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A nuclear target sequence
capture probe set for phylogeny
reconstruction of the charismatic
plant family Bignoniaceae
Luiz Henrique M. Fonseca
1
,
2
*, Mónica M. Carlsen
3
,
Paul V. A. Fine
4
and Lúcia G. Lohmann
1
,
4
*
1
Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil,
2
Systematic and Evolutionary Botany Laboratory, Department of Biology, Ghent University, Ghent,
Belgium,
3
Missouri Botanical Garden, Saint Louis, MO, United States,
4
University and Jepson Herbaria,
and Department of Integrative Biology, University of California, Berkeley, Berkeley, CA, United States
The plant family Bignoniaceae is a conspicuous and charismatic element of
the tropical flora. The family has a complex taxonomic history, with
substantial changes in the classification of the group during the past two
centuries. Recent re-classifications at the tribal and generic levels have been
largely possible by the availability of molecular phylogenies reconstructed
using Sanger sequencing data. However, our complete understanding of the
systematics, evolution, and biogeography of the family remains incomplete,
especially due to the low resolution and support of different portions of the
Bignoniaceae phylogeny. To overcome these limitations and increase the
amount of molecular data available for phylogeny reconstruction within this
plant family, we developed a bait kit targeting 762 nuclear genes, including
329 genes selected specifically for the Bignoniaceae; 348 genes obtained
from the Angiosperms353 with baits designed specifically for the family; and,
85 low-copy genes of known function. On average, 77.4% of the reads
mapped to the targets, and 755 genes were obtained per species. After
removing genes with putative paralogs, 677 loci were used for phylogenetic
analyses. On-target genes were compared and combinedintheExon-Only
dataset, and on-target + off-target regions were combined in the
Supercontig dataset. We tested the performance of the bait kit at
different taxonomic levels, from family to species-level, using
38 specimens of 36 different species of Bignoniaceae, representing: 1) six
(out of eight) tribal level-clades (e.g., Bignonieae, Oroxyleae, Tabebuia
Alliance, Paleotropical Clade, Tecomeae, and Jacarandeae), only
Tourrettieae and Catalpeae were not sampled; 2) all 20 genera of
Bignonieae; 3) seven (out of nine) species of Dolichandra (e.g., D.
chodatii,D. cynanchoides,D. dentata,D. hispida,D. quadrivalvis,D.
uncata,andD. uniguis-cati), only D. steyermarkii and D. unguiculata were
not sampled; and 4) three individuals of Dolichandra unguis-cati.Ourdata
reconstructed a well-supported phylogeny of the Bignoniaceae at different
taxonomic scales, opening new perspectives for a comprehensive
phylogenetic framework for the family as a whole.
OPEN ACCESS
EDITED BY
Carolina Machado,
Federal University of São Carlos, Brazil
REVIEWED BY
Loreta Brandão de Freitas,
Federal University of Rio Grande do Sul,
Brazil
Evandro Marsola Moraes,
Federal University of Sao Carlos, Brazil
*CORRESPONDENCE
Luiz Henrique M. Fonseca,
luizhmf@gmail.com
Lúcia G. Lohmann,
llohmann@usp.br
SPECIALTY SECTION
This article was submitted to
Evolutionary and Population Genetics,
a section of the journal
Frontiers in Genetics
RECEIVED 31 October 2022
ACCEPTED 12 December 2022
PUBLISHED 09 January 2023
CITATION
Fonseca LHM, Carlsen MM, Fine PVA
and Lohmann LG (2023), A nuclear
target sequence capture probe set for
phylogeny reconstruction of the
charismatic plant family Bignoniaceae.
Front. Genet. 13:1085692.
doi: 10.3389/fgene.2022.1085692
COPYRIGHT
© 2023 Fonseca, Carlsen, Fine and
Lohmann. This is an open-access article
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms.
Frontiers in Genetics frontiersin.org01
TYPE Original Research
PUBLISHED 09 January 2023
DOI 10.3389/fgene.2022.1085692
KEYWORDS
bait kit, probe design, sequence capture, target enrichment, HybPiper, phylogenomics
1 Introduction
The plant family Bignoniaceae is a conspicuous and
charismatic element of the tropical flora. The family has
85 genera and 850 species of shrubs, lianas, and trees (http://
www.theplantlist.org) and a history of diversification that was
broadly influenced by the colonization of different habitats and
biogeographic regions (Lohmann et al., 2013;Olmstead, 2013;
Thode et al., 2019;Francisco and Lohmann, 2020;Calió et al.,
2022;Ragsac et al., 2022). The family is known for its
conspicuous flowers, which are variable morphologically due
to specialized plant-animal interactions associated with
pollination (Gentry, 1990), and diverse fruit morphology
associated with different dispersal systems (Zjhra et al., 2004;
Farias-Singer, 2007;Ragsac et al., 2021). The great diversity and
high homoplasy of Bignoniaceae’s reproductive morphology, the
main characters used to circumscribe genera and supra-generic
groupings within the family, led to considerable taxonomic
confusion (reviewed by Gentry, 1980;Lohmann, 2006;
Lohmann and Taylor, 2014). The availability of broad-scale
phylogenies for the Bignoniaceae recently allowed for a re-
circumscription of lineages in the family and the recognition
of monophyletic tribes (Olmstead et al., 2009) and genera
(Lohmann, 2006;Grose and Olmstead, 2007a).
To date, phylogenetic inference in this plant group has
mainly relied on plastid markers (e.g., matK, ndhF, rbcL,
rpl32-trnL, trnL-F) or a few nuclear regions, such as the
multi-copy nuclear ribosomal (ITS) or the low copy gene
PepC (e.g.; Lohmann, 2006;Olmstead et al., 2009;Fonseca
and Lohmann, 2015;Francisco and Lohmann, 2020;Calió
et al., 2022;Ragsac et al., 2021;Ragsac et al., 2022). Robust
phylogenies were recovered for the family and infra-familial
levels based on these markers, providing a framework to test
tribal and generic limits within this plant clade. Traditionally
recognized tribes such as Tecomeae emerged as paraphyletic
(Spangler and Olmstead, 1999;Olmstead et al., 2009), while
traditionally recognized genera were also shown to not represent
monophyletic groupings, leading to extensive taxonomic
changes, especially in the tribes Bignonieae (Lohmann, 2006;
Lohmann and Taylor, 2014), and Tecomeae (Grose and
Olmstead, 2007a;Grose and Olmstead, 2007b). However, the
limited number of informative sites provided by the DNA regions
traditionally used to reconstruct phylogenetic relationships
within the Bignoniaceae prevents a thorough understanding of
phylogenetic relationships within this family. Recalcitrant
relationships and poorly supported branches are observed in
deeper phylogenetic nodes or even within more recently
diverging lineages (e.g., Lohmann, 2006;Olmstead et al.,
2009), highlighting the need for a higher number of
informative characters for phylogeny reconstruction at
different taxonomic levels. While a few Bignoniaceae
phylogenetic studies have incorporated plastome data (e.g.,
Fonseca and Lohmann, 2018;Thode et al., 2019;Fonseca and
Lohmann, 2020), or thousands of nuclear loci (Dong et al., 2022)
to reconstruct phylogenetic relationships within individual
Bignoniaceae genera, no phylogenetic study to date has used
genomic data to reconstruct a robust phylogeny of the family as a
whole.
High throughput DNA sequencing technologies coupled
with advances in bioinformatics are revolutionizing the field
of evolutionary biology (Soltis et al., 2013) and nuclear
genomes are increasingly available for non-model species
(Fonseca and Lohmann, 2018;Anderman et al., 2020).
Despite those advances, complete genomes are still not
affordable, and the computational time necessary to analyze
the massive amounts of genomic data remains prohibitive
(McKain et al., 2018;Anderman et al., 2020). To circumvent
these limitations, strategies of genome reduction that allow the
incorporation of hundreds of thousands to millions of base pairs
have been described. For example, multiplex PCR (Urive-
Convers et al., 2016), RAD-seq (Davey and Blaxter, 2010;
Eaton and Ree, 2013), RNA-seq (Wen et al., 2013), and target
capture-based approaches (Faircloth et al., 2012;Weitemier et al.,
2014) are allowing researchers to focus their sequencing efforts
on loci that are useful to address different taxonomic,
evolutionary, or biogeographical questions. These genome
reduction approaches increase the cost-effectiveness of
projects and improve phylogenetic resolution by allowing the
inclusion of the most informative data and an increase in the
number of taxa (Soltis et al., 2013;McKain et al., 2018;Anderman
et al., 2020).
Target sequence capture approaches are a promising tool
for studying evolutionary relationships in non-model
organisms, enabling researchers to maximize the number of
informative characters despite limited genomic resources
(Anderman et al., 2020). This approach is cost-effective
when compared to genome sequencing, allowing hundreds
of pre-selected target loci to be obtained for dozens of
specimens at once. Gene capture approaches have been
quite successful in multiple plant phylogenetic studies, and
customized bait kits are now available for those groups
(Weitemier et al., 2014;Heyduk et al., 2016;Carlsen et al.,
2018;Chau et al., 2018;Bagley et al., 2020;Jantzen et al., 2020;
Christe et al., 2021;Eserman et al., 2021;Ogutcen et al., 2021;
Yardeni et al., 2022). A universal bait kit designed to tackle
conserved genes is also available for angiosperms as a whole
(i.e., Angiosperms353; Johnson et al., 2019). The choice
between clade-specific or universal bait kits depends on the
questions being addressed and the degree of divergence
among the studied taxa (Yardeni et al., 2022), with custom
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Fonseca et al. 10.3389/fgene.2022.1085692
kits being more appropriate to resolve phylogenetic
relationships at shallow scales (Eserman et al., 2021;
Yardeni et al., 2022). The inclusion of universal kits in the
design of custom probe sets (e.g., Jantzen et al., 2020;Baker
et al., 2021;Christe et al., 2021;Eserman et al., 2021;Ogutcen
et al., 2021), opens up the possibility of integrating data across
flowering plants, while resolving phylogenetic relationships at
different taxonomic scales.
Here, we developed and tested the first nuclear target
sequence capture probe set for phylogenetic inference across
the entire family Bignoniaceae. We obtained sequence data for
762 genes, representing 329 nuclear genes (1307 putative exons)
selected following the Hyb-Seq protocol (Weitemier et al., 2014),
348 nuclear genes from the Angiospems353 bait set (Johnson
et al., 2019), and 85 low-copy functional genes with implications
for reproductive and vegetative organ development, and
biochemical synthesis. Of the 762 genes selected, 677 are
putatively single or low-copy nuclear genes. We tested the
utility of the new bait set for phylogeny reconstruction at
different taxonomic scales, including tribal, generic, and
species levels. For the broadest taxonomic scale, we sampled
species from six (out of eight) Bignoniaceae tribal-level clades
(i.e., Bignonieae, Oroxyleae, Tabebuia Alliance, Paleotropical
Clade, Tecomeae, and Jacarandeae), only Tourrettieae and
Catalpeae were not sampled (Olmstead et al., 2009). We also
sampled all 20 genera recognized in the tribe Bignonieae
(Lohmann and Taylor, 2014;Fonseca and Lohmann, 2019).
Furthermore, we selected the genus Dolichandra Cham. as a
test case for resolving species-level relationships within the family
and sampled seven (out of nine) species of Dolichandra, only D.
unguiculata (Vell.) L.G. Lohmann and D. steyermarkii
(Sandwith) L.G. Lohmann were not sampled (Fonseca and
Lohmann, 2015). This study aimed to: 1) test the efficiency of
the designed bait set to capture targeted regions throughout
Bignoniaceae; and 2) test whether the analyses of the captured
loci improve phylogenetic resolution at three different
evolutionary scales: the family Bignoniaceae, the tribe
Bignonieae, and the genus Dolichandra.
2 Materials and methods
2.1 Sampling
We used the genomic resources available for four species of
Bignoniaceae to select low to single-copy genes. The
transcriptomes of Kigelia africana (Lam.) Benth, Mansoa
alliacea (Lam.) A.H. Gentry, and Tabebuia umbellata (Sond.)
Sandwith were obtained from the 1 KP project (Matasci et al.,
2014). The partial nuclear genome of Handroanthus
impetiginosus (Mart. ex DC.) Mattos was also used (Silva-
Junior et al., 2018; GenBank: NKXS01000000). To evaluate the
bait set designed here, we sampled 38 species of Bignoniaceae.
Plant materials used for this study were collected in the wild and
dried in silica gel or collected from living collections available at
the Universidade de São Paulo (São Paulo, Brazil). Because our
sampling scheme aimed to evaluate the usefulness of our probe
set across the Bignoniaceae at different taxonomic levels, we
sampled species from five out of the eight recognized tribal-level
clades (Olmstead et al., 2009), as follows: 1) Oroxyleae (1 sp.); 2)
Tabebuia Alliance (6 spp.), 3) Tecomeae (2 spp.), 4) Jacarandeae
(1 sp.), and 5) Bignonieae (28 spp.). All 20 genera of tribe
Bignonieae currently recognized were sampled, including
Dolichandra for which seven out of the nine known species
(Fonseca and Lohmann, 2015) were included (accession
information available in Supplementary Table S1).
2.2 Selection of target loci
Loci were selected targeting regions consisting of orthologous
low-copy nuclear protein-coding genes and aiming to
reconstruct relationships at three evolutionary scales: tribal,
generic, and species levels. Genes were selected using the
Hyb-Seq pipeline (Weitemier et al., 2014). The probe design
was based on data from the draft genome of H. impetiginosus
combined with the three transcriptomes. Contigs from the draft
nuclear genome were matched against those sharing sequence
similarities from the transcriptomes individually using the
program BLAT (Kent, 2002). The similarity threshold used for
K. africana,M. alliacea, and T. umbellata were 0.92, 0.9, and 0.95,
respectively. The Hyb-Seq protocol was originally designed to use
genomic and transcriptomic data from the same species and used
an original threshold value of 0.99% of similarity. To overcome
this limitation, we tested different threshold values from 0.99 to
0.85 (0.01 of difference between steps). The values selected were
based on the number of loci selected at the end of the pipeline.
Thousands of exons and genes were obtained from the three
transcriptomes. To reduce the size of the dataset, we selected
genes with less than 10 introns and less than 2040 bp (equivalent
to 34 probes for 2× tiling). We only kept genes recovered in at
least two different datasets/transcriptomes using CD-HIT-EST
4.5 (with–c 0.9) (Fu et al., 2012). This step also maximizes the
chance that the selected loci are shared within different species of
the family. A total of 329 low to single-copy genes were selected.
The 353 universal loci available for Angiosperms were
included using the original alignments (available at: https://
github.com/mossmatters/Angiosperms353)(Johnson et al.,
2019). Sequences of all Bignoniaceae transcriptomes were
retained in each of the 353 original alignments, while
sequences from other plant families were discarded. For
338 loci, at least one sequence of Bignoniaceae was found.
When more than one Bignoniaceae species was found for a
specific gene, the longest sequence was retained. For the 15 genes
without Bignoniaceae sequences, one sample from a closely
related family in Lamiales was included. All genes were
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Fonseca et al. 10.3389/fgene.2022.1085692
investigated to evaluate the number of copies using the genome
of H. impetiginosus and BLAT (Kent, 2002). Originally, all genes
were included with one reference, although only 348 were
assembled. Using Bignoniaceae as a reference to design
specific baits for the Angiosperms353 panel, we saved
thousands of baits in our final set.
Functional genes were selected using Arabidopsis thaliana
(L.) Heynh. or closer relatives of the Bignoniaceae as reference
(mainly from Solanum lycopersicum L.) (Supplementary Material
S2). First the number of gene copies was evaluated in BLAT
(Kent, 2002) using the genome of H. impetiginosus as reference.
Putative orthologous sequences of the focal genes were examined
using BLAT (Kent, 2002) and the three transcriptomes available.
The longest and most similar sequences (threshold of 0.85)
available were selected for each reference. For three genes
(ALCATRAZ, FRIGIDA, and INDEHISCENT) no
Bignoniaceae sequences were recovered and the original
Arabidopsis thaliana (L.) Heynh. or Solanum lycopersicum L.
sequences were used (Supplementary Material S2). Initially, a
total of 89 low to single-copy functional genes were selected;
however, four genes failed during the assembly, and 85 genes
were used in subsequent analyses.
To evaluate if different datasets (e.g., Hyb-Seq,
Angiosperms353, and functional) shared loci, we used CD-
HIT-EST 4.5 (with–c 0.9) (Fu et al., 2012). Two genes were
shared between the Angiosperms353 and the functional datasets;
only the references from the former were maintained. The gene
set was sent to Arbor Biosciences and used as a basis to synthesize
the 80 bp 2× tiled bait set. The Bignoniaceae bait set is available
from Arbor Biosciences (Ann Arbor, Michigan, United States),
and the gene sequences used to generate the bait set are provided
on GitHub (https://github.com/luizhhziul/BigBait). We selected
771 putative low to single-copy nuclear loci for our probe set.
Nine genes failed to be assembled, leading to a final number of
762 genes with at least one copy assembled.
2.3 Library preparation, sequencing, and
gene assembly
Total genomic DNA was extracted from silica-dried or fresh
leaf tissue using the Invisorb®Spin Plant Mini Kit (Invitek,
Berlin, Germany). To isolate enough DNA for sequencing,
multiple extractions were performed for some samples,
pooled, and concentrated by vacuum centrifugation. Total
DNA was quantified using Qubit BR assay. DNA quality was
evaluated using NanoDrop 2000 (Thermo Fisher Scientific) and
gel electrophoresis. Library preparation and sequence capture
were performed by the QB3 Genomics facility (University of
California, Berkeley) using their high-throughput workflow. All
samples were prepared into standard-sized libraries using Kapa
Biosystems library preparation kits (using Covaris sonicator to
fragment gDNA) and custom Unique Dual Indexes. Samples
were multiplexed, and the captured fragments obtained using the
Bignoniaceae bait kit pooled and sequenced on an Illumina
NovaSeq 6000 S4 with 150 bp paired-end reads.
Illumina reads were demultiplexed at the sequencing facility,
and low-quality reads were trimmed using Trimmomatic 0.35
(Bolger et al., 2014) with the SLIDINGWINDOW:10:20 and
MINLEN:40 parameters. HybPiper 1.3.1 (Johnson et al., 2016)was
used to obtain the gene sequences using the “reads_first.py”Python
script through the following steps: quality filtering; map reads to target
the gene references provided using BWA 0.7.17 (Li and Durbin, 2009);
de novo contig assembly using SPAdes 3.6.1 (Bankevich et al., 2012);
and, return supercontigs (exons + introns/intergenic-regions) and
exon-only sequences per gene using Exonerate 2.4.0 (Slater and
Birney, 2005). We retrieved FASTA files of exon-only and
supercontig sequences containing all sampled species using the
“retrieve_sequences.py”script. Summary statistics were calculated
using the “hybpiper_stats.py”script. Three genes were excluded
due to their low representation (i.e., they appeared in less than
70% of the species sampled).
HybPiper flagged 202 genes that might contain paralogs.
Paralog warnings produced by the HybPiper pipeline for samples
with multiple long contigs were further investigated using the
HybPiper scripts paraloginvestigator.py and paralogretriever.py
(Johnson et al., 2016). To check if these sequences represented
true paralogs or alleles, we produced alignments using both
sequences retrieved for the 202 genes using MAFFT 7.450
(Katoh and Standley, 2013) and generated phylogenetic trees
using FastTree (Price et al., 2009) with default parameters
(Johnson et al., 2016). Of the 202 genes, 82 were flagged in more
than three specimens or showed phylogenetic evidence of paralogy;
these genes were excluded. The gene set used for downstream
analyses contained 677 single copy loci. Two datasets were
generated: 1) “Exon-Only”,withjustexons;and2)“Supercontig”,
with exons, complete or partial introns, and complete or partial
intergenic regions. The number of Parsimony Informative Sites
(PIS) for each gene alignment and combined alignments were
obtained using the R package “ips”(Heibl, unpublished data).
Saturation was evaluated using gene alignments, the function
“dist.dna”of the “ape”package (Paradis et al., 2004), and the
molecular model K80. The genetic distance between species of
Dolichandra was also evaluated using the function “dist.dna”and
the K80 model.
2.4 Phylogenomic analyses
Phylogenomic inferences were performed using the subset of
677 genes. Each step detailed here was conducted for either the
Exon-Only, or the Supercontig datasets. Each gene sequence was
aligned using MAFFT 7.450 (Katoh and Standley, 2013) with an
automatic selection of alignment strategy and a maximum of
1,000 iterations. Bases with more than 75% of the species as
missing data were deleted. Statistical properties of each alignment
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Fonseca et al. 10.3389/fgene.2022.1085692
TABLE 1 Summary statistics of sequencing success including the number of raw pair-end reads obtained, percentage of on-target reads, number of loci
obtained, percentage of gene recovery, and number of loci retained after paralogs removed.
Tribe Species Pair-end
reads
Percent of on-
target reads
Number of loci
obtained
Percent of recovery
length
Number of loci
retained
Bignonieae Adenocalymma
acutissimum1
18,401,073 76.1 757 93.3 676
Bignonieae Amphilophium
paniculatum
28,119,426 76.9 758 94.3 676
Bignonieae Anemopaegma arvense 8,486,773 69.7 757 92.8 675
Bignonieae Bignonia capreolata 4,863,524 80.1 759 94.5 676
Bignonieae Callichlamys latifolia 11,089,886 75.2 757 94.7 676
Crescentieae Crescentia cujete 6,770,767 80.4 759 96 676
Bignonieae Cuspidaria convoluta 7,953,194 65.3 758 93.7 676
Tecomeae Cybistax antisyphilitica 14,574,220 68.4 758 95.2 675
Bignonieae Dolichandra chodatii 3,077,426 68.7 757 93.8 676
Bignonieae Dolichandra
cynanchoides
11,401,675 71.3 758 95 676
Bignonieae Dolichandra dentata 9,614,141 79.4 758 94 676
Bignonieae Dolichandra hispida 8,467,292 79.2 759 94 677
Bignonieae Dolichandra
quadrivalvis
10,465,488 80.7 758 93.8 676
Bignonieae Dolichandra uncata 21,119,851 78.1 756 93 675
Bignonieae Dolichandra unguis-
cati1
15,336,452 80 755 92.1 674
Bignonieae Dolichandra unguis-
cati2
14,553,233 74.8 756 92.5 674
Bignonieae Dolichandra unguis-
cati3
15,517,980 49.1 757 92.5 675
Bignonieae Fridericia speciosa 4,029,504 76.8 755 93.5 674
Tecomeae Godmania aesculifolia 6,104,436 4.4 732 85.9 653
Tecomeae Handroanthus
catarinensis
13,546,481 66.6 758 94.9 675
Jacarandeae Jacaranda mimosifolia 5,051,624 68.6 741 82.7 661
Bignonieae Lundia longa 5,298,119 80.3 755 92.9 673
Bignonieae Manaosella cordifolia 27,279,881 77.8 756 94.3 674
Bignonieae Mansoa hirsuta 3,940,708 74.9 759 92.7 676
Bignonieae Martinella obovata 5,605,255 75.6 758 94.6 676
Oroxyleae Nyctocalos cuspidatum 55,042,136 79.6 754 94.1 673
Bignonieae Pachyptera incarnata 7,932,766 75.6 756 94.1 674
Crescentieae Parmentiera cereifera 71,238,833 80.8 754 93.1 673
Bignonieae Perianthomega vellozoi 5,814,004 81.5 755 95 675
Bignonieae Pleonotoma
jasminifolia
7,456,086 78.3 756 94.2 675
(Continued on following page)
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were evaluated using R (R Core Team, 2020). Alignments were
concatenated using AMAS (Borowiec, 2016) to generate a super-
matrix with all data combined. Maximum likelihood analyses
were implemented in IQ-TREE 1.6.1 (Nguyen et al., 2015). For
individual genes, model selection was performed before tree
search in IQ-TREE using the command–m MPF and greedy
algorithm (Lanfear et al., 2012). For combined Exon-Only and
Supercontig super-matrices, the option–m TESTMERGE was
used to select the best partition scheme before tree search.
Ultrafast bootstrap (UFBoot) replicates were inferred for all
analyses with 1,000 reanalyzes and–bnni option, performing
additional steps to further optimize UFBoot trees using the
nearest neighbor interchange (NNI) algorithm.
Super-matrix approaches can be inconsistent due to discordance
in gene trees. These discordances can result from multiple processes
and are commonly attributed to either incomplete lineage sorting
(ILS) and/or hybridization (Pamilo and Nei, 1988;Galtier and
Daubin, 2008), or phylogenetic errors (Shen et al., 2021). To
infer the species tree from the set of gene trees available, a
coalescent approach was performed using ASTRAL-III 5.6.3
(Mirarab et al., 2014;Zhang et al., 2018). Low-support branches
(i.e., >30%) were collapsed to improve accuracy (Zhang et al., 2018).
Branch support in ASTRAL-III was calculated using Local Posterior
Probabilities (LPP). Gene congruence was evaluated using the gene
concordance factor (gCF; Minh et al., 2020) using IQ-TREE 1.6.1
(Nguyen et al., 2015).
3 Results
3.1 Target enrichment with bait
hybridization
We recovered an average of 14,064,035.4 pair-end raw
sequence data, with a maximum of 71,238,833 pair-end reads
and a minimum of 3,077,426 pair-end reads. After the first
quality filtering, we retained an average of 93.7% of the raw
reads, with a maximum of 98.1% and a minimum of 76%. Raw
reads for all accessions are available in GenBank Sequence Read
Archive (SRA) under BioProject ID PRJNA909066. Baits showed
high accuracy, and most raw reads mapped to a target gene
(Table 1). An average of 77.4% of the reads mapped targets, with
a maximum of 82.1% and a minimum of 4.4% (Table 1). Our bait
set originally targeted 771 genes covered by 21,416 baits. Of the
771 genes originally targeted, 329 were specific for Bignoniaceae,
353 corresponded to genes in the Angiosperms353 bait kit, and
89 were known functional genes selected from the literature for
this study (names of selected genes and references in
Supplementary Material S2). We assembled 732–759 genes,
with an average of 755 per specimen (Figure 1;Table 1). Nine
genes failed for all species, including five genes from the
Angiosperms353 set and four genes from the functional set.
Recovery of the total reference gene length was 93.2%, with a
maximum of 96% and a minimum of 82.7% (Table 1). A final
array of 677 genes was used for gene alignments and phylogenetic
tree searches after the removal of putative paralogs and genes not
assembled in less than 70% of the species sampled.
The mean length of the 677 genes was 192 bp to 6,316 bp
long. Custom selected genes ranged from 940 bp to 2012 bp,
while Angiosperms353 genes ranged from 192 bp to 4,324 bp,
and functional genes ranged from 338 bp to 6,316 bp
(Supplementary Figure S1). The Exon-Only alignment
considering custom-selected genes, Angiosperm353 genes,
and the functional genes was 875,075 bp long, of which
203,576 bp were parsimony informative at the family level,
121,268 bp at the tribal level, and 22,251 bp at the generic level
(Table 2). The alignment considering the custom-selected
genes exclusively was 348,264 bp long, of which 76,056 bp
were parsimony informative at the family level, 64,790 bp at
the tribal level, and 11,926 bp at the generic level (Table 2).
TABLE 1 (Continued) Summary statistics of sequencing success including the number of raw pair-end reads obtained, percentage of on-target reads, number
of loci obtained, percentage of gene recovery, and number of loci retained after paralogs removed.
Tribe Species Pair-end
reads
Percent of on-
target reads
Number of loci
obtained
Percent of recovery
length
Number of loci
retained
Tecomeae Podranea ricasoliana 16,729,542 78.7 755 91.9 675
Bignonieae Pyrostegia venusta 5,081,154 66.8 757 91.7 675
Bignonieae Stizophyllum
perforatum
12,840,685 79.8 758 94.6 676
Tecomeae Tabebuia roseoalba 9,587,299 82.1 756 95.6 675
Bignonieae Tanaecium jaroba 23,112,074 76.1 758 94 676
Tecomeae Tecoma stans 21,512,775 61.4 755 91.7 674
Bignonieae Tynanthus polyanthus 11,207,016 80.2 757 93.7 675
Bignonieae Xylophragma pratense 6,210,566 75 754 93.4 673
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The alignment considering the Angiosperms353 genes
exclusively was 403,626 bp long, of which 94,933 bp were
parsimony informative at the family level, 36,576 bp at the
tribal level, and 6,740 bp at the generic level (Table 2). The
alignment considering the functional genes exclusively was
123,185 bp long, of which 32,587 bp were parsimony
informative at the family level, 19,902 bp at the tribal level,
and 3,585 bp at the generic level (Table 2). Individual genes
included 17 to 1,786 parsimony informative sites
(Supplementary Figure S2). The pairwise genetic distance
between species of Dolichandra ranged from 1% to 3.4%,
while the pairwise genetic distance within Dolichandra
unguis-cati L. ranged from 0.3% to 1% (Table 3).
3.2 Non-targeted sequences
Off-targeted regions from the “splash-zone”(i.e., protein-
coding regions plus the complete or partial introns and intergenic
regions) were 928 bp to 13,453 bp long. Custom selected genes
ranged from 1,683 bp to 6,939 bp, while Angiosperms353 genes
ranged from 928 bp to 10,816 bp, and functional genes ranged
from 1,299 bp to 13,453 bp (Supplementary Figure S1). The
Supercontig alignment was 2,811,957 bp long, of which
1,114,993 bp were parsimony informative at the family level,
688,408 bp at the tribal level, and 179,384 bp at the generic level
(Table 2). The alignment containing the custom-selected genes
exclusively was 1,366,090 bp long, of which 509,096 bp were
parsimony informative at the family level, 312,505 bp at the
tribal level, and 47,493 bp at the generic level (Table 2). The
alignment with the Angiosperms353 genes exclusively was
1,084,724 bp, of which 462,527 bp were parsimony informative
at the family level, 283,030 bp at the tribal level, and 42,457 bp at
the generic level (Table 2). The alignment with the functional
genes exclusively was 361,143 bp long, of which 143,370 bp were
parsimony informative at the family level, 92,873 bp at the tribal
level, and 14,880 bp at the generic level (Table 2). Individual
genes included 160 to 5,544 parsimony informative sites
(Supplementary Figure S2). The pairwise genetic distance
between species of Dolichandra ranged from 1.5% to 5.3%,
while the pairwise genetic distance within D. unguis-cati
ranged from 0.4% to 1.6% (Table 3). Only 14 alignments were
flagged with linear regression values below 0.7% using the
molecular model K80 when Supercontig data was evaluated
for saturation. Trees obtained for all 14 regions flagged were
FIGURE 1
Recovered sequence heatmap for all the 771 genes targeted. Each row corresponds to a different specimen sampled, and each column
correspond to a gene. Colors represent the length of the recovered sequence relative to the template sequence.
TABLE 2 Number of aligned and parsimony informative bases for each
dataset in each taxonomic scale.
–Informative sites
Size Family Tribe Dolichandra
Angiosperms353 403,626 94,933 36,576 6,740
custom genes 348,264 76,056 64,790 11,926
functional 123,185 32,587 19,902 3,585
exon-only 875,075 203,576 121,268 22,251
Angiosperms353 1,084,724 462,527 283,030 42,457
custom genes 1,366,090 509,096 312,505 47,493
functional 361,143 143,370 92,873 14,880
supercontig 2,811,957 1,114,993 688,408 179,384
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TABLE 3 Comparison of within-genus variation for all species of Dolichandra. Values below the diagonal are pairwise sequence divergences for the
Supercontig dataset. Values above the diagonal are pairwise sequence divergences for the Exon-Only dataset. All values are in percentages.
D. chod D. cyna D. dent D. hisp D. quad D. unca D. ung.1 D. ung.2 D. ung.3
D. chodatii –2.5 3.1 3.1 2.8 3.4 3.2 3.2 3.1
D. cynanchoides 3.8 –2.6 2.6 2.2 2.9 2.7 2.7 2.6
D. dentata 4.8 4 –1 2.7 2 1.6 1.6 1.5
D. hispida 4.8 4 1.5 –2.7 2 1.6 1.6 1.6
D. quadrivalvis 4.3 3.5 4.1 4.1 –3 2.8 2.8 2.7
D. uncata 5.3 4.5 3.1 3.1 4.6 –2.1 2.1 2
D. unguis-cati1 4.9 4.1 2.4 2.5 4.2 3.2 –0.3 1
D. unguis-cati2 4.9 4.1 2.3 2.4 4.2 3.1 0.4 –1
D. unguis-cati3 4.9 4 2.3 2.4 4.2 3.1 1.6 1.6 –
FIGURE 2
Tanglegram comparisons of phylogenies obtained using a supermatrix approach and IQ-TREE, and a coalescent approach and ASTRAL-III of
the Exon-Only dataset. The supermatrix result includes ultra-fast bootstrap proportion (UFBoot) support values. The coalescent result is labeled with
local posterior probabilities (LPP). Values above branches with maximum support of the UFBoot and LPP are not shown. Branches labeled on the
supermatrix tree are discussed in the text. Shaded boxes enclose the entire Bignoniaceae family; the tribe Bignoniaeae; and the genus
Dolichandra.
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highly concordant with the concatenated tree and the species
trees. As a result, the 14 genes were kept as part of the 677 set,
allowing comparisons between the Exon-Only and Supercontig
datasets due to the equivalent number of genes sampled.
3.3 Phylogenomic reconstructions
3.3.1 Topologies inferred using the Exon-Only
dataset
The single ML tree derived from the analysis of the
concatenated Exon-Only dataset with 677 genes and 875,075 bp
was well resolved, with most branches receiving high values of
UFBoot (>90%). The me an UFBoot value for the entire tree is
96.4%. The tree was rooted with Jacaranda mimosifolia D. Don
following previous phylogeny reconstructions of the Bignoniaceae
(Olmstead et al., 2009). Tribe Bignonieae (Figure 2,cladeE),tribe
Crescentieae (Figure 2, clade D), tribe Tecomeae, and the entire
Tabebuia Alliance clade emerged as monophyletic with maximum
UFBoot support. All branches at the tribal level received maximum
support, except for the clade composed of Bignonieae and
Oroxyleae (Nyctocalos cuspidatum Miq.) with 97% of support.
Most branches within tribe Bignonieae received maximum
support from UFBoot. Exceptions were branches on the
“backbone”of the tree or small clades with few genera, which
are among the shortest branches of the tree. Dolichandra emerged
as monophyletic with maximum support. All branches within the
genus received maximum UFBoot support, including the
relationships among the three terminals of D. unguis-cati
(Figure 2).
The species trees obtained using a coalescent approach, and
the supermatrix tree recovered similar topologies. The mean LPP
value for the entire tree is 0.936. Differences in tree topology were
usually poorly supported by both UFBoot and LPP, except for
clade B (Figure 2), which showed maximum support in the
concatenated tree, and 0.71 support in the coalescent tree.
Phylogenetic relationships at the tribal-level, generic-level
within clades F, G, and H, and species-level were concordant
(Figure 2). All branches within Dolichandra received maximum
support, including branches within D. unguis-cati. Gene tree
congruence/conflict was evaluated visually and quantitatively for
each node.
3.3.2 Topologies inferred using the supercontig
dataset
The ML tree derived from the analysis of the concatenated
Supercontig dataset with 677 genes and 2,811,957 bp is well
resolved, with most branches supported by high UFBoot
values (>90%). The mean UFBoot value for the entire tree is
97.2%. Strongly supported branches recovered the same
relationships as the Exon-Only dataset. When tribal-level
clades are considered, the clade composed of Bignonieae plus
N. cuspidatum (Oroxyleae) is the only one with UFBoot of 96%.
Most branches within tribe Bignonieae received maximum
UFBoot support, except for nodes on the “backbone”of the
tree, or small clades with few genera; these poorly supported
branches are associated with the shortest branches of the tree.
Dolichandra emerged as monophyletic with maximum support.
All branches within the genus received maximum UFBoot
support, including the phylogenetic relationship among the
three terminals of D. unguis-cati (Figure 3).
The species tree derived from the coalescent approach and
concatenated tree recovered a similar topology. The mean LPP
value for the entire tree is 0.954. Differences in tree topology were
usually poorly supported by both UFBoot and LPP. Nyctocalos
cuspidatum now emerged as sister to clade C, being the only
branch among tribal-level clades without maximum support of
LPP (0.96). Most branches within tribe Bignonieae received
maximum support of LPP. As recovered by the Exon-Only
analyses analyses and the combined Supercontig analysis, the
short branches of the “backbone”of Bignonieae are poorly
supported. Clades F, G, and H were recovered with maximum
support of LPP. Phylogenetic relationships within these clades
also received maximum support of LPP, including the genus
Dolichandra (Figure 3).
3.3.3 Gene conflicts
For the Exon-Only dataset, phylogenetic relationships
among tribal-level clades were supported by more than
300 genes in all cases. The only exception is the Bignonieae +
Oroxyleae clade, which was supported by 155 congruent genes.
Conflicts were frequent on the “backbone”of Bignonieae, with
six branches showing 15 or fewer genes that were congruent with
the species tree. Clade H was supported with maximum values of
UFBoot and LPP, however only 94 regions were congruent with
this branch. Most of the genes were uninformative within this
clade. Within Dolichandra, four branches were supported by
more genes than the main alternative topology. In all cases, the
species tree was congruent with at least 226 genes concordant
with that branch (Figure 4).
For the Supercontig dataset, conflicts among genes were
common in the “backbone”of Bignonieae, with eight clades
supported by only 77 or less genes, with a higher number of genes
supporting minor conflicts. Conflicts were also common within
clade F, with three out of five clades having a higher number of
genes supporting other resolutions of the quartet (Figure 4).
4 Discussion
Hybridization capture-based technologies are enabling the
retrieval of hundreds of nuclear loci from diverse plant
lineages (e.g., Soltis et al., 2013;McKain et al., 2018;
Johnson et al., 2019;Anderman et al., 2020). Target
enrichment approaches have been used to resolve
phylogenetic relationships at many different levels, from
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universal bait kits for all flowering plants (Johnson et al.,
2019), to custom kits designed for specific clades. Custom kits
for targeted plant families such as Gesneriaceae (Ogutcen
et al., 2021), Orchidaceae (Eserman et al., 2021), or
Sapotaceae (Christe et al., 2021) and less comprehensive
clades, such as the genus Burmeistera H. Karst. & Triana
(Bagley et al., 2020), or Dioscorea L. (Soto Gomez et al.,
2019) are available to date. The specificity of custom bait
kits usually allows for a higher recovery rate in the targeted
regions, with recovery values reaching up to 99.6% in
Dioscorea (Soto Gomez et al., 2019). Here we provide the
first bait kit for the tropical plant family Bignoniaceae,
recovering up to 677 single-copy nuclear genes for
phylogenetic analyses. The efficiency of our targeted
sequence capture baits was extremely high, with a mean
value of 98% of the genes recovered (Table 1). The kit
included baits targeting three different gene sets. The first
set is composed by 329 custom selected genes obtained using
available genomic resources and the protocol described by
Weitemier et al. (2014). The second set includes genes
previously selected by the Angiosperms353 group (Johnson
et al., 2019), with probes designed here specifically for
Bignoniaceae. The last set is composed of low to single-
copy functional genes. The bait kit was applied to
38 species of Bignoniaceae and aimed to resolve
phylogenetic relationships from tribe to species-level. Most
clades recovered on different levels of the tree received
maximum support.
4.1 Capture efficiency
Our Bignoniaceae bait kit enabled the sequencing of
762 genes and up to 959,346 targeted base pairs (Table 1).
FIGURE 3
Tanglegram comparisons of phylogenies obtained using a supermatrix approach and IQ-TREE, and a coalescent approach and ASTRAL-III of
the Supercontig dataset. The supermatrix result includes ultra-fast bootstrap proportion (UFBoot) support values. The coalescent result is labeled
with local posterior probabilities (LPP). Values above branches with maximum support of the UFBoot and LPP are not shown. Branches labeled on the
supermatrix tree are discussed in the text. Shaded boxes enclose the entire Bignoniaceae family; the tribe Bignoniaeae; and the genus
Dolichandra.
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The proportion of on-target reads, and the number of genes
recovered for each specimen sampled can measure the
efficiency of the capture reaction. Here, we obtained 73%
(4.4–82.1) of on-target reads on average. This is a robust
result compared to other angiosperms clades (e.g., 31.6% in
Dioscorea,Soto Gomez et al., 2019; 48.6% in Euphorbia,
Villaverde et al., 2018), revealing an efficient selection of
targeted DNA fragments. The percentage of genes recovered
is even higher, with a mean value of 98% and only two
specimens obtaining less than 97.8% of the genes (Figure 1;
Table 1). Nine genes (1.4% of total genes) failed to be
assembled for all the species, including five genes from the
Angiosperms353 set, and four genes from the functional set.
Of the nine genes, eight used references outside Bignoniaceae
due to a lack of sequences within the family. The average
recovery of total length was of 93.2%. The species with the least
data obtained was J. mimosifolia, with 82.5% of the reference
recovered. Godmania aesculifolia (Kunth) Standl, the species
with the fewest on-target reads (4.4%), recovered 85.9% of the
reference size (Table 1). The result obtained here is excellent
when compared to other studies (e.g., 78.6% in Dioscorea,Soto
Gomez et al., 2019; 73% in Euphorbia,Villaverde et al., 2018).
This result shows how the bait set is robust to capture a large
number of genes, even when the enrichment reaction did not
meet expectations (e.g., for G. aesculifolia with 4.4% of on-
target reads). Here we applied a great depth of sequence,
higher than that applied in other studies (e.g., Soto Gomez
et al., 2019;Jantzen et al., 2020;Sanderson et al., 2020;
Eserman et al., 2021). The sequencing depth and laboratory
protocols applied during library preparation and enrichment
FIGURE 4
Phylogenies obtained through a coalescent approach and Exon-Only and Supercontig datasets . Gene concordance factor (gCF) values shown
as pie charts. For gCF pie charts, blue represents the proportion of gene trees concordant with that branch, purple represents the proportion of gene
trees concordant with the first alternative quartet, orange represents the proportion of gene trees concordant with the second alternative quartet,
and red represents the gene discordance support due to polyphyly.
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reaction are relevant variables controlling the number of reads
on target, the number of genes recovered, or the total length of
the genes compared to the reference (Johnson et al., 2016;
Anderman et al., 2020). Comparisons between studies are
limited because of the many variables involved during wet
lab steps and sequencing; however, our results illustrate that
we have designed an extremely efficient bait kit for molecular
phylogenetic studies of Bignoniaceae.
4.2 The paralogs
The number of genes used for phylogenetic analyses was
reduced to 677 after removing paralogs and genes with sequences
present in less than 70% of the species (Table 1). HybPiper
flagged 202 genes that represented putative paralogs, a number
that is similar to that recovered by other studies (135 of 681 genes
in Burmeistera,Bagley et al., 2020; 219 of 830 genes in
Gesneriaceae, Ogutcen et al., 2021). These genes were
evaluated using phylogenetic trees. We found little evidence of
paralogy, with most trees showing sequences from the same
sample grouped together. Of the 202 genes, 82 were removed due
to paralogy or because they showed more than two specimens
flagged as paralogous. Many putative alleles were obtained, which
can be explained by the great gene coverage obtained for all
species (Johnson et al., 2016;Table 1).
The decision to exclude genes with paralogs can be
considered conservative, removing a substantial amount of
sequence data that could be used in phylogenetic inferences.
Strategies to select and incorporate paralogs are available (Yang
and Smith, 2014;Moore et al., 2018;Karimi et al., 2020;Morales-
Briones et al., 2021), allowing the expansion of the final gene set
using gene tree-guided orthology identification. Criteria such as
“Monophyletic outgroups”or “Rooted ingroups”could be used
to identify paralogs (Yang and Smith, 2014;Morales-Briones
et al., 2021). These different gene sequences could be used as
paralog-specific references in HybPiper, recursively recovering
orthologous sequences (e.g., Johnson et al., 2016;Karimi et al.,
2020). A pilot study using this strategy was applied to the
Bignoniaceae data generated here, adding 71 genes after two
rounds of iteratively selecting orthologous and paralogous
sequences. Although these data were not used in this paper
for phylogenetic analyses, it highlights the potential of the
genes flagged as paralogous by HybPiper for future
phylogenetic studies within the family.
4.3 Bignoniaceae phylogenomics
We inferred phylogenomic relationships for all samples
using two datasets: the “Exon-Only”, containing the targeted
regions, and the “Supercontig”, containing the targeted regions
+ non-targeted regions composedofintronsorintergenic
regions. We also applied two methods to infer trees: a
concatenation method using a supermatrix and a coalescent-
based species tree estimation. Gene tree incongruence due to
incomplete lineage sorting is a common pattern not accounted
for by concatenation methods, which could result in high
support for an incorrect topology (Degnan and Rosenberg,
2009). Coalescent approaches minimized this problem,
representing a fundamental tool in phylogenomic studies
using nuclear data (Karimi et al., 2020;Morales-Briones
et al., 2021).
The trees obtained using both datasets and methods were
generally very similar to each other (Figures 2,3)and
resembled previous phylogenetic findings. The phylogenetic
relationships at the tribal-level were evaluated using
representatives of five different tribes or clades. Jacaranda
mimosifolia (tribe Jacarandeae) was used to root the tree.
Tribes Bignonieae, Crescentieae, Tecomeae, and the Tabebuia
Alliance clade emerged as monophyletic in all results with
maximum support of UFBoot or LPP (Figures 2,3). These
findings corroborate previous results using Sanger-generated
data (Lohmann, 2006;Olmstead et al., 2009;Ragsac et al.,
2021). Tribes Bignonieae and Tecomeae have a consistent
taxonomic history and are recognized by morphological
synapomorphies (Gentry, 1976;Lohmann and Taylor, 2014;
Ragsac et al., 2021). Tribe Tecomeae and the Tabebuia
Alliance clade emerged as monophyletic here,
corroborating the most recent phylogeny of the family
(Olmstead et al., 2009). Nyctocalos cuspidatum (tribe
Oroxyleae) emerged as sister of Bignonieae in most trees;
however, the UFBoot support was not maximum for both
datasets and the species tree obtained using the Supercontig
dataset recovered N. cuspidatum as sister to clade C. An
expanded sampling of Oroxyleae could help place this tribe
within the Bignoniaceae with higher certainty (Olmstead
et al., 2009). Other phylogenetic relationships at the tribal-
level received maximum support and were congruent
throughout the analyses (Figures 2,3) suggesting a robust
set of genes for phylogenetic studies at this level.
The robustness of the kit to resolve phylogenetic
relationships at tribal-level clades of Bignoniaceae is also clear
when the numbers of parsimony informative bases are
considered. The Exon-Only dataset had 203,576 bp (23.2%) of
informative sites at this level, while the Supercontig dataset
reached 1,114,993 bp (39.6%). When custom selected and the
Angiosperms353 genes are compared in terms of Exon-Only, the
proportion of informative sites was 21.8% and 23.5%,
respectively. For Supercontig, the proportions are 37.2% and
42.6% (Table 2). The proportions of phylogenetically informative
sites (PIS) between custom selected and the
Angiosperms353 datasets are similar, with a slight advantage
for the Angiosperms353. This finding resonates previous results
that showed that bait kits could be as informative as custom baits
at comprehensive levels of the tree (Yardeni et al., 2022).
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We sampled all 20 genera of tribe Bignonieae to evaluate the
performance of the gene set at this level. Considering the results
derived from the analyses of the Exon-Only and Supercontig
datasets, the trees were robust, congruent with hundreds of genes
(Figure 4), and showed branches that mostly received maximum
support for both UFBoot and LPP (Figures 2,3). The topology
largely resembles the most comprehensive phylogenetic result for
Bignonieae (Lohmann, 2006). Among the similarities are the
sister relationship between Perianthomega vellozoi Bureau
and the rest of the tribe. The clades “Multiples of Four”and
“Fridericia and Allies”also emerged as monophyletic
(Lohmann, 2006;Lohmann and Taylor, 2014); these
phylogenetic relationships received maximum support for
both metrics (Figures 2,3). Other phylogenetic relationships
were revealed for the first time by the new data, such as the
clade that included Dolichandra as sister to Manaosella
cordifolia (DC.) A.H. Gentry. Both genera are composed
of lianas and conspicuous flowers with membranaceous
calyces and infundibular corollas (Fonseca and Lohmann,
2015). Poorly supported clades recovered in previous studies
(Lohmann, 2006) are also poorly supported here.
Furthermore, new phylogenetic relationships were
recovered in the “backbone”of the tree, but these
relationships were poorly supported (Figures 2,3).
Incomplete lineage sorting appears to be a reasonable
explanation for these recalcitrant regions of the tree (Suh
et al., 2015;Moore et al., 2018); however, the results from
ASTRAL-III were poorly supported for this region of the tree
and revealed significant underlying genomic conflicts
(Figures 2,3,4) suggesting that other processes might be
shaping the tree.
At the generic-level, the bait kit resolved most phylogenetic
relationships with maximum support, although some poorly
supported short branches revealed significant conflicts
between gene trees (Figure 4). This result highlights how
diversification over short periods of evolutionary time may
impact phylogeny reconstruction, despite the abundant
molecular data available (Table 2). The Exon-Only dataset
had 121,268 bp (13.8%) informative sites at the genus-level,
and the Supercontig dataset had 688,408 bp (24.5%). For the
Exon-Only dataset, the proportions of the custom-selected and
Angiosperms353 genes were 18.6% and 9%, respectively. For the
Supercontigs, the proportions were 22.9% and 26%, respectively.
Overall, our findings indicate that the target genes of the
Angiosperms353 bait kit are less variable at the genus-level;
however, when non-targeted data are included, this bait kit
has genes with higher PIS. The same trend is observed in
Dolichandra, where the Exon-Only recovered 3.4% and 1.7%
of PIS for the custom-selected and Angiosperms353 datasets,
while the Supercontig recovered 3.4% and 3.9%, respectively
(Table 2). These results are in line with earlier findings in
Buddleja L. (Chau et al., 2018), Burmeistera (Balgley et al.,
2020), Cyperus L. (Larridon et al., 2020), and Orchidaceae
(Yardeni et al., 2022), where the universal bait kit worked as
well as the custom kit or even better at the generic and infra-
generic levels.
Within Dolichandra, all analyses recovered congruent
results that were fully supported by UFBoot and LPP
(Figures 2,3). These findings corroborate earlier
phylogenetic hypotheses obtained using plastid data
(i.e., ndhFandrpL32-trnL). Interestingly, the topology of
Dolichandra previously recovered based on a nuclear marker
(i.e., pepC; Fonseca and Lohmann, 2015)isnotfully
concordant with the topology obtained here. The number
of informative sites just for Dolichandra reached 22,251 bp
for the Exon-Only data and 179,384 bp for the Supercontig
dataset (Table 2). To evaluate the potential of the 677 genes
used in this study for shallow taxonomic scales, we also
evaluated within-genus pairwise distances. All within-genus
comparisons showed values that were greater than 1% of
sequence divergence. Even within species, 0.3% and 1% of
sequence divergence was recovered for the Exon-Only, while
0.4% and 1.6% of sequence divergence was recovered for the
Supercontig dataset. For these analyses, three specimens of
D. unguis-cati from different localities were used
(Supplementary Table S1). These findings highlight the
potential utility of the bait kit for species delimitation
and population studies, which is consistent with earlier
findings based on the Angiosperms353 kit (Slimp et al.,
2021).
4.4 To Bignoniaceae and beyond
The datasets “Exon-Only”and “Supercontig”recovered
similar trees in all phylogenetic strategies (Figures 2,3,4),
showing the presence of phylogenetic signal in both protein
coding and the “splash zone”regions. The absence of saturated
markers also reveals the utility of protein coding and non-
coding regions at different levels, reaching the family level of the
tree. Gains in UFBoot (96.4 vs. 97.2) and LPP (0.936 vs. 0.954)
were marginal when both “Exon-Only”and “Supercontig”
datasets were compared. Dolichandra was also recovered as
monophyletic with all branches receiving maximum support in
all scenarios. These results could suggest redundancy between
datasets, however the addition of the thousands of base pairs
from the “splash zone”will certainly be relevant to resolve
phylogenetic relationships at shallow levels of the tree (Bagley
et al., 2020;Ogutcen et al., 2021).
The use of the bait kit beyond Bignoniaceae is
speculative, with possible applications inside the order
Lamiales (Ogutcen et al., 2021)assuggestedbythe12of
the 18 genes without Bignoniaceae references successfully
assembled. The steps used here to select low copy genes are
certainly reproducible in other plant groups. The Hyb-Seq
protocol is wide established, requiring few genomic
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resources to select single/low copy genes (Weitemier et al.,
2014). New pipelines could also be used to complement the
gene pool selected by Hyb-Seq or as alternatives, such as
MarkerMiner (Chamala et al., 2015), or AllMarkers (Kadlec
et al., 2017). The strategy used to select family/clade specific
genes from the 353 universal loci pool is also reproducible,
with more than a thousand of transcriptomes covering the
majority of angiosperm diversity available for gene selection
(Johnson et al., 2019).
5 Conclusion
Here we provide the first bait kit designed to capture low to
single-copy nuclear genes for the plant family Bignoniaceae. The
kit incorporates novel markers designed specifically for this plant
family using the Hyb-Seq protocol; the gene set of the
Angiosperms353, with baits designed specifically for
Bignoniaceae; and functional genes with regulatory roles at
different stages of flower, fruit, and leaf development, as well
as roles in biochemical synthesis (Supplementary Material S2).
Our bait kit enables the capture of 762 genes, among which
329 are specific for Bignoniaceae, 348 are from the
Angiosperms353 bait kit designed by Johnson et al. (2019),
and 85 correspond to functional genes. We tested the
effectiveness of the enrichment steps using 38 samples of
Bignoniaceae from 36 different species. These taxa spanned
five different tribes, 20 different genera within the Bignonieae,
and seven species of the genus Dolichandra. Gene recovery was
exceptionally high, enabling near complete on-target data in all
different levels evaluated.
The approach implemented here validated the bait kit from
tribal to species-level, recovering informative regions and robust
phylogenetic relationships through different time scales.
Resolving phylogenetic relationships with highly supported
branches is a prerequisite for many downstream applications
such as diversification, biogeographic, and evolutionary studies.
Phylogenomic results could also update classifications and
contribute to taxonomic studies. The Bignoniaceae-specific kit
will be implemented in phylogenetic studies at species-level
within the family. We aim to clarify the evolutionary history
of morphological traits, biogeographic history, timing of origin,
and many other open questions to be addressed in the family.
The kit will also allow for data reuse and will contribute to
ongoing efforts to assemble the plant tree of life using
the Angiosperms353 kit (Baker et al., 2021). The kit
developed here will also allow evo-devo and physiological
studies, especially through the use of the set of 85 functional
genes selected. Indeed, the newly developed probe set will allow
many evolutionary questions to be addressed within the
Bignoniaceae using a reliable phylogenomic framework.
Data availability statement
Sequence alignments and phylogenetic trees presented in
this study can be found on Github: https://github.com/
luizhhziul. Raw reads for all accessions are available in
GenBank Sequence Read Archive (SRA) under BioProject
ID PRJNA909066.
Author contributions
LF, MC, PF, and LL conceived and designed the experiment.
LF performed the experiments, assembled sequences, and
analyzed the data. LF and LL collected the materials, and
wrote the paper. LF, MC, PF, and LL edited the text and
agreed with the final version of the manuscript.
Funding
This work was supported by CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior) CNPq
(Conselho Nacional de Desenvolvimento Científico e
Tecnológico-Grant Pq1B-310871/2017-4), and FAPESP
(Fundação de Amparo à Pesquisa do Estado de São
Paulo–Grants: 2011/09160-5, 2012/50260-6, 2018/23899-2,
and 2019/13624-9).
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fgene.
2022.1085692/full#supplementary-material
Frontiers in Genetics frontiersin.org14
Fonseca et al. 10.3389/fgene.2022.1085692
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