Phylogenomics of Tetraopes longhorn beetles unravels their evolutionary history and biogeographic origins

Abstract

Tetraopes longhorn beetles are known for their resistance to milkweed plant toxins and their coevolutionary dynamics with milkweed plants (Asclepias). This association is considered a textbook example of coevolution, in which each species of Tetraopes is specialized to feed on one or a few species of Asclepias. A major challenge to investigating coevolutionary hypotheses and conducting molecular ecology studies lies in the limited understanding of the evolutionary history and biogeographical patterns of Tetraopes. By integrating genomic, morphological, paleontological, and geographical data, we present a robust phylogeny of Tetraopes and their relatives, using three inference methods with varying subsets of data, encompassing 2–12 thousand UCE loci. We elucidate the diversification patterns of Tetraopes species across major biogeographical regions and their colonization of the American continent. Our findings suggest that the genus originated in Central America approximately 21 million years ago during the Miocene and diversified from the Mid-Miocene to the Pleistocene. These events coincided with intense geological activity in Central America. Additionally, independent colonization events in North America occurred from the Late Miocene to the early Pleistocene, potentially contributing to the early diversification of the group. Our data suggest that a common ancestor of Tetraopini migrated into North America, likely facilitated by North Atlantic land bridges, while closely related tribes diverged in Asia and Europe during the Paleocene. Establishing a robust and densely sampled phylogeny of Tetraopes beetles provides a foundation for investigating micro- and macroevolutionary phenomena, including clinal variation, coevolution, and detoxification mechanisms in this ecologically important group.

Full text

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Phylogenomics of Tetraopes
longhorn beetles unravels
their evolutionary history
and biogeographic origins
Nayeli Gutiérrez‑Trejo
1,2*, Matthew H. Van Dam
3,4, Athena W. Lam
4,
Gonzalo Martínez‑Herrera
5, Felipe A. Noguera
6, Thomas Weissling
7, Jessica L. Ware
1,
Víctor H. Toledo‑Hernández
8, Frederick W. Skillman Jr.
9, Brian D. Farrell
10,
Oscar Pérez‑Flores
11, Lorenzo Prendini
1 & James M. Carpenter
1
Tetraopes longhorn beetles are known for their resistance to milkweed plant toxins and their
coevolutionary dynamics with milkweed plants (Asclepias). This association is considered a
textbook example of coevolution, in which each species of Tetraopes is specialized to feed on one
or a few species of Asclepias. A major challenge to investigating coevolutionary hypotheses and
conducting molecular ecology studies lies in the limited understanding of the evolutionary history
and biogeographical patterns of Tetraopes. By integrating genomic, morphological, paleontological,
and geographical data, we present a robust phylogeny of Tetraopes and their relatives, using three
inference methods with varying subsets of data, encompassing 2–12 thousand UCE loci. We elucidate
the diversication patterns of Tetraopes species across major biogeographical regions and their
colonization of the American continent. Our ndings suggest that the genus originated in Central
America approximately 21 million years ago during the Miocene and diversied from the Mid‑Miocene
to the Pleistocene. These events coincided with intense geological activity in Central America.
Additionally, independent colonization events in North America occurred from the Late Miocene
to the early Pleistocene, potentially contributing to the early diversication of the group. Our data
suggest that a common ancestor of Tetraopini migrated into North America, likely facilitated by North
Atlantic land bridges, while closely related tribes diverged in Asia and Europe during the Paleocene.
Establishing a robust and densely sampled phylogeny of Tetraopes beetles provides a foundation for
investigating micro‑ and macroevolutionary phenomena, including clinal variation, coevolution, and
detoxication mechanisms in this ecologically important group.
Beetles are the largest group of animals on earth, with more than 385,000 species described1. ey have evolved
an astonishing heterogeneity of trophic niches, behavior, and morphological diversity. Many beetles have devel-
oped very specialized life histories contributing to this diversity. For example, the Phytophaga beetle lineage is
hyperdiverse and has members who oen specialize on a single plant lineage2,3. One such example is the genus
Tetraopes Dalman in Schönherr, 1817, a lineage of 26 species distributed in North and Central America (Fig.1).
OPEN
1Division of Invertebrate Zoology, American Museum of Natural History, New York City, NY, USA. 2Richard Gilder
Graduate School, American Museum of Natural History, New York City, NY, USA. 3Entomology Department,
Institute for Biodiversity Science and Sustainability, California Academy of Sciences, San Francisco, CA,
USA. 4Center for Comparative Genomics, Institute for Biodiversity Science and Sustainability, California Academy
of Sciences, San Francisco, CA, USA. 5The Graduate Center of the City University of New York, New York City,
NY, USA. 6Estación de Biología Chamela, Instituto de Biología, Universidad Nacional Autónoma de México, San
Patricio, JAL, México. 7Department of Entomology, University of Nebraska-Lincoln, Lincoln, NE, USA. 8Centro
de Investigación en Biodiversidad y Conservación, Universidad Autónoma del Estado de Morelos, Cuernavaca,
MOR, México. 9P. O. Box 375, Pearce, AZ, USA. 10Museum of Comparative Zoology, Department of Organismic
and Evolutionary Biology, Harvard University, Cambridge, MA, USA. 11Laboratorio Nacional de Análisis y Síntesis
Ecológica, Escuela Nacional de Estudios Superiores, Universidad Nacional Autónoma de México, Morelia, MICH,
Mexico. *email: ngutierrez@amnh.org
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is group is known as milkweed longhorn beetles because of their highly specialized feeding habits on plants
of the genus Asclepias L. and other species of Apocynaceae Juss. plants4.
Studies on Tetraopes have thus far focused on addressing the gene mutations conferring resistance to milk-
weed’s toxins in the most common of its 26 species (T. tetrophthalmus Forster, 1771; throughout the document,
‘T.’ will refer to the genus Tetraopes) or assessing the coevolution of a subset of mainly North American spe-
cies with its host plants5–11. e evolutionary history of Tetraopes remains poorly understood. One crucial but
oen overlooked component of coevolution is concordant biogeographical histories. Dierent biogeographical
hypotheses have been proposed for the origin of the genus. Based on the distribution and species richness of
Asclepias and Tetraopes, Linsley hypothesized that Tetraopes has southern anities and is a descendant of the
Sonoran fauna12. Later, Chemsak proposed the Alleghenian region as the center of origin of Tetraopes. Chemsak
considered it more likely that Tetraopes derived from an Asian ancestor which colonized North America than
to have originated in South America and later migrated into North America4. Aer mapping the distribution of
Tetraopes, Farrell & Mitter (1998) proposed a tropical lowland origin for Tetraopes and subsequent colonization
of temperate upland territories. Finally, Farrell suggested the mid-Tertiary as the time for contemporaneous
diversication of both Asclepias and Tetraopes11. Nevertheless, the temporal and spatial changes of the genus
have yet to be thoroughly investigated in a macroevolutionary framework.
ere are several reasons to expect Central America (CA) to have played an important role in the diversi-
cation of Tetraopes. First, CA Tetraopes comprise 50% of the clade. Second, events of intense geological activity
coincide with previously proposed times of origin of Tetraopes during the Neogene11. ird, the geological
history of this region triggered the diversication of entire biomes and clades13,14. We hypothesize that adding
Central American lineages to the analysis will help resolve deep and shallow nodes and discern biogeographical
patterns within the genus.
We employ state-of-the-art methods to address the evolutionary history of Tetraopes beetles and their closest
relatives by integrating genomic, morphological, paleontological, and geographical data. We emphasize sam-
pling of CA species, where most of the species of the genus are distributed. us, our goals are: (1) to generate
a phylogeny of Tetraopes and evaluate node stability through a sensitivity analysis; (2) employ paleontological
evidence to date the divergence of Tetraopes and closely related lineages; and (3) address the biogeographical
history at a local scale including Tetraopes species divergences, as well as in a broader scale by studying species
from closely related tribes to understand patterns in deep time.
Figure1. Adult Tetraopes femoratus (A), T. cleroides (B), T. melanurus (C), and T. discoideus (D). All
photos from inaturalist. Photographers: omas Schultz, Gil Torres, Christine Young, and Miranda Kersten,
respectively.
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Results
Genome assembly and UCE capture
e assembly of T. tetrophthalmus comprised 771 Mbp distributed across 61,094 scaolds with an N50 of
30,733bp. BUSCO analyses found 88.1% complete genes, with 84.6% representing complete and single-copy
genes, 3.5% complete and duplicated, 6.6% fragmented, and 5.3% missing. e remaining genome assemblies
ranged in size from 36 to 715Mb. Gene completeness was very dissimilar among species, ranging from 3 to 88%.
GC content ranged from 30 to 55%. N50 values ranged from 669 to 7276 (Supplementary Table3).
e custom Lamiinae probe set selected 17,086 loci among the eight taxa included in the design, with an
average of 10,855 UCEs (ranging from 512 to 13,081) recovered from the 36 species. We conrmed previous
ndings on the decreasing numbers of loci captured in relation to increasing phylogenetic distance in insects,
using the Coleoptera probe set 1.1Kv115–17. To enhance capture success, a more comprehensive species sampling
of the focal taxon seems necessary. We found that specimen preservation method aected both UCE number and
length, with strategies such as obtaining extra sequences for dry pinned samples potentially improving outcomes,
albeit mainly in mean length quality.
Phylogenomics
We analyzed three UCE matrices varying in loci number aer ltering by completeness and by PIS (Table1): a
50% complete matrix (12,158 loci), 75% complete matrix (10,025 loci), and 90% complete matrix (2859 loci).
e nine topologies obtained from dierent completeness matrices and three phylogenetic inference methods,
recovered the same phylogeny, with three major monophyletic lineages: Astathini, Tetropini, and Tetraopini,
the latter with genus Phaea placed sister to Tetraopes. All analyses recovered Tetraopes as monophyletic with
high support (Fig.2, Table2). Furthermore, all topologies recovered Phaea mankinsi within Tetraopes, being the
basalmost species of the clade. T. ineditus Chemsak & Giesbert, 1986 and T. cleroides omson, 1860 formed
a clade sister to the rest of the species. T. discoideus LeConte, 1858, T. umbonatus LeConte, 1852, T. skillmani
Chemsak & Noguera, 2004, and T. batesi Chemsak, 1963 formed a clade, also recovered in the morphological
tree described in the next section. e next species to diverge are T. crinitus Chemsak & Noguera, 2004, T. linsleyi
Chemsak, 1963, and T. elegans Horn, 1894, followed by the clade comprising T. texanus Horn, 1878, T. melanurus
Schönherr, 1817, and T. quinquemaculatus Haldeman, 1847. In the sister clade, the rst groups to diverge were
T. thermophilus Chevrolat, 1861, T. tetrophthalmus, and T. mandibularis Chemsak, 1963, and the clade compris-
ing T. annulatus and T. pilosus. Next, T. subfasciatus Bates, 1881 diverged as the sister to the remaining species,
distributed in two main groups; T. varicornis Castelnau, 1840 and T. paracomes Chemsak, 1963; and T. basalis
LeConte, 1852, T. femoratus LeConte, 1847, and T. sublaevis.
e only conict in topologies was the position of T. crinitus which could either be sister to all other species
in the largest Tetraopes clade or part of the clade comprising T. discoideus, T. umbonatus, T. skillmani, and T.
batesi. e rst option was strongly supported by coalescent-based analysis with over 90% agreement from gene
trees in the three matrices, while the alternative topology was better supported by ML and BI analyses (Table2).
e ML analyses in RAxML resulted in topologies with 100% support for all clades in all three matrices.
Bayesian Inference in Exabayes received good convergence statistics with ASDSF values < 1%, ESS values > 200,
and PSRF values < 1.1. e three topologies from dierent completeness matrices were identical with a marginal
probability of 1 in all nodes. Multi-species coalescent phylogenies had an LPP of 1 in all nodes, except for an
internal Tetraopes node, and a group sister to the clade of T. discoideus, T. umbonatus, T. skillmani, and T. batesi.
e nal normalized quartet score indicated low discordance in the gene trees, ranging from 90.6 to 90.7, and
normalized quartet support (NQS) varied from 36–37 to 99% across completeness matrices.
Divergence‐time estimate analysis
e parsimony analysis resulted in a single tree with 126 steps. Tetrops rottensis and Tetrops praeustus formed a
clade sister to all Tetraopini species. e morphology-based phylogeny included two major Tetraopes clades: one
comprised T. discoideus, T. skillmani, T. umbonatus, and T. batesi, while the other comprised T. tetrophthalmus, T.
femoratus, T. annulatus, and T. pilosus Chemsak, 1963. e position of Phaea as the sister group of Tetraopes was
also conrmed. Convergence diagnostics conrmed that independent runs in MCMCTree reached convergence,
and a comparison of prior and posterior densities of node ages from the independent rate model revealed no
substantial truncation eects.
Ancestral range estimations
Ancestral range estimation in BioGeoBEARS at a global geographical scale recovered DEC + J as the best-t
model to explain the diversication of Astathini, Tetraopini, and Tetropini clades (LnL = −40.01, AICc = 86.03)
(Table3). Furthermore, the analysis focused on Tetraopes species recovered BAYAREALIKE + J model as the
most appropriate for the dataset (LnL = −110.15, AICc = 167.5) (Table4).
Table 1. Number of loci aer ltering by completeness and by parsimony informative sites (PIS).
Filtering criteria 50% 75% 90%
By completeness 13,137 11,700 3329
By PIS 12,158 10,025 2859
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e time-calibrated phylogeny estimated the origin of tribe Astathini at 33mya (HPD 21–38Ma), and its
ancestor was estimated to have occurred in Asia. e following lineage to diverge was the tribe Tetropini 24mya
(HPD 23–24Ma) in the late Oligocene, with Europe as the ancestral geographical range inferred with the highest
probability. Tribe Tetraopini, which comprises the genera Phaea and Tetraopes, was estimated to have diverged
in the early Oligocene, around 34mya (HPD 28–40Ma), and the most likely ancestral range of this clade is
Central America (Fig.3).
e crown group of Tetraopes was estimated to have originated in the last ~ 21Ma (HPD 17.1–24Ma) during
the late Oligocene and early Miocene. e Mexican Transition Zone and Mesoamerica (MTZ-M) were estimated
as the ancestral geographical range for the clade with the highest probability. Tetraopes mankinsi, T. ineditus, and
T. cleroides, which diverged 21mya (HPD 17.1–24), and 8mya (HPD 4.8–11.4), retained a MTZ-M distribution.
Subsequent northward dispersal by the remaining Tetraopes clades along with the crown group diversica-
tion during the Miocene and Pleistocene. e largest Tetraopes clade with 17 species diverged ~ 12mya (HPD
9.6–14.6) and is represented by a mixture of biogeographic lineages, which include areas such as the Mesoameri-
can, Alleghany, and the Arctic (Fig.4).
Figure2. Dated phylogeny of Tetraopes and relatives. Support values from Maximum Likelihood, Bayesian
Inference, and Coalescence are indicated on each node as Bootstrap (0–100)/MP (0–1)/NQS (0–100). Colors
indicate the geographical distribution of species in the map. Image source:19. e map was generated in R
(version 4.3.2; https:// www.r- proje ct. org), with ggplot2 (https:// ggplo t2. tidyv erse. org) using freely accessible
vector map 1:50m data (https:// www. natur alear thdata. com) without requiring any permission from external
sources.
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Discussion
Tetraopes comprises 26 species distributed from southern Canada, throughout the United States, as far south
as Costa Rica18,19. e taxonomic history of Tetraopes begins with its description by Dalman in an unpublished
work, which Schönherr later published in 181720. Guérin-Méneville (1844) designated Tetraopes tetrophthalmus21
as the type species of the genus4. Aer that, the most prolic period of new species discovery was the nineteenth
century, in which 58% of the 26 species currently known, were described. In the twentieth century, 27% of the
species were described whereas, in the twenty-rst century, 13% were described18. e rst revision of the genus
by Casey in 1913 included 28 species described at that time (not all valid today)22. e most comprehensive
morphological study of Tetraopes to date was published by Chemsak in 19634. Chemsak reviewed the taxonomy
and ecology of the 22 species recognized at the time, providing a key to the adults, along with geographical
distribution maps and detailed illustrations.
Table 2. Sensitivity analysis indicating clade support for each subset of data and inference method.
Clade
Maximum
likelihood Bayesian inference Coalescence
Bootstrap MP NQS
50% 75% 90% 50% 75% 90% 50% 75% 90%
Tetraopes (P. mankinsi + rest of Tetraopes spp.) 100 100 100 1 1 1 76.78 77.31 76.27
T. ineditus, T. cleroides 100 100 100 1 1 1 94.21 93.70 92.96
(T. crinitus (T. discoideus (T. u mbo nat us (T. batesi. T. skillmani) 100 100 100 1 1 1 NA NA NA
(T. discoideus (T. um bon atu s (T. batesi. T. skillmani)NA NA NA NA NA NA 97.21 96.93 96.82
(T. crinitus (T. linsleyi (T. elegans (T. texanus (T. melanurus. T.
quinquemaculatus)NA NA NA NA NA NA 47.26 47.54 46.40
(T. linsleyi (T. elegans (T. texanus (T. melanurus. T. quinquemacu-
latus)100 100 100 1 1 1 NA NA NA
(T. thermophilus (T. tetrophthalmus. T. mandibularis) 100 100 100 1 1 1 95.02 94.75 94.87
T. annulatus, T. pilosus 100 100 100 1 1 1 98.45 98.34 98.35
(T. subfaciatus (T. varicornis, T. paracomes) (T. basalis (T. femoratus,
T. sublaevis)100 100 100 1 1 1 61.26 60.42 59.96
Phaea (P. a. marthae (P. lauriae (P. parallela, P. quadrimaculata) 100 100 100 1 1 1 82.96 83.69 84.31
Astathini (B. fortunei (E. ava, T. simulator) (A. biplagiata. T.
dimidiatus))) 100 100 100 1 1 1 97.10 96.14 96.45
Tetropini (Tetrops starkii, Tetrops praeustrus) 100 100 100 1 1 1 89.21 89.75 89.66
Table 3. Results for the six biogeographical models tested in BioGeoBEARS at global geographical scale. Best-
t models are highlighted.
Model LnL Parameters d e j AICc AIC_wt
DEC −52.01 2 0.0110 4.4e−03 0.000 108.00 1.1e−05
DEC+J −40.01 3 0.0037 0.0e+00 0.027 86.03 6.7e−01
DIVALIKE −54.62 2 0.0150 5.0e−03 0.000 113.20 8.0e−07
DIVALIKE+J −41.09 3 0.0040 0.0e+00 0.029 88.18 2.3e−01
BAYAREALIKE −65.77 2 0.0130 2.7e−02 0.000 135.50 0.0e+00
BAYAREALIKE+J −41.92 3 0.0033 1.0e−07 0.032 89.84 1.0e−01
Table 4. Results for the six biogeographical models tested in BioGeoBEARS for Tetraopes only analysis. Best-
t models are highlighted.
Model LnL Parameters d e j AICc AIC_wt
DEC −77.32 2 0.0470 2.8e−02 0.000 158.6 0.0020
DEC+J −75.29 3 0.0340 1.1e−02 0.070 156.6 0.0055
DIVALIKE −78.34 2 0.0580 2.9e−02 0.000 160.7 0.0007
DIVALIKE+J −77.42 3 0.0460 1.8e−02 0.039 160.8 0.0007
BAYAREALIKE −80.19 2 0.0700 1.4e−01 0.000 164.4 0.0001
BAYAREALIKE+J −70.10 3 0.0094 1.0e−07 0.110 146.2 0.9900
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irty-ve years aer Chemsak’s revision, an attempt to analyze possible coevolutionary diversication in
Asclepias and Tetraopes generated the rst phylogeny of the genus based on allozyme data for 13 species10. A
parsimony analysis of the data supported the monophyly of the genus, although it had several weakly resolved
nodes. In a subsequent study, cytochrome oxidase I (COI) sequences of the species analyzed previously were
used to establish a molecular clock that estimated a genus age of 15 million years11. Both phylogenetic analyses of
Tetraopes included only 50% of the species of the genus and were based on only a limited number of characters.
Recent taxonomic changes in Tetraopes species include the transfer of T. mankinsi Chemsak & Linsley, 1979 to
the genus Phaea Newman, 1840, apparently because it did not match the diagnosis of Tetraopes, although few
details were provided23. e species was initially described as T. mankinsi in 1979 based on specimens from
Honduras and El Salvador24. Also, T. huetheri was described and subsequently synonymized with T. annulatus
LeConte, 184725,26.
Since previous molecular studies only sampled approximately 50% of the genus, comparing previous topolo-
gies to our more complete sampling eort is only partially instructive. Phylogenetic relationships of Tetraopes
based on fewer taxa and characters10,11 were only partially conrmed by our results. Previous topologies diered
substantially in the most recently divergent clade. Similarly to the previous hypotheses, our results recovered T.
discoideus and T. umb onatus, and T. texanus, T. melanurus, and T. quinquemaculatus as closely related species.
Figure3. BioGeoBEARS best-t model (DEC+J) for the tribes Astathini, and Tetropini, and Tetraopini. e
map was generated in R (version 4.3.2; https:// www.r- proje ct. org), with ggplot2 (https:// ggplo t2. tidyv erse.
org) using freely accessible vector map 1:50m data (https:// www. natur alear thdata. com) without requiring any
permission from external sources.
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Relationships between the T. annulatus + T. pilosus and T. tetrophthalmus + T. mandibularis as sister groups were
also conrmed by this work. Interestingly, previous phylogenetic hypotheses based on allozymes10, COI11, and our
results with over 12 thousand UCE loci obtained dierent combinations for one of the most recently divergent
clades, which includes T. sublaevis, T. femoratus, T. varicornis, and T. basalis. e major discrepancy concerned
the position of T. basalis, recovered as sister to T. sublaevis10, to T. varicornis, T. femoratus, and T. varicornis11,
and to T. sublaevis and T. femoratus (this work). is clade also includes T. paracomes in our analyses.
In addition to conicting topologies, the COI analyses yielded diering divergence time estimations com-
pared to this work. According to our results, the crown age of Tetraopes is older than estimated based on COI
(21 and 15mya, respectively)11. However, the ages of divergence of internal nodes are, in some cases, younger
than previously proposed11. is incongruence in age estimates is likely due to dierences in the datasets (300
loci alignment vs. mtDNA-only), taxon sampling (36 species vs. 13), and calibration strategies. More taxa and
data resolved the relationships and provided more precise divergence time estimations.
Regarding the taxonomic changes based on the phylogenetic analyses, Phaea mankinsi appears to belong to
Tetraopes. e stability of the node in which P. mankinsi was placed within Tetraopes was tested with a sensitiv-
ity analysis that explored how parameters aect phylogenetic hypotheses27. is node has maximum bootstrap
and marginal support from ML and Bayesian analyses and 76-77 of NQS from a coalescent-based study (Fig.2).
Also, the DNA of the species used in this work was obtained from the holotype. Here, we provide evidence to
transfer P. mankinsi to Tetraopes: Tetraopes mankinsi Chemsak & Linsley, 1979, new status.
With respect to tribe classication of Tetraopini, the tribe containing Tetraopes, the most recent phylogeny of
Lamiinae recovered Tetraopini, Tetropini, and Astathini as clades in analyses with Maximum Likelihood (ML)
and Bayesian inference with high support values28. e need to redene the classication of the three tribes has
Figure4. Map summarizing (A) the inferred colonization scenario for the tribes Astathini, and Tetropini, and
Tetraopini. North Atlantic land bridges are shown as the most likely dispersal route of the ancestor of Tetraopini.
Map was modied in GIMP (version 2.10; https:// www. gimp. org) from84, published under the Creative
Commons Attribution- NonCommercial 4.0 International License (CC BY-NC) open-access license without
requiring any permission from external sources.
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been recognized, as their morphology is rather uniform and they have been separated based mainly on their
geographical distributions (Astathini in Asia, Tetropini in Europe, and Tetraopini in the Americas)28. In this
work, we studied the type genera of the three tribes (Tetrophthalmus, Tetrops, and Tetraopes) and conrmed their
monophyly29. In addition, this represents the rst biogeographical analysis to shed light on how and when these
tribes diverged to archive their current distributions. Our results provide robust support for synonymizing two
of the tribes into the nominotypical tribe, which would be the subject of future study.
e body of knowledge about Tetraopes beetles has accumulated over two hundred years. is has included
knowledge about their taxonomy and distribution, and in particular, has focused on understanding its conspicu-
ous relationship with host plants of the genus Asclepias. However, a key piece to integrating all this information
had been missing, a comprehensive analysis of their phylogenetic relationships and biogeographical history.
Here, we present a phylogeny and biogeographical history of 23 species of the genus Tetraopes and their closely
related genera. On a broader geographical scale, ancestral range estimations of the tribes Astathini, Tetropini,
and Tetraopini suggest the occurrence of at least two founder-event speciations during the evolutionary his-
tory of the tribes (Fig.3). According to Matzke30, founder events imply long-distance colonization that founds
a population genetically isolated from the ancestral population. e two founder events in the history of the
three tribes are: (1) Tetropini species colonizing Europe from Asia during the Late Oligocene/Early Miocene; (2)
Tetraopini ancestor colonizing North America from Europe during the Late Eocene and Early Oligocene. Our
dating analyses estimated divergence times of the later colonization event (34Ma, HPD 28–40Ma) correspond
well with the onset of the Bering Land Bridges (BLB) and the North Atlantic land bridges (NALB)31.
Although our estimation of divergence times suggest that the ancestor of Tetraopini may have colonized
North America from Europe both via BLB and NALB, the most parsimonious biogeographical scenario con-
sidering the present-day occurrence of Tetrops and the fossil record is that the ancestor of Tetraopini dispersed
from Europe to North America via NALB. Only one of the 17 species in the genus is distributed in Asia, with
most of the diversity concentrated in South and Central Europe19,32. Also, the fossil species Tetrops rottensis was
described as part of the insects found in the Rott lagerstatten in Germany, which implies an ancient occurrence
of early-divergent lineages of Tetrops in Europe, supporting an early NALB migration route rather than the BLB33.
Our results suggest an origin of Tetraopes ~ 21mya during the Miocene (probably late Oligocene, considering
the wide 95 HPD of the divergence times of the node). e Mesoamerican and Mexican Transition Zone were
recognized as the ancestral ranges of the clade (Fig.4). Both regions experienced intense geological activity
during that period, with the formation of the mountain systems of Mexico and Central America from the mid-
Miocene to the Pliocene. As a result, new habitats and climatic conditions were generated and impacted the
diversication of species in the region, as reported for other taxa13,14. One of the biomes that could have been
produced by the formation of the Sierra Madre Occidental and the Neovolcanic belt is the dry forest, where
several Tetraopes species are distributed. us, rapid cladogenesis of Tetraopes species could have been triggered
by the establishment of modern biomes in western and central Mexico, resulting from the formation of mountain
systems in the region34–36.
Aer the origin of the major clades of Tetraopes in the Mesoamerican and Mexican Transition Zone, there
were at least two independent colonization events in the Western and Alleghany areas. One occurred in the late-
Miocene ~ 9mya in the lineage leading to the largest Tetraopes clade, currently including 16 species. is coloni-
zation coincides with a global decrease in temperature and humidity during the Late Miocene, which gave rise
to postglacial dispersal northward in some taxa37–39. e second colonization event to northern Mexico and the
southwestern US involved a lineage that gave rise to T. skillmani. is colonization constitutes one of only three
divergences during the Pleistocene (T. batesi and T. skillmani; T. annulatus and T. pilosus; T.quinquemaculatus and
T. melanurus). During this time, desert formation in North America started in the Miocene and continued into
the Pleistocene, as well as Holocene climatic shis, which connected and disconnected the eastern and western
deserts of North America40,41. erefore, the current distribution of Tetraopes species results from colonization
of its northern range from southern regions in the vicinity of the MTZ and the M areas.
e existence of a robust and densely sampled phylogeny of Tetraopes, along with an exploration of its diversi-
cation across major biogeographical regions and biomes, will signicantly enhance our understanding of evolu-
tionary processes, including coevolution and insect-plant interactions. Furthermore, it provides a framework for
comprehending micro- and macroevolutionary processes, such as clinal variation, speciation, and diversication.
Methods
Specimen collection
A total of 36 specimens were used in this study, including 23 species of Tetraopes and 13 species from the
Tetropini and Astathini tribes. Adult specimens were collected from Mexico and the United States. Samples
were preserved in liquid nitrogen or in 98% ethanol and stored at −70°C. Specimens from the Czech Republic,
Slovakia, Indonesia, the Philippines, and Japan were also included.
Specimens from entomological collections were assessed for molecular and morphological work. Specimens
were identied using taxonomic keys4,23, and the identity of non-American species was corroborated by experts
(Petr Švácha, Academy of Sciences of the Czech Republic; Karl Adlbauer, Austria; and Junsuke Yamasako Japa-
nese Institute for Plant Protection). For a complete list of specimens and collecting localities, see Supplementary
Table1.
DNA extraction and library preparation
DNA was extracted from body tissue and legs; legs were punctured to facilitate the action of proteinase-k. MagAt-
tract HMW DNA Kit was used to isolate high molecular weight genomic DNA from T. tetrophthalmus following
the manufacturer’s protocol for solid frozen tissue and recommendations from 10× Chromium DNA Extraction
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from Single Insects42. OmniPrep DNA Extraction kit, DNeasy Blood & Tissue Kit, and Qlamp mini kit were
used for DNA extractions of other specimens. DNA fragment size was quantied using Qbit 2.0 uorometric
quantication (Invitrogen, USA), a Bioanalyzer, and 0.5% agarose gel electrophoresis. In some cases, DNA was
sheared before library preparation in a Covaris M220 (Covaris Inc., USA).
Aer DNA extraction, samples were divided into two groups for library preparation and sequencing. e rst
included 30 specimens for which library preparation was performed at the Center for Comparative Genomics
of the California Academy of Sciences. Library preparation for these samples was conducted using NEBNext
Ultra II DNA Library Preparation kit (New England Biolabs Inc, USA) following the manufacturer’s protocol
(size selection protocol for fresh samples, and without size-selection for degraded DNA extracted from museum
samples) and later sequenced in Illumina Novaseq, 150bp paired-end reads. e second group included six
samples for which an external company performed library preparation. A 10× Genomics Chromium linked-read
library was prepared for T. tetrophthalmus, whereas standard Illumina libraries were generated for the other
samples. Libraries were sequenced on Illumina HiSequ, 2 × 250 base-pairs (bp), and generated linked reads for
T. tetrophthalmus and short paired-end Illumina reads for the other species (Supplementary Table2). Reads were
subject to quality control on Fastp 0.23.243 and removal of Illumina universal adapters.
Genome assembly
e genome of T. tetraophthalmus was assembled in Supernova 2.144 with default settings. Remaining genomes
were assembled using SPAdes 3.1545–47 with k-mer values of 21, 33, 55, 77, 99, and 127, as recommended for
read lengths of 150bp.
Ultraconserved Element (UCE) custom probe set design
An in-silico test of the Coleoptera UCE probe set version 1.1Kv1 was performed on six Tetraopes genomes with
the PHYLUCE tutorial III16,48. e probe set captured only 380–390 (~ 33%) of the 1172 UCEs in the Coleoptera
set. However, as 17 museum specimens were to be included in the sampling (some of them over 70years old),
there was the possibility of the number of UCEs captured to be even lower because fragmented DNA decreases
the performance of the probes17,49.
As UCE probe sets customized for a focal group have resulted in a larger number of recovered loci in other
insect groups15,50–52, we designed a customized set of probes for Lamiinae using the PHYLUCE 1.7.1 pipeline48.
Eight species were included in the design (7 Lamiinae and one Cerambycinae), including T. tetrophthalmus
(generated in this work), Anoplophora glabripennis [53, Agla_2.0], Doliops geometrica Waterhouse, 1842 (Van
Dam, unpublished data), Aprophata a. notha (Newman, 1842) (Van Dam, unpublished data), Achriotypa
basalis Pascoe, 1875 (NHI Accession No. SRR15249232), Similosodus venosus (Pascoe, 1867) (NHI Accession
No.SRR15249233), Rhytiphora diva (omson, 1860) (NHI Accession No.SRR15249221), and Turano clytus
namaganensis (Heyden, 1885) (NHI Accession No.SRR16700842). e species used as a base taxon was A.
glabripennis because it corresponds to the same subfamily as Tetraopes beetles. At the time of the study, it was
the most complete genome available for the group. e other species belong to four Lamiinae tribes spanning
the phylogenetic diversity of the subfamily Lamiinae (Apomecynini, Lamiini, Tetraopini, and Pteropliini)28. S o
masked les were used following guidelines of probe design50,54.
UCE matrix generation and partitioning
We used the PHYLUCE48 pipeline with default settings to extract probes from the assembled genomes. Aer
aligning with MAFFT55 and trimming the conserved locus matrices, we ltered for completeness by generating
concatenated matrices in which the loci retained at least 50, 75, and 90% of the taxa. We conducted additional
ltering on each completeness matrix by calculating the number of parsimony-informative sites (PIS) using a
script implemented in Phyloch56,57. Informed by an examination of the loci distribution and their associated
PIS, we established a threshold to retain loci with 50 to 250 PIS (50 < PIS < 250). Loci with lower and higher PIS
were considered low-informative and highly-informative outliers, respectively, oen associated with increased
phylogenetic noise for topological inference and saturation in studies across dierent taxa58–60.
Each matrix was subsequently input to the Sliding-Window Site Characteristics (SWSC) method that
accounts for UCE heterogeneity and increases the model t61. Following this step, for each matrix, we used
PartitionFinder262 to nd the best-t models for the subsets created by SWSC with the following settings: linked
branch lengths, GTR, GTR+G, GTR+I+G models, AICc as criteria for model selection, and rclusterf search.
Phylogenomics
Phylogenetic analyses were performed on each completeness matrix (50, 75, and 90%) with dierent phylogenetic
inference methods (Maximum Likelihood, Bayesian Inference, and coalescent-based analysis). For Maximum
Likelihood analysis, RAxML-NG v. 0.8.063 with default settings was used, conrming convergence with the
default cuto for large data sets and mapping bootstrap values onto the best ML tree.
Bayesian inference in ExaBayes 1.5.164 was performed by two independent Markov chain Monte Carlo
(MCMC) searches, each with one cold and one heated chain, with default parameters, a 25% burn-in, and link-
ing all partitions into a single branch length parameter. Non-partitioned data were used for these analyses. e
Average Standard Deviation of Split Frequency (ASDSF) was used to evaluate convergence, with ESS and PSRF
values also examined. A consensus tree was generated from sets of trees.
For the multi-species coalescent approach, gene trees were generated with IQ-TREE 2.0.365 ultrafast bootstrap
with 1000 replicates for ASTRAL 5.7.866 input. ASTRAL was run with default settings, and support values, local
posterior probabilities (LPP), and normalized quartet support (NQS) values were mapped in the tree.
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Divergence time estimation
e fossil Palaeoncoderes piacentinii (59.2Ma) was used as minimum bound as it is the most recent Lamiinae
fossil described67. Cretoprionus liutiaogouensis, the earliest known cerambycid beetle, served as the maximum
bound for the root (124Ma)68.
e fossil species Tetrops rottensis33,69 was used as a prior to time-calibrate the Tetropini clade. Its phylogenetic
position was unconrmed, so the species was rst included in a phylogenetic analysis to conrm that it would
be an appropriate calibration point70. A morphological matrix was generated, including Tetrops rottensis, Tetrops
praeustus Linné, 1758, eigth Tetraopes species, one Phaea species, and Anoplophora glabripennis as outgroup.
e morphological matrix included 50 characters and 12 taxa (Supplementary Table1 and Supplementary
List 2). Observations and measurements of the specimens were conducted using a Nikon SMZ 1500 microscope.
e ‘body length’ character was coded as continuous and standardized in TNT 1.6 with the nstates stand com-
mand. A parsimony analysis was conducted in TNT using new technology search strategies (10 random seeds,
nd minimum length 20 times, and trees collapsed aer search). Symmetric resampling and bootstrap support
were performed with a removal probability of (p) 1⁄4 33, 1000 pseudoreplicates, collapsing the nodes below P
1⁄4 5071–73.
Aer conrming the phylogenetic position of Tetrops rottensis, normal age distributions were generated in
MCMCtreeR74 (root: 59.2–124mya, Tetropini node: 24.2–23mya) to account for the uncertainty of the fossil
record. Input les for MCMCTree75 consisted of a MAFFT55 alignment of the 300 most PIS loci and the ASTRAL
phylogeny as the starting topology. e same data was employed to estimate the substitution rate in baseml75.
Using a relaxed-clock model with independent distributed rates, branch lengths, gradient, and Hessian were
estimated, followed by the estimation of divergence times76. Sampling included a burn-in of 50,000 generations
followed by 500,000 posterior samples every 50 generations. A replica with random seeds was also run. Conver-
gence diagnostics were calculated, including comparing the posterior means among the dierent runs to evaluate
whether they converged and generating several convergence statistics (posterior mean, ESS, posterior variance,
and standard error of posterior means)77. Finally, prior densities of node ages were compared to posterior densi-
ties aer sampling from the prior with a replica and convergence evaluation.
Ancestral range estimation
e R package BioGeoBEARS78 was employed for ancestral range estimation of the time-calibrated phylogeny,
which served as a xed topology. Presence-absence matrices were generated based on geographical records from
two Cerambycidae databases18,19. e analysis included an areas adjacent matrix indicating adjacent (1) and
nonadjacent (0) areas. Six models of evolution were evaluated (1) Dispersal-Extinction-Cladogenesis (DEC)79,
(2) DEC+founder-event speciation (“jump”; DEC+J), (3)Dispersal-Vicariance Analyses (DIVALIKE)80, (4)
DIVALIKE+J, (5) Bayesian inference of historical biogeography for discrete areas (BAYAREALIKE)81, and (6)
BAYAREALIKE+J. e Akaike Information Criterion (AICc)82 was used to determine the likelihood of the
dataset given each model.
Two geographical scales were used, global with all the species and the American scale focusing on Tetraopes
species. For the former, four biogeographical areas were dened, based on previous studies, as Asia (A), Europe
(E), North America (NA), and Central America (CA)83,84. e two species of Tetrops included in this dataset
occur in Europe. However, the genus also includes species from northern Asia. e second analysis focused
on Tetraopes species. Outgroups were excluded using the drop.tip function from the R package ape85. Six bio-
geographical areas were dened as Arctic (A), Western (W), Alleghany (AL), Mexican Transition Zone (MT),
Mesoamerican (M) based on previous studies86,87.
Data availability
Data and scripts relevant for this project are available at: htt ps:// github. com/ Nayel iGuti errez/ 2023_ Macro evolT
etrao pes. git. Genome assemblies and UCE custom probes are be available at: https:// datad ryad. org/ stash/ landi
ng/ show? id= doi% 3A10. 5061% 2Fdry ad. gmsbc c2vh.
Received: 29 October 2023; Accepted: 21 March 2024
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Acknowledgements
We gratefully acknowledge James E. Wappes, Rafael Cerón-Gómez, Fernanda Valverde, Josef Vlasak, Steven W.
Lingafelter, Norman E. Woodley, and David Molina for their support during eldwork. We thank Petr Švácha
for providing specimens of Astathini and Tetropini, as well as oering valuable feedback throughout the project’s
development; Robert Naczi and Cristopher O. Cervantes for providing specimens to include in this study. We
extend our thanks to Karl Adlbauer and Junsuke Yamasako for their expertise in corroborating the taxonomic
identication of Astathini species. We acknowledge Dean Bobo and the Bioinformatics core, as well as the sta
of the Sackler Institute for Comparative Genomics at the AMNH, for their assistance with lab and bioinfor-
matics work. We thank Miles Zhang for his advice and suggestions during the initial phases of the UCE work.
Special thanks go to Bruno Melo and Edson Abreu for their advice on the UCE and biogeographical sections.
We acknowledge the following curators and sta of public and private collections who facilitated the study of
specimens: Lee Herman and Corey Smith (American Museum of Natural History Coleoptera Collection), James
Wappes (American Museum of Coleoptera, James Wappes Private Collection, now part of the Florida State Col-
lection of Arthropods), Christopher Grinter (California Academy of Sciences Entomology Collection), Víctor
Toledo (Colección de Insectos, Universidad Autónoma del Estado de Morelos), Steve Lingafelter (Lingafelter
Private Collection), Crystal Maier (MCZ Museum of Comparative Zoology Coleoptera Collection), Frederick
Content courtesy of Springer Nature, terms of use apply. Rights reserved

13
Vol.:(0123456789)
Scientic Reports | (2024) 14:7285 | https://doi.org/10.1038/s41598-024-57827-z
www.nature.com/scientificreports/
W. Skillman, Jr. (Skillman Private Collection),Felipe A. Noguera (EBCC Colección Entomológica de la Estación
de Biología Chamela, UNAM), and Patrick Sullivan (Sullivan Private Collection). is project was funded by
the Richard Gilder Graduate School at the American Museum of Natural History (AMNH), the eodore
Roosevelt Memorial Fund (AMNH), the Sydney Anderson Travel award (AMNH), the Maxwell/Hanrahan Award
(AMNH), and the Ernst Mayr Grant (Museum of Comparative Zoology, Harvard University) awarded to N.G.T.
Author contributions
N.G.T. conceptualization, data generation, formal analysis, writing—original dra, and writing—review & edit-
ing. J.M.C., M.V.D., J.L.W., L.P., and F.A.N. contributed to conceptualization and provided feedback of previous
versions of manuscript. N.G.T., G.M.H. and V.H.T.H. did eldwork. M.V.D. designed UCE probes. N.G.T. and
A.W.L. generated the genomic libraries. M.V.D. and G.M.H. assisted bioinformatics analyses. J.L.W. assisted with
divergence dating analyses. T.W., F.W.S., B.D.F., and O.P.F. provided specimens used in the study. All authors
contributed to review and editing of the nal manuscript, and approved the nal version of the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 024- 57827-z.
Correspondence and requests for materials should be addressed to N.G.-T.
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