Research Article |
Corresponding author: Wendy Moore ( wmoore@arizona.edu ) Academic editor: Lyubomir Penev
© 2022 Raine M. Ikagawa, Wendy Moore.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Ikagawa RM, Moore W (2022) Molecular phylogeny and revision of species groups of Nearctic bombardier beetles (Carabidae, Brachininae, Brachinus ( Neobrachinus)). ZooKeys 1131: 155-171. https://doi.org/10.3897/zookeys.1131.85218
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Bombardier beetles of the genus Brachinus Weber are notorious for their explosive defensive chemistry. Despite ongoing research on their defense mechanism, life history, and ecology, the group lacks a robust molecular-based phylogeny. In this study, three loci from mitochondrial and nuclear genomes (COI, CAD, 28S) are used to reconstruct the phylogeny of the large subgenus Neobrachinus, and test species group boundaries hypothesized by
molecular phylogenetics, systematics
Bombardier beetles of the genus Brachinus Weber are famous for their explosive defensive chemistry; when provoked, they generate a 100 °C cloud of benzoquinones and aim the explosion towards their enemy (
Species of the Brachinus subgenus Neobrachinus Erwin have historically been described as difficult to identify. George
Dorsal habitus view of representatives from several species groups of the subgenus Neobrachinus Erwin A B. azureipennis Chaudoir B B. gebhardis Erwin C B. elongatulus Chaudoir D B. mexicanus Dejean E B. cibolensis Erwin F B. costipennis Motschulsky G B. hirsutus Bates H B. lateralis Dejean I B. favicollis Erwin. Scale bar: 1 cm.
The vast majority of species examined in
Erwin’s work transformed brachinine taxonomy and provided a dichotomous key for identifying brachinine genera and North and Central American Neobrachinus species. However, identification of Neobrachinus species remains challenging. This is largely due to highly conserved morphology; the maintenance of “the Brachinus habitus” seems to have been favored over the course of multiple speciation events (e.g., Fig.
Neobrachinus are abundant members of riparian arthropod communities in the southwestern US (
With morphologically challenging taxa, molecular sequence data are often used to determine species boundaries and relationships, and these studies also help to reveal cryptic diversity. This study aims to address the morphological challenges of Neobrachinus by using molecular sequence data to infer the phylogeny of the species and to test proposed species groups.
Challenges associated with Neobrachinus identification led us to limit our taxon sampling to expertly identified specimens in museum collections (Suppl. material
Efforts were made to sample several species from as many species groups as possible, especially within the large fumans group which we hypothesized may not be monophyletic. We also downloaded all available sequences of Neobrachinus species from the Barcode of Life Database (BOLD) and GenBank and tested their species identities against sequence data from expertly identified specimens.
Total genomic DNA was extracted from the right middle leg of specimens using the Qiagen DNeasy Blood & Tissue Kit (Valencia, CA) following the manufacturer suggested protocol. Extractions on older specimens were conducted in the Schlinger Ancient DNA Laboratory at the University of Arizona Insect Collection using the QIAamp DNA Micro Kit (Qiagen Inc., Valencia, CA) following the manufacturer suggested protocol. The concentration of total genomic DNA in extraction products was measured on a Qubit 3.0 Fluorometer (Thermo Fisher, USA). Samples with quantifiable DNA were used in subsequent PCRs.
The gene regions CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase) and COI (cytochrome c oxidase subunit I) have been shown to be phylogenetically informative in Neobrachinus by
Sequence data were also obtained for the D2-3 region of large subunit ribosomal gene (28S) from the total genomic DNA extracted for
PCR products were quantified, normalized, and sequenced in forward and reverse directions using Sanger sequencing at the University of Arizona Genetics Core (UAGC) using an Applied Biosystems 3730 DNA Analyzer (ThermoFisher Scientific). Chromatograms were assembled into contigs, and initial base calls were made using Phred (
Three single gene matrices (COI, CAD, and 28S) were assembled. Each matrix contained sequences generated specifically for this study as well as all homologous sequences of Neobrachinus publicly available on BOLD and GenBank (databases searched January 2021) (Suppl. material
IQ-Tree ModelFinder and ModelFinder Plus identified the following models of evolution for each character partition: COI codon 1 = K2P+I+G4; COI codon position 2, CAD codon 1, CAD codon 2 = 2K2P+I; COI codon 3 = TIM2+F+G4; CAD codon 3 = HKY+F; and 28S = GTR+F+I+G4.
The three-gene IQ-Tree analysis resulted in the phylogeny shown in Figs
The maximum-likelihood three-gene molecular phylogeny of Neobrachinus with clades collapsed. Clades are colored by species group. Clades in solid blue were formerly placed within the fumans species group. Nodes with bootstrap values > 0.90 are denoted with grey circles. Clades present in South America are denoted with “SA.”
In all analyses the fumans species group proposed by
Revised classification of Nearctic Brachinus. New species groups indicated with a triangle. Species groups and species not present in molecular phylogeny are indicated with an asterisk. Species present in South America are indicated with (SA). Incertae sedis taxa not considered in
aabaaba species group | fumans species group△ | incertae sedis |
B. aabaaba Erwin | B. fumans (Fabricius) | B. conformis Dejean* |
B. sonorous Erwin* | B. favicollis Erwin | B. cyanipennis Say |
alternans species group | B. imperialensis Erwin | B. gebhardis Erwin |
B. alternans Dejean | B. perplexus Dejean | B. kavanaughi Erwin |
B. rugipennis Chaudoir* | B. puberulus Chaudoir* | B. mexicanus Dejean |
B. viridipennis Dejean* | B. velutinus Erwin* | B. neglectus LeConte |
brunneus species group* | galactoderus species group△ | B. oaxacensis Erwin* |
B. brunneus Laporte* | B. galactoderus Erwin | B. ovipennis LeConte |
B. melanarthrus Chaudoir* | grandis species group* | B. patruelis LeConte* |
cinctipennis species group△ | B. grandis Brullé SA | B. quadripennis Dejean |
B. cinctipennis Chevrolat | hirsutus species group | B. tenuicollis LeConte |
B. cibolensis Erwin | B. hirsutus Bates | Brachinus sp. C SA |
cordicollis species group | B. pallidus Erwin | Brachinus sp. E SA |
B. cordicollis Dejean | kansanus species group* | B. atramentarius Mannerheim○SA* |
B. americanus (LeConte) | B. kansanus LeConte* | B. bilineatus Laporte○SA* |
B. alexiguus Erwin* | lateralis species group | B. bruchi Liebke○SA* |
B. capnicus Erwin* | B. lateralis Dejean | B. fulvipennis Chaudoir○SA* |
B. cyanochroaticus Erwin | B. adustipennis Erwin | B. fuscicornis Dejean○SA* |
B. fulminatus Erwin | B. aeger Chaudoir SA | B. genicularis Mannerheim○SA* |
B. ichabodopsis Erwin* | B. arboreus Chevrolat* SA | B. hylaenus Reichardt○SA* |
B. janthinipennis (Dejean) | B. bilineatus Castelnau* | B. immarginatus Brullé○SA* |
B. medius T.W. Harris | B. chalchihuitlicue Erwin* | B. intermedius Brullé○SA* |
B. microamericanus Erwin* | B. chirriador Erwin* | B. limbiger Chaudoir○SA* |
B. mobilis Erwin | phaeocerus species group△ | B. marginellus Dejean○SA* |
B. oxygonus Chaudoir* | B. phaeocerus Chaudoir | B. marginiventris Brullé○SA* |
B. sublaevis Chaudoir | B. azureipennis Chaudoir | B. niger Chaudoir○SA* |
B. vulcanoides Erwin* | B. consanguineus Chaudoir* | B. nigricans Chaudoir○SA* |
costipennis species group | B. imporcitis Erwin | B. nigripes G.R. Waterhouse○SA* |
B. costipennis Motschulsky | B. javalinopsis Erwin | B. olidus Reiche○SA* |
explosus species group* | texanus species group | B. pachygaster Perty○SA* |
B. explosus Erwin* | B. texanus Chaudoir* | B. pallipes Dejean○SA* |
B. elongatulus Chaudoir | B. vicinus Dejean○SA* | |
B. geniculatus DejeanSA | B. xanthophryus Chaudoir○SA* | |
sallei species group* | B. xanthopleurus Chaudoir○SA* | |
B. sallei Chaudoir* |
This study used molecular data to test previous hypotheses of species group membership and phylogenetic relationships in the subgenus Neobrachinus that were proposed based on morphological data (
The shape of the virga was not found to be phylogenetically informative as envisioned by
Erwin’s fumans group contained 26 morphologically diverse species and was defined by a troughed virga. Considering the molecular evidence that supports splitting the fumans group, the troughed virga could be an ancestral or convergent form among Neobrachinus.
Species subgroups of the fumans group were also polyphyletic in the molecular phylogeny, highlighting potential convergent character states of the male genitalia. Members of Erwin’s quadripennis subgroup of the fumans group were recovered throughout the Neobrachinus tree: in the phaeocerus species group (Fig.
Erwin’s fumans species group also contained seven monotypic species subgroups, of which four were included in this study. Two of these, B. cyanipennis and B. ovipennis, formed a clade and are now placed together in the cyanipennis species group (Fig.
Erwin postulated all 84 species of Neobrachinus evolved from a single most recent common ancestor that crossed the Bering Land Bridge. The molecular phylogeny supports a Nearctic origin of the Neobrachinus, as predicted by
Among the previously published sequences downloaded from public databases, molecular phylogenetic analysis revealed several cases where specimens were likely misidentified. Some specimens from North Dakota were identified as B. medius (BETN1837-18, BETN9260-20, BETN9117-20, BETN9121-20), however the sequences were in a well-supported clade, separate from B. medius from the same region (Fig.
Other potential misidentifications exist yet are difficult to confirm. For example, within the new fumans species group, there are several clades of the species B. fumans and B. perplexus (Fig.
Another example is the clade containing B. kavanaughi and B. mexicanus (Fig.
Bootstrap support for and against clades of Neobrachinus. Each column has maximum likelihood bootstrap values as percentages for or against each clade recovered in each dataset: the three-gene concatenated matrix (3G), and the single-gene datasets, 28S, CAD, and COI. Positive values indicate support while negative numbers indicate support for the contradictory clade with the highest support. Cells with bootstrap values ≥ 90 are in black, values between 75 and 89 in dark grey, and values between 50 and 74 in light grey. Cells in red have bootstrap values for the contradictory clade ≥ 50.
This research presents a molecular test of
Considering the challenges of morphological identification to the species level among Neobrachinus, molecular sequence data offer an accurate, alternative path to identification. Continued contribution of sequences from expertly identified specimens to libraries within databases such as BOLD and GenBank, will facilitate rapid, accessible, and accurate species identification. As sequencing technologies become cheaper and more readily available, acquiring sequence data for comparison in such databases is increasingly cost- and time-effective.
The present study elucidates the species group classification of more than half of the species of Neobrachinus detailed in
This systematic study of Neobrachinus emphasizes the importance of continued taxonomic and phylogenetic work to better understand their species boundaries, biogeography, and evolutionary history, and will enable future efforts to better understand these remarkable beetles.
It is our pleasure to dedicate this work to Dr. Terry L. Erwin [1940–2020], virtuoso coleopterist, biodiversity explorer, scholar, and gentleman. As a former colleague, mentor, and friend his enthusiasm for carabid systematics was contagious and his inspiration timeless. This work is in partial fulfilment of RI’s Master’s degree in the Graduate Interdisciplinary Program in Entomology and Insect Science (GIDP-EIS) at the University of Arizona (UA) and is a product of the Arizona Sky Island Arthropod Project based in W.M.’s laboratory. We thank those who helped collect specimens in the field including Davide Bergamaschi and Carlos Martinez. A special thanks to Jacob Simon, who contributed to collecting specimens in the field and molecular data acquisition. We also thank Jason Schaller for early advice on how to collect and identify Neobrachinus. We thank Daniel Shpeley (University of Alberta) and Evan Waite (Arizona State University) for loans of Brachinus specimens included in our molecular phylogeny. We thank Drs. David Kavanaugh and David Maddison for their insightful comments that improved the quality of the manuscript. Funding for this project came from the UA’s GIDP-EIS, as well as from T&E, Inc. and is gratefully acknowledged.
Supplementary data
Data type: occurences, morphological, phylogenetic
Explanation note: IQ-Tree maximum-likelihood phylogeny on concatenated dataset containing 282 specimens of Neobrachinus and outgroups with bootstrap values. IQ-Tree maximum-likelihood phylogeny on 28S dataset containing 54 specimens of Neobrachinus and outgroups with bootstrap values. IQ-Tree maximum-likelihood phylogeny on CAD dataset containing 70 specimens of Neobrachinus and outgroups with bootstrap values. IQ-Tree maximum-likelihood phylogeny on COI dataset containing 270 specimens of Neobrachinus and outgroups with bootstrap values. Voucher specimens. Voucher number, collection information, and GenBank or BOLD accession numbers are provided for each specimen.