The genomic signatures of evolutionary stasis

Abstract

Evolutionary stasis characterizes lineages that seldom speciate and show little phenotypic change over long stretches of geological time. Although lineages that appear to exhibit evolutionary stasis are often called living fossils, no single mechanism is thought responsible for their slow rates of morphological evolution and low species diversity. Some analyses of molecular evolutionary rates in a handful of living fossil lineages have indicated they exhibit slow rates of genomic change. Here, we investigate mechanisms of evolutionary stasis using a dataset of 1,105 exons for 481 vertebrate species. We demonstrate that two ancient clades of ray-finned fishes classically called living fossils, gars and sturgeons, exhibit the lowest rates of molecular substitution in protein coding genes among all jawed vertebrates. Comparably low rates of evolution are observed at four-fold degenerate sites in gars and sturgeons, implying a mechanism of stasis decoupled from selection that we speculate is linked to a highly effective DNA repair apparatus. We show that two gar species last sharing common ancestry over 100 million years ago naturally produce morphologically intermediate and fertile hybrids. This makes gars the oldest naturally hybridizing divergence among eukaryotes and supports a theoretical prediction that slow rates of nucleotide substitution across the genome slows the accumulation of genetic incompatibilities, enabling hybridization across deeply divergent lineages and perhaps slowing the rate of speciation. Our results help establish molecular stasis as a barrier to speciation and phenotypic innovation and provide a mechanism to explain the low species diversity in living fossil lineages.

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The genomic signatures of evolutionary stasis
Chase D. Brownstein, 1, 2*† Daniel J. MacGuigan, 3† Daemin Kim,1 Oliver Orr,4 Liandong Yang,5
Solomon R. David,6 Brian Kreiser,7 Thomas J. Near1, 8
1Department of Ecology and Evolutionary Biology, Yale University, New Haven CT, USA
2Stamford Museum and Nature Center, Stamford CT, USA
3Department of Biological Sciences, University at Buffalo, Buffalo NY, USA
4The Metropolitan Museum of Art, New York NY, USA
5Institute of Hydrobiology, Chinese Academy of Sciences, Beijing, China
6Department of Fisheries, Wildlife and Conservation Biology, University of Minnesota
7School of Biological, Environmental, and Earth Sciences, University of Southern Mississippi, MS, USA
8Peabody Museum, Yale University, CT, USA
*Corresponding author
†Co-lead authors
Email: chase.brownstein@yale.edu
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Abstract.
Evolutionary stasis characterizes lineages that seldom speciate and show little phenotypic change
over long stretches of geological time. Although lineages that appear to exhibit evolutionary stasis
are often called living fossils, no single mechanism is thought responsible for their slow rates of
morphological evolution and low species diversity. Some analyses of molecular evolutionary rates in
a handful of living fossil lineages have indicated they exhibit slow rates of genomic change. Here, we
investigate mechanisms of evolutionary stasis using a dataset of 1,105 exons for 481 vertebrate
species. We demonstrate that two ancient clades of ray-finned fishes classically called living fossils,
gars and sturgeons, exhibit the lowest rates of molecular substitution in protein coding genes among
all jawed vertebrates. Comparably low rates of evolution are observed at four-fold degenerate sites
in gars and sturgeons, implying a mechanism of stasis decoupled from selection that we speculate is
linked to a highly effective DNA repair apparatus. We show that two gar species last sharing
common ancestry over 100 million years ago naturally produce morphologically intermediate
and fertile hybrids. This makes gars the oldest naturally hybridizing divergence among eukaryotes
and supports a theoretical prediction that slow rates of nucleotide substitution across the genome
slows the accumulation of genetic incompatibilities, enabling hybridization across deeply divergent
lineages and perhaps slowing the rate of speciation. Our results help establish molecular stasis as a
barrier to speciation and phenotypic innovation and provide a mechanism to explain the low species
diversity in living fossil lineages.
Keywords: Living Fossils; Phylogenetics; Genomics; Evolutionary Rates; Gars; Fishes.
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Introduction.
Biodiversity is asymmetrically distributed across the Tree of Life. Understanding the drivers of
this variation is a central problem in evolutionary biology (Darwin 1859; Stanley 1975; Schluter 2000;
Gavrilets and Losos 2009). Long-lived, species-poor lineages represent a high proportion of unique
phenotypes and evolutionary history (Stanley 1975; Stein et al. 2018; Dornburg and Near 2021). Yet, the
factors that contribute to the persistence of the long-lived, phenotypically conservative, and species-
poor lineages known as living fossils remain unknown (Darwin 1859; Stanley 1975; Turner 2019; Lidgard
and Love 2021).
Speciation resulting from the reproductive isolation of populations is theoretically a function of
the mutation rate; isolated populations will accumulate mutations that are incompatible with mutations
found in the genomes of individuals in other populations (Orr and Turelli 2001; Coyne and Orr 2004).
Although observations highlight the importance of mutation rates and underlying genomic potential for
generating bursts of speciation and morphological diversification (Schluter 2000; Gavrilets and Losos
2009; McGee et al. 2020), molecular mechanisms for the origins and persistence of living fossils have not
been observed in many classic examples of this phenomenon (Selander et al. 1970; Avise et al. 1994;
Hay et al. 2008; Casane and Laurenti 2013; Chalopin et al. 2014). Studies of living fossil lineages have
variously found that they possess low (Amemiya et al. 2013; Venkatesh et al. 2014; Braasch et al. 2016;
Du et al. 2020; Thompson et al. 2021) to rapid (Hay et al. 2008) molecular evolutionary rates compared
to other lineages. Thus, only equivocal evidence exists for a molecular counterpart to morphological
stasis in living fossils that could explain their low rates of speciation.
Ray-finned fishes include a high proportion of living fossil vertebrates (Darwin 1859; Stanley
1975, 1979; Grande 2010; Brito et al. 2017; Dornburg and Near 2021; Lidgard and Love 2021). Gars
(Lepisosteidae; Fig. 1) are a clade of seven living species of ray-finned fishes (Fig. 1a) that form part of
the sister lineage of Teleostei, which includes over 35,000 species and represents half of all extant
vertebrate diversity (Grande 2010; Dornburg and Near 2021).
Gars are notable for their low anatomical variation (Fig. 1b) (Grande 2010). The earliest fossil
gars from the Jurassic are nearly identical to living species (Brito et al. 2017), and recognizable members
of living genera appear in the fossil record as early as the middle Cretaceous (Grande 2010; Brownstein
et al. 2023). Observations of the fossil record of lepisosteids led Darwin (1859:107) to identify them as
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living fossils, and the label has persisted since (Wiley and Schultze 1984; Braasch et al. 2016; Dornburg
and Near 2021).
Here, we investigate whether there are molecular analogs to the patterns of slow morphological
evolution observable in gars and other living fossil jawed vertebrates. Several studies have found
evidence that gars may have slower rates of molecular evolution than other ray-finned fishes (Braasch et
al. 2016; Thompson et al. 2021), but large-scale comparisons have not been made across vertebrates.
Nor has evidence of incomplete reproductive isolation across the deepest divergence in living gars (Bohn
et al. 2017) been tested using genome-scale data or integrated with observations of molecular evolution
in Lepisosteidae. We demonstrate that the molecular evolutionary rates of gars and another clade of
living fossil fishes, Acipenseriformes (sturgeons and paddlefishes), are the lowest among vertebrates
and associated with evidence of incomplete reproductive isolation across geological time scales in
species-poor living fossil lineages. By confirming the existence of fertile, morphologically intermediate
hybrids in wild populations of the two extant gar lineages Atractosteus and Lepisosteus, which diverged
during the Early Cretaceous (Fig. 1a; Grande, 2010; Brownstein et al., 2023), we link the slow rates of
molecular evolution in gars with the production of hybrids among species with ancient (>100 million
year) common ancestry. Our results show that hybrid viability broadly decreases with older parental
divergence times and higher rates of molecular evolution across jawed vertebrates. Consequently, our
findings support the hypothesis that low molecular evolutionary rates are coupled with low species
diversity and stagnant phenotypic evolution over long stretches of geologic time in living fossil lineages.
Materials and Methods
Molecular evolutionary rates among major lineages of jawed vertebrates.
In order to test whether slow rates of morphological evolution are paired with low rates of
molecular evolution in living fossils like gars, sturgeons, and paddlefishes, we estimated molecular rate
variation across 1,105 exons from a sample of 471 jawed vertebrate species. We identified orthologous
exon sequences from the genomes of selected species in the NCBI database for the following major
jawed vertebrate lineages: Acipenseriformes, Aves, Crocodylia, Chondrichthyes, Lepidosauria,
Lissamphibia, Marsupialia, Placentalia, Polypteridae, and Teleostei (Figure S1a). The HMM protocol
available in HMMER 3.1 (Wheeler and Eddy 2013) was used to search each of the downloaded genomes
for orthologous exons. These exon sequences were extracted using Python scripts from a phylogenomic
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analysis of ray-finned fishes using these loci (Hughes et al. 2018). For each group of the exon sequences,
we aligned them using MAFFT v.7.3 (Katoh and Standley 2013) with default parameters. Exon
alignments for the seven living species of gars (Lepisosteidae) used in a phylogenomic study (Brownstein
et al., 2023) were included in the comparative analysis. Each exon was separately aligned among the
species in a given vertebrate lineage, resulting in a maximum of 1,105 alignments sampled for each
lineage. Fourfold degenerate (4D) sites were extracted from all exons and concatenated in every
vertebrate clade except Polypteridae and Acipenseriformes. This was because we could not find
orthologous 4D site sequences for all the available genome assemblies for these two clades; sampling
4D sites for all three species would be needed to sample the common ancestors of Polypteridae and
Acipenseriformes, as we were only able to include three species of each in our exon rate estimate
analyses.
We estimated and compared posterior molecular substitution rates at each exon across all
major vertebrate clades using fixed input trees in Bayesian molecular clock analyses. We used previously
published time calibrated phylogenies for Teleostei (Hughes et al. 2018), Acipenseriformes (Kumar et al.
2017), Polypteridae (Near et al. 2014), Lepisosteidae ( Brownstein et al. 2023), Chondrichthyes (Kumar
et al. 2017), Testudines (Shaffer et al. 2017), Amphibia (Kumar et al. 2017), Lepidosauria (Pyron and
Burbrink 2014), Aves (Prum et al. 2015), Crocodylia (Green et al. 2014), Marsupialia (Upham et al. 2019),
and Placentalia (Upham et al. 2019). The time tree of vertebrates used in the branch rate analysis of
coelacanths and lungfish were taken from the literature (Wang et al. 2021) and timetree.org (Kumar et
al. 2017). We used these time-calibrated molecular phylogenies in BEAST 2.5.2 (Bouckaert et al. 2019)
by inserting them in Newick format into the ‘Starting Tree’ tab in the BEAUTi terminal. The following
operators were turned off to ensure the input tree remained fixed: tree scaler, tree root scaler, uniform
operator, subtree slide, narrow and wide exchange, and Wilson-Balding. In turn, we neither estimated
tree topology or divergence times. Custom scripts for inserting the trees, along with xml files containing
the tree topologies used, are in the Supplementary Data.
BEAST 2.5.2 (Bouckaert et al. 2019) was used to estimate the Bayesian posterior nucleotide
substitution rate for each of the 1,105 exons and fourfold degenerate sites separately from each
vertebrate clade. The computer program BEAUTi (Bouckaert et al. 2019) was used to construct
individual xml files from each exon alignment and the pooled fourfold degenerate sites for each clade
with the clade-specific time-calibrated phylogeny. The time-calibrated phylogeny was fixed such that
BEAST did not estimate topology or divergence times. Because of the large number of BEAST analyses,
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we took advantage of the Yale High-Performance Computing cluster and built custom scripts to produce
and run xml files along this pipeline. First, we produced a template xml for each clade that specified all
input parameters with reference to a single xml. We then used a custom batch script (Supplementary
Information) to fit the specifications given by the template xml file with each gene to produce individual
xml files for every single exon sampled in a given vertebrate clade. We used a Yule (pure-birth)
branching model as the tree prior, a relaxed Lognormal clock model as the clock prior to allow
independent rates for each branch, and an HKY model of nucleotide evolution to allow for unequal
frequencies and transition rates, and ran each analysis ran for 10 million generations. Upon completion
of the analyses, we confirmed sufficient MCMC mixing (ESS>200) for each BEAST run using the program
Tracer v. 1.7 (Rambaut et al. 2018) and the R package “coda” v.0.19-4 (Makowski et al. 2019).
To test whether our rate estimates were unaffected by tree model choice, we reran exons in
lepidosaurs and teleosts with the five fastest, five slowest, and five middlemost rate estimates under a
Birth-Death tree model. Theoretically, tree model choice should not affect our results because we fixed
the time tree in each analysis, but we chose to test this outright. Parameter choices were otherwise the
same as the original runs. We then compared absolute rate estimates between runs using the Yule and
Birth-Death models. Next, we tested to see whether the number of species sampled for different clades
biased estimated rates. Among the six clades with more than seven species sampled that had average
estimated substitution rates higher than the average in gars (Figure 2), we subsampled seven species of
lepidosaurs and teleosts (the number of species sampled for gars) that captured the common ancestry
of major subclades (i.e., Squamata, Toxicofera, Euteleostei, Acanthomorpha) out of our exon dataset
and reran the analyses under the original parameter specification (i.e., under a Yule model). We then
compared absolute rate estimates between runs using the full and reduced sampling of lepidosaurs and
teleosts.
Estimation of branch specific molecular evolutionary rates for living fossils.
We investigated the rates of molecular substitution in candidate living fossil lineages including
paleognathous birds, the Hoatzin Opisthocomus hoazin, the Tuatara Sphenodon punctatus, the
Salamanderfish Lepidogalaxias salamandroides, the African Coelacanth Latimeria chalumnae, the
Australian Lungfish Neoceratodus forsteri, and the West African Lungfish Protopterus annectens. We
extracted the estimated posterior molecular substitution rates for the corresponding terminal branches
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in our fixed time trees. For the two lungfishes and African Coelacanth, we constructed a time tree that
included these taxa and the species of Lissamphibia for which exon data was available. We included
Lissamphibians to ensure that the time tree accommodated the paraphyly of sarcopterygian “fishes.”
This was accomplished by taking the fixed tree of Lissamphibia (see ‘Input Tree Selection’) and manually
adding Latimeria chalumnae, the Australian Lungfish Neoceratodus forsteri, and the West African
Lungfish Protopterus annectens as progressive outgroups to Amphibia (following (Amemiya et al. 2013;
Meyer et al. 2021; Wang et al. 2021)) and fixing divergence times following TimeTree.org searches.
BEAST analyses were run using the same parameters as in the whole-clade rate estimation runs and
extracted branch-specific rates for L. chalumnae and Dipnoi.
Hybridization among deeply divergent gar lineages Lepisosteus and Atractosteus.
Next, we investigated signatures of hybridization across deeply divergent gar lineages in order
to test whether slow rates of molecular evolution might be associated with incomplete reproductive
isolation across deep time in living fossil lineages. To check for regions where both extant gar genera are
currently sympatric, we downloaded occurrence data for species of Lepisosteus and Atractosteus spatula
from FishNet2 (http://fishnet2.net/). We pruned erroneous and duplicate records. The clean occurrence
data files are included in the Supplementary Data.
Natural hybrids have been reported among wild populations of Atractosteus spatula and
Lepisosteus osseus in Texas and Oklahoma (Bohn et al. 2017; Taylor et al. 2020), and so we examined
genomic evidence of hybridization in these populations. We obtained tissue samples from 206
specimens of Atractosteus spatula, Lepisosteus osseus, and hypothesized A. spatula X L. osseus hybrids
from across Gulf of Mexico coastal river systems (Table S1) to test for both the commonality of hybrids
and the presence of both F1s and F2s in the Brazos river system. The Brazos and Trinity systems and
Choke Canyon Reservoir were targeted because previous studies have convincingly demonstrated the
presence of hybrid Atractosteus spatula X Lepisosteus osseus individuals in this region (Bohn et al. 2017;
Taylor et al. 2019). We chose a subset of five A. spatula and seven L. osseus isolations with high DNA
concentrations based on Qubit fluorometer (Life Technologies, Carlsbad, CA, USA) readings to test for
naturally occurring hybrids between species in these genera. These were compared with tissue samples
from individuals that showed morphological intermediacy consistent with being a hybrid, as well as their
identification in a previous study (Bohn et al. 2017).
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We used double digest restriction site associated DNA sequencing (ddRADseq) to obtain a large
dataset of single nucleotide polymorphisms (SNPs) to investigate whether sympatric populations of
Lepisosteus osseus and Atractosteus spatula show evidence of hybridization. ddRADseq was performed
following a modified version of frequently used protocol (Peterson et al. 2012). Additional details are
described in the Supplementary Information. Pooled libraries were size-selected for 300-500 bps using a
BluePippin sequenced by the University of Oregon Genomics & Cell Characterization Core Facility on an
Illumina HiSeq 4000. We assembled the gar ddRAD dataset using iPyrad v.0.9.68 (Eaton and Overcast
2020) with the Lepisosteus oculatus genome (NCBI accession number:GCF_000242695.1) (Braasch et al.
2016) as a reference, resulting in a total of 256,750 loci shared by at least four samples. For analysis of
hybridization, ddRAD SNPs were filtered using VCFTools v.0.1.15 (Danecek et al. 2011) so that only
biallelic SNPs with minor allele counts >1 and <5% missing data were retained. To minimize the effects
of linkage among markers, one random SNP per 10,000 bp window was retained, resulting in a dataset
containing 2,097 SNPs (2.2% missing data). In addition, we used a custom R script to identify 1,223 SNPs
(2.1% missing data) that were fixed between the parental species (Atractosteus spatula and Lepisosteus
osseus). Demultiplexed ddRAD Illumina reads are deposited at the NCBI SRA (PRJNA1077910).
We assessed genomic signals of hybridization between Atractosteus spatula and Lepisosteus
osseus. First, missing genotypes were imputed with the “impute” function (method = “random”) in the R
package LEA v.3.4.0 (Frichot and François 2015). To examine patterns of genetic variation, we then
performed principal component analysis (PCA) using the 2,097 filtered SNPs with the “dudi.pca”
function in the R package ade4 v.2.1.4 (Dray and Dufour 2007; Thioulouse et al. 2018). In addition, we
estimated genomic ancestry coefficients for each individual with sparse nonnegative matrix factorization
implemented in the R package LEA. Ten replicate analyses were performed with between two and ten
ancestral populations (K). We examined cross-entropy scores to determine the optimal value of K
(Figure S3a).
Hybrid classification was performed using the dataset of 1,223 fixed SNPs. First, the
“find.clusters” function from the Adegenet v.2.1.4 R package (Jombart and Ahmed 2011) was used to
assign all individuals to two genetic clusters by performing 10 million search iterations of the K-means
algorithm with 1,000 random starting centroids. Starting with the K-means group memberships, we used
the Adegenet “snapclust” function (Beugin et al. 2018) to estimate probability of membership to
parental, F1, or backcross classes. A maximum of ten million generations for 1,000 replicate runs of the
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expectation–maximization were performed. Additionally, we estimated the ancestry index and
interspecific heterozygosity for each individual using the R package HIest v.2.0 (Fitzpatrick 2012).
To better understand patterns of hybridization, including skewed sex ratios among parental
Atractosteus spatula and Lepisosteus osseus, we interrogated mitochondrial DNA (mtDNA) data for a
sample of 201 gars including all living species in Lepisosteidae. Because mitochondrial DNA is maternally
inherited, inferring a phylogenetic tree using mitochondrial sequence data can illuminate skewed sex
ratios in hybrid crosses. We included two specimens of Amia calva to serve as outgroups in the
phylogenetic analysis. Sampling locations of specimens used for genetic analyses are listed in
Supplemental File Table_S1.csv. DNA was extracted from 95% ethanol-preserved tissues using a
standard DNeasy Qiagen Blood and Tissue Kit (QIAGEN, Valencia, CA, USA). To minimize downstream
enzymatic inhibition, we purified DNA extractions with an ethanol precipitation: 3M sodium acetate (pH
= 5.2) was added equal to 10% of the total volume of the DNA extraction followed with 100% ethanol
equal to 2.5 times the total volume of DNA. After mixing, extractions were incubated for 10 minutes at -
80C. Samples were centrifuged for 30 minutes at 8,000 RCF, the supernatant was carefully poured off,
and the DNA pellet was washed with 250 uL of cold 70% ethanol. Samples were centrifuged again for 5
minutes at 8,000 RCF, supernatant was poured off, the pellet was allowed to air dry for ~15 minutes,
and the DNA pellet was resuspended with the desired amount of DNAse-free water.
The mtDNA gene tree of gars (Figure 5f) was inferred with a phylogenetic analysis of the
mitochondrial encoded cytochrome b (cytb) gene. The molecular phylogenetic analysis included 33
specimens of Atractosteus spatula, two specimens of A. tristoechus, eight specimens of A. tropicus, 65
specimens of Lepisosteus osseus, five specimens of L. platostomus, 44 specimens of L. oculatus, 23
specimens of L. platyrhincus, 21 specimens of Atractosteus spatula X Lepisosteus osseus hybrids, and a
single specimen of both Amia calva and A. ocellicauda to serve as outgroups. The cytb gene was
amplified using previously published PCR primers and cycling conditions (Wright et al. 2012).
Amplification products were prepared for DNA sequencing using a polyethylene glycol precipitation.
Contiguous sequences were assembled from individual DNA sequencing reactions using the computer
program Geneious v.7.2 (Kearse et al. 2012). New cytb sequences were aligned by eye to those
previously generated in early studies of gar phylogeny (Wright et al. 2012). The optimal data partitioning
scheme, among the three codon positions of the cytb gene, and molecular evolutionary models were
determined using the Bayesian information criterion in the computer program Partitionfinder v. 2.1
(Lanfear et al. 2017). The mitochondrial gene tree was inferred from the aligned cytb sequences using
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the optimal molecular evolutionary models and partitioning scheme using the computer program
MrBayes v. 3.2 (Ronquist et al. 2012), where posterior probabilities for the phylogeny and parameter
values were estimated using Metropolis-couple Markov chain Monte Carlo (Larget and Simon 1999;
Huelsenbeck et al. 2001). The MrBayes analysis was run for 107 generations with two simultaneous runs
each with four chains. Convergence of the MC3 algorithm and stationarity of the chains was assessed by
monitoring the average standard deviation of the split frequencies between the two runs, which was
less than 0.005 after 3 X 106 generations. In addition, the likelihood score and all model parameter
estimates were plotted against the generation number to determine when there was no increase
relative to the generation number in the computer program Tracer v. 1.7 (Rambaut et al. 2018). The first
50% of the sampled generations were discarded as burn-in, and the posterior phylogeny was
summarized as a 50% majority-rule consensus tree. All cytb gene sequences generated for this study are
available at GenBank PP331004 - PP331204.
Geometric morphometric analyses of gar hybrids.
To quantify how the phenotypes of hybrid individuals of the two extant gar genera compared to
those of their parental lineages, we used a dataset sampled from 25 specimens of Atractosteus spatula,
Lepisosteus osseus, and A. spatula x L. osseus from the Brazos River system in Texas. The skull and
mandible were selected as regions of study because there is morphological variation in these traits
among lineages of extant and extinct gars (Wiley 1976; Kammerer et al. 2006; Grande 2010; Brito et al.
2017) and both the skull and mandible contain key apomorphies of both Atractosteus and Lepisosteus
(Wiley 1976; Grande 2010). These include features like the orientation and contact of the dentary
symphyses and the width of the skull, which differ among Lepisosteus and Atractosteus and might
appear distinct in hybrid individuals. A total of 7 meristic counts and proportions were measured.
We included 24 morphometric landmarks: 12 on the skull in dorsal view and 12 on the mandible
in ventral view (Figure S6). Landmarks were placed based on previously defined borders between major
craniomandibular elements (Grande 2010). We digitized landmark coordinates and defined scales for
each skull using tpsUtil64 v. 1.7 and tpsDIG2 v. 2.26. We ran analyses in both the R package geomorph
(Adams and Otárola-Castillo 2013) and the program MorphoJ (Klingenberg 2011). In both programs, we
applied a generalized Procrustes superimposition to exclude size, positional, and orientation effects
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before conducting principal components analyses on the data. We checked the resulting Procrustes fit
graph for outliers and then ran principal components analyses.
Assessing the morphological disparity of gars in deep time.
To quantify variation in gar morphology over deep time, we collected data for individual
specimens of extant and extinct gar species spanning over 75 million years of time to compare the shape
of the skull across the gar crown clade. We opted for quantitative comparison of these features in crown
gars rather than analysis of the rates of trait evolution through time given the small sample size of this
clade (n=12 species; Fig. 1) and focused on two-dimensional measurements and meristic counts to
mitigate the effects of post-mortem compression and deformation of gar fossils.
Our measurements and meristic dataset includes all currently described extinct species
confidently placed in the gar crown group (Fig. 1), as well as a new sample of Atractosteus spatula X
Lepisosteus osseus hybrid crosses. Hybrids examined included one alcohol preserved head (KU
uncatalogued), three alcohol preserved full fish (KU 18407, 18408, 18560), and three skeletonized skulls
also used in our geometric morphometric analyses (KU 18558, 18409, 18559). KU 18560 is labeled as the
holotype of “Atractosteus macrobeccus” in the University of Kansas collections. These specimens were
collected from the same river systems from which our sequence data establishes the presence of
hybrids: the Trinity River ~1.6 km north of the US Route 90 Bridge in Liberty, Texas (KU 18407), near the
mouth of the Trinity River (KU 18560, 18558, 18559), and at the mouth of the Trinity River near Trinity
Bay (KU 18408, 18409).
Measurements taken included dimensional ratios of all gar skull roof bones, as well as standard
length, head length, lateral line scale, and fin ray counts. All measurements were taken using digital
calipers. The sample was combined with the total sample of crown gars in Grande (2010) for a total of
n=124 specimens, and the dataset was analyzed and plotted in R using ggplot2. All measurements and
meristic data are available in the Supplement.
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Detection of introgression among living lineages of gars.
Lastly, we assessed whether living species of Lepisosteidae exhibit signatures of introgression
over deeper phylogenetic time scales. First, we used MrBayes 3.2.7 (Ronquist et al. 2012) to generate
Bayesian gene trees for the subset of 770 orthologous exons identified by Brownstein et al. (2023) to be
variant in gars sampled for all extant gars and two teleost outgroups (Megalops cyprinoides and
Osteoglossum bicirrhosum) . These represent the variable exons in gars sampled to reconstruct our tip-
dated Bayesian phylogenomic hypothesis. We used an HKY+I+G molecular evolutionary model as
implemented in computer program MrBayes v. 3.2 (Ronquist et al. 2012) and estimated posterior
probabilities for the phylogeny and parameter values were using Metropolis-couple Markov chain
Monte Carlo (Larget and Simon 1999; Huelsenbeck et al. 2001). MrBayes was run for 1.5 x 106
generations with two simultaneous runs each with four chains for each exon. Convergence of the
posteriors was checked in Tracer v. 1.7 (Rambaut et al. 2018). Finally, we used the program DensiTree
(Bouckaert 2010) in order to assess concordance among the resulting gene trees (Fig. 1, Fig. 4a).
Previous studies (e.g., Maddison, 1997; Mallet et al., 2016; Edelman et al. 2019) have noted
that evidence of topological discordance across gene tree topologies may be reflective of both
incomplete lineage sorting and introgression. As such, we conducted a secondary test of introgressive
episodes across Lepisosteidae using PhyloNet 3.7.3 (Wen et al. 2018). Based on the 770 gene trees
generated in Mr. Bayes 3.2.7, we inferred phylogenetic networks from maximum pseudolikelihood
(MPL) with a reticulation of zero and then compared the log-probability of the reticulations up to six
with 200 replications for each reticulation scenario.
Comparison of hybridizing species pair MRCA ages and whole-clade rates.
We searched the literature for information on the most deeply divergent lineages that still
hybridize for the major vertebrate clades analyzed in this study (Figure 2). The ages of the oldest
hybridizing divergences were plotted against mean exon substitution rate estimates generated in this
paper. These data are included in the Supplemental File Table_S2.csv.
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Results and Discussion
The lowest genome-wide substitution rates in vertebrates.
In contrast to the high molecular rates associated with adaptive radiations (Schluter 2000;
Gavrilets and Losos 2009; McGee et al. 2020) it is unclear whether the slow rates of speciation and
phenotypic evolution in gars and other lineages thought to be undergoing evolutionary stasis reflect a
slower rate of genomic change (Braasch et al. 2016; Takezaki 2018). To test this, we estimated
nucleotide substitution rates for 1,105 orthologous exons (Hughes et al. 2018) sampled from 478
vertebrates (Fig. 2a), including new sequences for all extant species of gars.
The resulting rate estimates demonstrate that gar exons consistently evolve between 0.5 to 3
orders of magnitude more slowly than any other major vertebrate clade (Fig. 2b, Fig. 3; Table S2). This
pattern is consistent across genes, with nearly every locus in gars exhibiting the lowest or one of the
lowest rates among orthologs across vertebrates (Fig. 2; Fig. 3; Fig S1, Fig S6). The slow rates of gars are
likely not artifacts of different clade ages or species diversity, as both younger, more species-rich and
older, depauperate lineages in our analysis exhibit higher average substitution rates (Fig. 2; Fig. S1-S5).
Only the genomes of sturgeons and paddlefish (Acipenseriformes), turtles, and crocodylians approach
the slow evolutionary rates observed in gars. Crocodylian and turtle rates are also right-skewed,
reflecting high substitution rates for a number of exons in these clades when compared to gars (Fig. 2b).
Gar and acipenseriform exon substitution rates are also far lower than estimated exonic rates
from more diverse clades (teleosts, lepidosaurs), even when these species-rich clades are subsampled to
contain the same number of species as gars (Table S3). Finally, similar rate results are obtained for gars
and other clades even when different tree models (Yule vs Birth-Death) are used (Table 1), verifying that
these prior choices do not affect our results. Thus, across every iteration of our analyses, gars and
acipenseriforms have the lowest estimated exon substitution rates in jawed vertebrates by several
orders of magnitude.
The genomic rates of gars and acipenseriforms are also much slower than other putative living
fossils, including the Tuatara Sphenodon punctatus, the Coelacanth Latimeria chalumnae, lungfishes, the
Elephant Shark Callorhinchus milii, and the Hoatzin Opisthocomus hoazin (Fig. 3a). This contrasts with
previous estimates of vertebrate genomic evolutionary rates, which have suggested that L. chalumnae
and C. milii possess genomic rates comparable to or even slower than holostean fishes (Braasch et al.
2016; Takezaki 2018; Du et al. 2020; Thompson et al. 2021). Such distinctions may be a result of the
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lower number of species and genes sampled in earlier studies (Braasch et al. 2016; Takezaki 2018; Du et
al. 2020; Thompson et al. 2021). The sluggish tempo of gar genomic evolution also contrasts with the
exceptionally high molecular evolutionary rates of teleost fishes, which have the highest average rate of
the vertebrate clades sampled in our study (Fig. 2b). This high evolutionary rate has been linked to the
whole-genome duplication event that occurred early in the evolutionary history of teleosts (Brunet et al.
2006; Ravi and Venkatesh 2018). In contrast, polypterids, the sister lineage to all other ray-finned fishes
and potential living fossils (Near et al. 2014), show genome-wide substitutions rates similar to most
other vertebrate clades (Fig. 2b).
Molecular evolution is so slow in gars and acipenseriforms that sister species with times to
common ancestry exceeding 20 million years (Fig. 1a; Luo et al., 2019; Brownstein et al., 2023) show no
nucleotide differences in an appreciable proportion of sampled exons; this is visible in boxplots showing
a multimodal distribution of exon rates in terminal branches leading to individual gar species (Fig. 3b-e;
Figure S3, also see Figure S7). These slow rates of genomic evolution in gars and acipenseriforms (Fig.
2b, Fig. 3b-e) validate low rates of transposable element and proteomic evolution reported in species
from both lineages (Braasch et al. 2016; Du et al. 2020), as well as evidence suggesting slower
substitution rates in Holostei (gars and the Bowfin Amia calva) relative to other vertebrates (Takezaki
2018; Thompson et al. 2021). The slow rate of evolution in sturgeons is present despite the major
structural changes to the genome, including whole-genome duplications, that have occurred in this
clade (Du et al. 2020).
We also estimated substitution rates at fourfold degenerate (4D) sites for each clade of
vertebrates. Fourfold degenerate sites are thought to be shielded from selection because every
mutation at these sites is a synonymous substitution that does not change the amino acid translated (Li
1993). The differences in the rates of changes at 4D sites among vertebrate lineages were largely
comparable to the estimated substitution rates for all changes in the exons (Fig. 2c; Fig. S6), with gars
exhibiting much slower rates than all other vertebrates except crocodylians (Fig. 2c). This demonstrates
that gar genomic rates are consistently stagnant across types of nucleotide substitutions that are under
varying degrees of selective pressure, establishing a similar rate pattern between selectively constrained
and neutral sites across the genomes of vertebrates.
For every clade except gars, the 4D site substitution rate estimates were consistently lower than
the mean exonic substitution rate estimates (Fig. 2). This is surprising considering that 4D sites should be
under relaxed selection relative to other exonic sites. However, 4D sites have been shown to be under
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selection due to codon-usage bias, which could lower their substitution rate (Chamary and Hurst, 2004).
Additionally, our method for estimating substitution rates for 4D sites is not directly comparable to the
approach we used to estimate exonic substitution rates. Exonic substitution rates were estimated
independently for each exon. However, any given exon contained only a few 4D sites, so we analyzed a
concatenated dataset of all 4D sites for each clade. Thus, our finding of lower substitution rates for 4D
sites versus all exonic sites may be the product of both biology and methodology. Regardless, in both
datasets, we observe the same pattern where gars have the lowest estimated rate of molecular
evolution. Low molecular evolutionary rates observed in exons and 4D sites in gars coupled with
previous studies of transposable elements (Braasch et al. 2016) and gene order rearrangements
(Thompson et al. 2021) demonstrate that low evolutionary rates are found throughout the genome in
these ancient ray-finned fishes.
The oldest hybridizing divergences in eukaryotes.
An indication that slow molecular evolutionary rates are associated with low species diversity in
living fossil lineages is the generation of hybrids of species with ancient divergence times. These include
artificial crosses of Atractosteus spatula with Lepisosteus osseus and Paddlefish Polyodon spathula with
the Russian Sturgeon Acipenser gueldenstaedtii (Herrington et al. 2008; Káldy et al. 2020). Relaxed clock
analyses estimate 105 Mya as the divergence time between Atractosteus and Lepisosteus (Fig. 1a;
Brownstein et al., 2023). Despite this ancient divergence time, Alligator Gar are reported to hybridize
with Longnose Gar and Spotted Gar (L. oculatus) in the Brazos, Trinity, and Red river systems, Choke
Canyon Reservoir (Frio River), and Aransas Bay in Texas and Oklahoma, USA (Bohn et al. 2017; Taylor et
al. 2019).
To understand the patterns of hybridization across Mesozoic divergences in gars, we assembled
a dataset of single nucleotide polymorphisms (SNPs) using double digest restriction-site associated DNA
sequencing. Principal component analysis of our 1,223 SNP dataset shows that suspected Atractosteus
spatula X Lepisosteus osseus specimens form a tight cluster midway along the first principal component
axis that distinguishes the genotypes of individuals from the parental species (Fig. 4c, Fig. S8-11). Hybrid
gars show markedly high heterozygosity relative to individuals of the parental species (Fig. 4c). Hybrid
classification analyses confidently identify these individuals as naturally occurring F1 hybrids between
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sympatric populations of A. spatula and L. osseus (Fig. 4c-f; Figure S15). We also identified a backcross
between hybrids and A. spatula, confirming that some F1 hybrids are fertile (Fig. 4d-f).
Geometric morphometric analysis of both cranial and mandibular shape found that hybrids
consistently cluster as intermediates between Alligator Gar and Longnose Gar (Fig. 4b; Fig. S12-14).
Thus, patterns of morphological intermediacy found in hybrids of lineages that share much younger
common ancestry (Zou et al. 2007) are recapitulated in the aspects of gar anatomy that vary among
Alligator Gar and Longnose Gar (Grande 2010). As hybridization is an important driver of phenotypic
innovation (Arnold and Hodges 1995; Seehausen 2004), the intermediacy of Alligator Gar and Longnose
Gar hybrids (Fig. 4b) and the overlap between the morphology of these hybrids and other gar species
spanning 100 million years of time (Fig. 5a; Brownstein et al., 2023) illustrates a potential barrier to the
production of novel phenotypes in this clade.
The probability of viable hybridization decreases exponentially with divergence in animals
(Edmands 2002; Coyne and Orr 2004; Bolnick and Near 2005; Matute et al. 2010) and extensive
hybridization in the wild between species with such an ancient time to common ancestry is currently
unknown for any other vertebrate lineage. Our results demonstrate the ages of divergences able to
hybridize decline precipitously with increasing molecular evolutionary rate, suggesting the ‘snowball
accumulation’ model of genetic incompatibilities over time holds across vertebrates (Fig. 5b). Previous
to this study, the oldest-diverging lineages known to hybridize in the wild are species of Cystopteris and
Gymnocarpium ferns, which last share common ancestry approximately 58 million years ago (Rothfels et
al. 2015). In turn, the existence of hybrids and backcrosses establish Atractosteus and Lepisosteus as the
most deeply divergent naturally hybridizing eukaryotes (Figure S16).
Deep-time introgression and species boundaries in gars.
Natural hybridization between Atractosteus and Lepisosteus is associated with stagnant
nucleotide substitution rates (Fig. 2b,c), which may explain low rates of lineage diversification (Fig. 1a)
and minimal phenotypic change over more than 100 million years in gars (Grande 2010; Rabosky et al.
2013; Clarke et al. 2016). One possibility is that consistent episodes of hybridization among sympatric
and divergent lineages of gars have limited genetic and phenotypic divergence in this clade. Given that
Atractosteus and Lepisosteus have lived in sympatry in North America since the Paleocene (Grande
2010), we tested whether gar genomes show signatures of historical introgression. First, we assessed
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the presence of incomplete lineage sorting among extant gar species by examining discordance among
gene trees representing the 770 variable exons sampled for this clade. We observed limited incomplete
lineage sorting within Lepisosteus, but no patterns of discordance between species of Atractosteus and
Lepisosteus suggestive of introgression between these clades (Fig. 5c; Figure S17). Second, we
conducted phylogenetic network analyses (Wen et al. 2018) to assess the presence of reticulate
evolution in gars. These resolved no clear episodes of reticulate evolution between lineages of
Atractosteus and Lepisosteus but resulted in spurious scenarios that suggested introgression across
Holostei and Teleostei. Third, we reconstructed a mitochondrial gene tree of n=199 extant gars, which
unambiguously resolved Atractosteus and Lepisosteus as separate lineages (Fig. S18). Although they
have coexisted for more than 55 million years in North America (Grande 2010), hybridization has not
promoted widespread gene flow between species of Atractosteus and Lepisosteus (Fig. 5d-e).
Despite their capacity to hybridize, there are no signals of ancient introgression in gars. Instead,
barriers to gene flow among species of Lepisosteus and Atractosteus may result from behavioral and life
history differences among these clades (Echelle and Grande 2014). To investigate, we analyzed whether
the A. spatula X L. osseus hybrids resolve exclusively within one of the parental lineages in a
mitochondrial gene tree. All hybrids sampled possess A. spatula mitochondrial DNA (Fig. 5f), indicating
that A. spatula is the maternal parent for all of the wild, genotyped hybrids. This suggests asymmetry in
hybrid viability in gars may be related to the sex of the parental species (Turelli and Moyle 2007).
Although differences in relative rates of nuclear and mitochondrial evolution in L. osseus and A. spatula
(Bolnick et al. 2008; Moran et al. 2021) could result in the observed asymmetry, there is no detectable
difference in the genomic substitution rates of these species (Fig. 3a-c) or the branch lengths subtending
specimens of these species in the mitochondrial DNA gene tree (Figure 5f). The asymmetry is not
attributable to a higher degree of infertility in the species with sex-specific chromosomes (Turelli 1998),
as gars and their living sister species Amia calva lack a heterogametic sex (Thompson et al. 2021). There
is no indication of genomic incompatibilities associated with the sex of the parental species in
hybridizing gars because artificial crosses of male A. spatula and female L. osseus have produced viable
offspring (Herrington et al. 2008). Instead, gar hybrid asymmetry could be attributable to the
significantly higher fecundity of A. spatula relative to Lepisosteus (Smith et al. 2020) or forced sharing of
spawning habitat during years without floodplain inundation in river systems inhabited by both lineages.
These observations illustrate that the divergence of Atractosteus and Lepisosteus has been maintained
by subtle ecological and behavioral factors and hint at explanations for the higher number of F1 than F2
hybrids or backcrosses from regions where Atractosteus and Lepisosteus exist in sympatry.
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Genomic mechanisms of evolutionary stasis.
What is responsible for the exceptionally low genomic substitution rates and linked patterns of
incomplete reproductive isolation, low species diversity, and stagnant phenotypic evolution in gars? Our
results, which show that comparably slow rates of molecular evolution are present at both selectively
constrained and neutral sites in the exons of these fishes (Fig. 2b,c, Fig. 3), imply a mechanism of
evolutionary stasis detached from extrinsic causes such as the absence of ecological competition
(Darwin 1859; Stanley 1975). Nor are these extraordinarily slow evolutionary rates attributable to
continuous gene flow among sympatric populations of gars with incomplete reproductive isolation.
Instead, the evidence favors molecular processes underlying stasis, perhaps associated with DNA repair
mechanisms. Recent work suggests sturgeons possess highly effective DNA repair mechanisms (Gazo et
al. 2021). This may be attributable to differential activity of genes such as xpc (Gazo et al. 2021), which
forms part of the single nucleotide repair mechanism throughout vertebrates (Puumalainen et al. 2016;
Kusakabe et al. 2019). We speculate that DNA repair mechanisms might work to promote low rates of
nucleotide substitution across the genome in gars and sturgeons, though future work will be needed to
thoroughly demonstrate this.
Exceptionally slow evolutionary rates provide a mechanism for evolutionary stasis.
The presence of intrinsic features responsible for prolonged evolutionary stasis and the
existence of living fossils are both contentious (Schopf 1984; Eldredge et al. 2005; Casane and Laurenti
2013; Lidgard and Love 2018). One primary critique is the lack of an explanation for the coupling of low
rates of lineage diversification and phenotypic change in clades thought to exhibit stasis. Indeed, our
analyses confirm that classic living fossil lineages, such as coelacanths and rhynchocephalians, have
rates of molecular evolution similar to most other vertebrate clades (Chalopin et al. 2014; Gemmell et
al. 2020) (Fig. 3a, Fig. S2), matching their higher ancient phenotypic disparity (Friedman and Coates
2006; Herrera-Flores et al. 2017) and contrasting with previous estimates of genomic evolutionary rates
in these clades based on fewer sampled species and loci.
Our results validate the theoretical prediction that exceptionally slow genomic substitution rates
in gars and acipenseriforms act as a mechanism for incomplete reproductive isolation, allowing deeply
divergent lineages to produce viable and fertile hybrids. This fits the null hypothesis of the Dobzhansky-
Muller model of speciation (Coyne and Orr 2004): low mutation rates are associated with incomplete
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reproductive isolation in gars over more than 100 million years. We do not find extremely slow genomic
rates in other vertebrates previously characterized as living fossils, such as coelacanths, lungfishes, and
the Tuatara (Fig. 2a), suggesting that extrinsic factors like stable ecologies or isolation on islands might
play a role in the persistence of these old lineages. Nonetheless, our results show that continuous gene
flow across deeply divergent species, low species diversity, and low morphological disparity are paired
with slow rates of genomic evolution in several major ancient fish groups. With the identification of the
relationship between extraordinarily low rates of genomic change and evolutionary stasis in these
ancient living fossil fishes, work can begin on assessing if mechanisms of DNA damage repair and
nucleotide mismatch editing underlie the dramatic stasis in these lineages that extends over hundreds
of millions of years in evolutionary time.
Conclusions.
Evolutionary stasis, a phenomenon in which a lineage generates little phenotypic or species
diversity over time, may explain why some branches on the Tree of Life are much less species-rich and
morphologically disparate than others. However, whether molecular rates of evolution are slower in
living fossil lineages has not yet been confidently established. Here, using a sample of 1,105 exons, we
show that several classic living fossil lineages, among them gars (Lepisosteidae) and sturgeons and
paddlefishes (Acipenseriformes), possess exceptionally low genomic substitution rates. By analyzing
SNP, mitochondrial, and geometric morphometric data, we confirm that gar genera last sharing common
ancestry in the Early Cretaceous naturally hybridize. Incredibly, some hybrids appear to be fertile,
implying that barriers to gene flow have failed to manifest in gars despite a time to common ancestry
exceeding 100 million years. Together, these data suggest that slow rates of morphological evolution
and speciation are paired with low rates of molecular substitution, which may facilitate hybridization
over deep divergences by reducing the accumulation of genetic incompatibilities.
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Data availability: All generated data is in the manuscript, Supplementary materials, or the Dryad
Repository associated with this paper: https://doi.org/10.5061/dryad.15dv41p2q
Author Contributions: C.D.B. collected the data, conducted the analyses, and wrote and edited the
paper with input from the other authors. D.J.M. and T.J.N. collected the data, conducted the analyses,
and edited drafts of the paper. D.M.K. conducted analyses. L.Y. collected the data and conducted
analyses. O.O., S.R.D., and B.K. collected data.
Funding: C.D.B. was supported by the Society of Systematic Biologists miniARTs Award and the Yale
Peabody Museum Internship Program. L.Y. was supported by the Strategic Priority Research Program of
Chinese Academy of Sciences (Grant No. XDB31000000), the National Natural Science Foundation of
China (32170480, 31972866), Chinese Academy of Sciences Youth Innovation Promotion Association,
Chinese Academy of Sciences (http://www.yicas.cn), the Young Top-notch Talent Cultivation Program of
Hubei Province, and the Wuhan Branch, Supercomputing Center, Chinese Academy of Sciences, China.
T.J.N. was supported by the Bingham Oceanographic Fund of the Yale Peabody Museum.
Conflict of interest: The authors declare no competing interests.
Acknowledgements: We thank Andrew Bentley for providing high-quality photographs of delicate gar
skulls for use in the geometric morphometric analyses and access to the collections of the University of
Kansas Natural History museum, The University of Florida and Matt Thomas for providing high-quality
photographs of extant species, Lance Grande for providing maps of ancient lakes and photographs of
Green River gar specimens, members of the Near, Muñoz, and Donoghue labs of Yale E&EB for
discussions and feedback, and Greg Watkins-Colwell for collections access. Dan Daughtery (Texas Parks
& Wildlife) provided gar samples from Texas and facilitated gar sample acquisitions. C.D.B. thanks
Spencer Lott for help with a coding issue regarding taxon subsampling. Crocodylian, sturgeon, turtle,
shark, polypterid, amphibian, marsupial, and teleost silhouettes used throughout the paper are public
domain from phylopic.org.
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Table 1. Results of tests using reduced sampling.
Figure 1. Phylogenetic relationships, geographic distribution, and morphological stasis of gars. (A) Tip-
dated phylogenetic tree of gars based on three subsets of the 90 largest exons and with positions of
fossils fixed based on morphological phylogenies. The red circle marks the timing of divergence between
Atractosteus and Lepisosteus at approximately 105 million years ago. Phylogeny from Brownstein et al.
(2023). (B) Morphological stasis in gars is exemplified by nearly identical species pairs separated by over
50 million years. Photographs of gar fossils are by Lance Grande, and photographs of living gars are by
Zachary Miller, both used with permission.
Figure 2. Genomic substitution rates across vertebrates reveal the slow tempo of gar molecular
evolution. (A) Combined, annotated time-calibrated phylogeny of all 478 vertebrate species included in
the exon rates estimation analysis. For the purposes of combining the subtrees used into a single figure,
divergence dates between independently analyzed subclades are taken from Timetree.org. (B) Violin
and box plot showing distributions of estimated exon log substitution rates in different vertebrate clades
relative to the mean rate in gar (black line). (C) Estimated substitution rates at fourfold degenerate sites
in different clades of vertebrates. Silhouettes are public domain from Phylopic.org.
Figure 3. Patterns of genomic evolution in living fossils. (A) Estimated substitution rates at exons in
different clades of vertebrates considered to be living fossils, showing that lepisosteids and
acipenseriformes have slower rates of molecular evolution than other living fossil vertebrates. Plots of
extracted branch exon substitution rates against percent sequence divergence from their sister species
in our input trees for (B) Longnose Gar (sister species is Shortnose Gar L. platostomus), (C) Alligator Gar
(sister species is Cuban Gar A. tristoechus), (D) Sterlet (sister species is Chinese Sturgeon A. sinensis), and
(E) Chinese Sturgeon (sister species is Sterlet A. ruthenus). Note that there is no sequence divergence
from sister taxa for a large proportion of sampled exons in individual species of gars and sturgeons.
Silhouettes are public domain from Phylopic.org.
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Figure 4. Verification of naturally occurring Atractosteus x Lepisosteus hybrids. (A) Distribution of
extant gars (red=Atractosteus spp., blue=Lepisosteus spp.), showing the wide overlap in the ranges of
species of both extant genera. (B) Phenotypic intermediacy in the skull of putative hybrids. (C) First
principal component scores versus estimated genomic heterozygosity. (D) HIest joint estimates of
ancestry index and interspecific heterozygosity inferred. (E) LEA population structure results for the
optimal K=3, with individuals (columns) colored by estimated ancestry coefficients. (F) SNAPCLUST
assignment probabilities for parental, F1, and backcross classes. No individuals were strongly assigned to
either backcross class. (C) and (E) used 2,097 biallelic ddRAD SNPs, (D) and (F) used 1,223 biallelic
diagnostic SNPs differentially fixed between A. spatula and L. osseus. All datasets have <10% missing
genotypes per SNP.
Figure 5. Phenotypic intermediacy of gar hybrids and mechanisms of genomic stasis in gars. (A)
Grouped boxplot showing that hybrid gars do not expand the variance in cranial measurement ratios
observed in extant and extinct gar species. (B) Ages of the oldest hybridizing divergence in selected
vertebrate lineages plotted against the mean exon rates estimated for their respective crown clades,
showing a pattern of exponential decay consistent with the tempo of hybrid incompatibility predicted by
theory. (C) Plot of Bayesian-inferred gene trees of gar interrelationships using DensiTree, showing the
absence of incomplete lineage sorting across Atractosteus-Lepisosteus. Reconstructed introgression
events in the gar tree found in PhyloNet using (D) one and (E) six reticulations. 2 and 6 reticulations have
the highest associated log-likelihood scores yet produce unlikely scenarios. (F) Mitochondrial DNA gene
tree inferred from sequences of the cytochrome b gene using MrBayes 3.2. Numbers at nodes report
Bayesian posterior support and individuals identified as hybrids of Atractosteus spatula and Lepisosteus
osseus are marked with a purple circle. Photographs of living gars are by Zachary Miller and used with
permission, and photographs of the sturgeon and turtle are public domain.
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Table 1
Clade Name
Species Count
Mean Rate
Log10 Mean
Ln Mean
Standard Dev.
Gars
7
8.71E-05
-4.06
-9.35
4.97E-05
Acipenseriforms
3
1.90E-04
-3.72
-8.57
1.90E-04
Polypterids
3
9.03E-04
-3.04
-7.01
9.91E-04
Turtles
22
3.34E-03
-2.48
-5.7
1.37E-02
Crocodylians
4
3.90E-03
-2.41
-5.55
1.50E-02
Sharks
5
5.75E-03
-2.24
-5.16
1.81E-02
Amphibians
5
7.13E-03
-2.15
-4.94
1.69E-02
Teleosts1
7
7.24E-03
-2.14
-4.93
1.78E-02
marsupial
4
7.47E-03
-2.13
-4.9
1.67E-02
Lepidosaurs1
7
7.68E-03
-2.11
-4.87
1.81E-02
Lepidosaurs
36
1.50E-02
-1.82
-4.2
3.80E-02
Placentals
42
2.02E-02
-1.69
-3.9
4.85E-02
Birds
60
2.11E-02
-1.68
-3.86
4.72E-02
Teleosts
292
6.34E-02
-1.2
-2.76
4.58E-02
1Subsampling analysis.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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