFinder
 Plus using BIC, Akaike Information Criterion (AIC), and corrected Akaike
 Information Criterion (AICc). For analyses in IQ-TREE, models of both
 Gamma rate heterogeneity (TESTMERGE) and FreeRate heterogeneity
 (MFP+MERGE) were investigated. Collectively, this strategy allowed for an
 exploration of the sensitivity of the data to different partitioning
 schemes.   Phylogenetic analyses of paralogues,
 identified via HybPiper, were conducted with RAxML-NG and IQ-TREE using
 the aforementioned parameters. Trees from Angiosperms353 paralogues,
 Borage-specific paralogues, and both together were analyzed in an MSC
 framework using ASTRAL-Pro (Zhang et al. 2020), an approach similar to
 ASTRAL that can account for multiple paralogues per species. All trees
 were compared to identify congruent and incongruent areas of the phylogeny
 of Boraginaceae. Along with analyses of paralogous loci, three datasets of
 single-copy loci as identified by HybPiper were constructed: 25
 borage-specific loci, 209 Angiosperms353 loci, and 234 for both datasets
 collectively. These datasets were analyzed in MP and ML frameworks as
 individual loci and with concatenated datasets. All analyses used the same
 aforementioned parameters. This strategy was employed to recognize
 particular evolutionary patterns resolved solely by single-copy
 orthologues.   To further explore the evolutionary
 relationships of Boraginaceae, sequence data for 95 species of Boraginales
 (101 accessions) and 13 outgroup species (14 accessions) from Lamiidae
 from the Kew Tree of Life Explorer (Baker et al. 2022) and GenBank were
 accessed. These species have been enriched for the Angiosperms353 loci.
 Using the HybPiper parameters described above, sequences were mapped to
 loci, and DNA sequences were retrieved. The resulting sequences were
 combined with those from the Angiosperms353 loci from the present study
 into two different datasets: one with 297 loci that includes at least 100
 species and one with all Angiosperms353 loci, which includes loci that can
 have a much smaller number of species for each locus. With this dataset,
 MP and ML phylogenetic analyses were conducted as described above using
 individual genes, concatenated (partitioned and unpartitioned) datasets,
 and paralogous loci.
description: # Phylogenomics of Boraginaceae using lineage-specific and angiosperms353
 loci
 [https://doi.org/10.5061/dryad.f7m0cfz5d](https://doi.org/10.5061/dryad.f7m0cfz5d) The dataset focuses on DNA sequence data to investigate the Phylogenomics relationships of the plant family Boraginaceae and the order to which it belongs, Boraginales. Phylogenomic analyses were conducted using both Maximum Parsimony (MP) and Maximum Likelihood (ML) approaches for concatenated data and Multispecies Coalescent (MSC) methods with phylogenies of individual loci. For each method, multiple analyses were conducted to fully explore the various combinations of data and the impacts of different parameters on methods of analysis. ## Description of the data and file structure The dataset includes nuclear and organellar DNA sequence alignments and phylogenies from concatenated and multispecies coalescent analyses of species of Boraginaceae and Boraginales. -The Alignments folder includes aligned DNA sequence data of orthologues of Angiosperms353 loci, loci from Boraginaceae-specific probes, and organellar and nuclear ribosomal regions. -The Concatenation folder includes the phylogenies, from IQ-TREE and TNT, of concatenated DNA from Angiosperms353 and Boraginaceae-specific loci. The folder of IQ-TREE files includes results from BIC, AIC, and AICc model selection for partition and unpartitioned data, and partitioned data was analyzed with Gamma rate heterogeneity and FreeRate heterogeneity. Please see methods for additional information on phylogenetic search parameters. -The MSC folder includes the input phylogenies from MP (TNT) and ML (IQ-TREE and RAxML-NG) analyses of individual loci as well as results of multispecies coalescent analyses from ASTRAL (including weighted ASTRAL [waster]). -The Organellar and nrDNA folder includes the results of MP and ML analyses of the plastid and mitochondrial genomes and nrDNA. -The Paralogues folder includes alignments of Angiosperms353 and Boraginaceae-specific paralogous loci, phylogenies of ML (IQ-TREE and RAxML-NG) analyses of individual trees, and MSC phylogenies of the individual trees using ASTRAL-Pro. -The Boraginales folder includes alignments of Angiosperms353 loci, phylogenies of concatenated data analyzed with MP and ML approaches, input phylogenies from MP (TNT) and ML (IQ-TREE and RAxML-NG) analyses of individual loci as well as results of multispecies coalescent analyses from ASTRAL (including weighted ASTRAL [waster]), and alignments of Angiosperms353 and Boraginaceae-specific paralogous loci, phylogenies of ML (IQ-TREE and RAxML-NG) analyses of individual trees, and MSC phylogenies of the individual trees using ASTRAL-Pro. ## Sharing/Access information Raw reads are available at PRJNA1098857 and PRJNA1106503 at GenBank, and accession numbers are listed after the species for other taxa downloaded from GenBank for the Boraginales datasets. ## Code/Software See Methods for parameters used to align DNA sequence data and for phylogenetic analyses.