Taxon sampling and vouchers

The two above-mentioned goals of this paper were realized by analyzing two separate molecular datasets (Appendix 1 and 2). This approach was selected in order to avoid potential biases caused by the unevenness of taxon sampling between Amphidorini and the remaining tribes in the analysis, which tested the monophyly of the tribe.

Species identifications were performed by MAJ and ADS using recent treatments (Aalbu et al. 2012, Triplehorn and Thomas 2012, 2015, Triplehorn et al. 2015, Johnston et al. 2015, Johnston 2019) in conjunction with examination of primary types representing over 350 nominal taxa within Amphidorini. Species concepts largely follow the recent treatments (cited above) and those of Johnston (2018).

Monophyly of Amphidorini. In order to test the constitution of the tribe, 21 selected representatives of all currently recognised genera were subjected to an analysis together with 23 outgroups from a wide spectrum of other tenebrionid tribes representing Blaptinae and Tenebrioninae (Appendix 1), Namely: two species of Blaptini Leach, 1815, two of Platynotini Mulsant & Rey, 1853, five of Opatrini Brullé, 1832 all representing Blaptinae; two of Amarygmini Gistel, 1848, one of Centronopini Doyen 1989, two of Cerenopini Horn 1870, two of Eulabini, Horn 1870, one of Melanimonini Seidlitz, 1894, two of Scotobiini Solier, 1838, and three of Tenebrionini Latreille, 1802 all classified within Tenebrioninae. The above-mentioned tribes were selected based on historical aspects (taxa treated as closely related by previous authors, e.g. Kamiński et al. 2021), general morphological similarity of some of the representatives, and similar distributional patterns. The focus on assessing Amphidorini monophyly limits the use of this dataset for examining broader relationships among Tenebrionidae as a whole and instead tests clustering within the Blaptinae versus with other groups of tenebrionidae. The tribe Melanimonini was used to root these analyses as it has been recovered amongst the earliest diverging members of Tenebrioninae (see Kergoat et al. 2014, Kanda 2017).

Relations among Amphidorini. To test the generic and species group relationships within Amphidorini, three outgroups (Notibius puberulus LeConte, 1851 and Blapstinus fortis LeConte, 1878 of Opatrini and Blaps mucronata Latreille, 1804 of Blaptini) were chosen based on the tribal phylogenetic results in order to root a tree including 159 Amphidorini OTUs (Appendix 2). All currently recognized genera were included in the OTU sampling, namely: one Eleodimorpha, seven Embaphion, three Lariversius, three Neobaphion, and two Trogloderus. A total of 143 OTUs were used for the large genus Eleodes which span all currently recognized subgenera: three of Amphidora Eschscholtz, 1829; two of Ardeleodes Blaisdell, 1937; five of Blapylis Horn, 1870; eight of Caverneleodes Triplehorn, 1975; one of Chaseleodes Thomas, 2015; one of Cratidus LeConte, 1862; two of Discogenia LeConte, 1866; 29 of Eleodes s.s.; one of Heteropromus Blaisdell, 1909; nine of Litheleodes Blaisdell, 1909; eight of Melaneleodes Blaisdell, 1909; four of Metablapylis Blaisdell, 1909; five of Omegeleodes Triplehorn and Thomas, 2012; 15 of Promus LeConte, 1862; seven of Pseudeleodes Blaisdell, 1909; 28 of Steneleodes Blaisdell, 1909; three of Tricheleodes Blaisdell, 1909; and 12 of incertae sedis species.

DNA extraction and sequencing for ethanol-preserved specimens

Molecular protocols for extraction and sequencing of DNA follow recent studies of related darkling beetles (Kanda 2017, Kamiński et al. 2018, Johnston 2019, Lumen et al. 2020), and are summarized below. Genomic DNA was extracted from head capsules or thoracic musculature dissected from beetles, previously stored in ethanol at -20°C, using a DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany,). No cuticle was ground during the extraction process. Seven gene fragments were amplified: 12s mitochondrial ribosomal RNA (12s, 356bp from the open reading frame corresponding to the small subunit ribosomal RNA), 28s ribosomal RNA (28s, 1041bp from the open reading frame corresponding to the large subunit ribosomal RNA in the regions of D1-D3), CAD/rudimentary [CAD2 sensu Moulton and Wiegmann (2004), 750bp of a nuclear protein coding gene), cytochrome c oxidase subunit II (COII, 699bp of a mitochondrial protein coding gene), cytochrome c oxidase subunit I (COI, 822bp of a mitochondrial protein coding gene), histone 3 (H3, 348bp of a nuclear protein coding gene), and wingless (wg, 477bp of a nuclear protein coding gene). The seven loci produced a total alignment length of 4493bp. PCRs were performed using ExTaq (Takara) with primers and thermocycler protocols given in Wild and Maddison (2008), Kanda et al. (2015), and Lumen et al. (2020). PCR clean-up, quantification, and sequencing were performed by the Genetics Core Facility of the University of Arizona. Sequencing was performed on an Applied Biosystems 3730XL DNA Analyzer.

Museum sequencing

DNA from museum specimens representing five Amphidorini species Eleodes (Caverneleodes) easterlai Triplehorn, 1975, Eleodes (Caverneleodes) sprousei Triplehorn and Reddel, 1991, Eleodes (Pseudeleodes) granosa LeConte, 1866, Eleodimorpha bolcan Blaisdell, 1909, and Neobaphion alleni Triplehorn, 1989) was extracted under a UV-sterilized laminar flow hood with dedicated equipment using QIAamp DNA Micro kits (Qiagen) following the manufacturer's protocol with the addition of carrier RNA (for details see Kanda et al. 2015). Extractions were made from abdomens, without grinding the cuticle, to minimize damage to specimens. As the obtained DNA was already fragmented, no further fragmentation was required before library preparation. Libraries were built using the NEBNext Ultra II DNA Library Prep kits (New England BioLabs), following the manufacturer's protocols and barcoded using NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1). Libraries were sequenced using a HiSeq3000 (Illumina) maintained by Oregon State University Center for Genomic Research and Biocomputing (CGRB). Each sample was given roughly a tenth of a 150 bp end lane.

Phylogenetic inference methods

All loci were aligned using MAFFT v. 7.130b (Katoh and Standley 2013) as implemented through Mesquite v. 3.11 (Maddison and Maddison 2018), visually inspected for accuracy, and placed into coding frame for protein coding genes to inspect for stop codons or possible paralogs and to allow for testing partition strategies discussed below (Kanda et al. 2015, Kanda 2017). Alignments were further verified by generating gene trees for each locus to identify and remove any highly anomalous branches potentially corresponding to sequencing errors or paralogs. The final alignments made across all taxa were then used to create two datasets to test Amphidorini tribal composition along with diverse tenebrionid outgroups and intra-tribal generic and species relationships, respectively.

Monophyly of Amphidorini. This dataset was separated into two partition schemes using the best scheme recovered by PartitionFinder (version 2, Lanfear et al. 2017) under both linked and unlinked branch-length constraints. Phylogenetic inference was performed using both maximum likelihood and Bayesian inference on each of the two partition schemes. RAxML 8.2 (Stamatakis 2014) was used to perform 10 replicates to find the best-scoring tree and to generate 1000 rapid bootstrap replicates, with the bootstrap scores mapped to each branch of the best tree. MrBayes 3.2 (Ronquist and Huelsenbeck 2003) was run using four chains for 10 million generations and sampled every 1000 generations with the following parameters unlinked across partitions: transition/ transversion rate, substitution rates, character state frequencies, proportion of invariable sites, and gamma shape. The first 25% of trees were discarded as burnin. The inferred tribal relationships were identical for both schemes but the four-partition unlinked branch-length scheme is preferred and presented below to be consistent with the within-Amphidorini analyses discussed below.

Relationships among Amphidorini. This dataset was similarly analyzed using PartitionFinder under linked and unlinked branch-length constraints and using both resultant schemes for both RAxML and MrBayes analyses as outlined above for the first dataset.

Data availability

Sequence alignments, partition analysis, and phylogenetic trees from all inferences are available for download as a published dataset (Johnston 2022). All newly generated sequence data are available through NCBI's GenBank (OM745750–OM745894, OM746966–OM747533).

Amphidorini monophyly and composition

Maximum likelihood and Bayesian analyses recovered highly congruent topologies for the tribal monophyly dataset (Fig. 1) across partitioning schemes (see Johnston 2022). Amphidorini was recovered in two distinct clades. First, the North American components formed an expected monophyletic lineage sister to the palearctic Blaptini within the reinstated concept of Blaptinae (Kamiński et al. 2021). Nycterinus was recovered in a strongly supported clade, which we here refer to as the ‘scotobiine clade’, including the tribes Scotobiini, Eulabini, Cerenopini, and Tenebrionini (in part).

Amphidorini generic composition and relationships

Congruent topologies were similarly recovered between maximum likelihood and Bayesian analysis for generic and species relationships within the tribe.

Analyses were sensitive to partitioning scheme, particularly in regard to the branching pattern of the deepest nodes within Amphidorini. The general trend is one of numerous generally well-supported clades which more or less correspond to current genera and subgenera whose relationships to each other are represented by shorter branches with poor support. Topology incongruence primarily revolved around the unsupported early-diverging relationships. Here, and for the purpose of the discussion, we present the topology inferred from the unlinked branch length partition scheme (Figs. 2–4). Four higher level groupings, highlighted in Figure 2, broadly correspond to clades recovered from phylotranscriptomic analyses (Johnston 2018). These are the “embaphion clade,” “blapylis clade,” “metablapylis group,” and “core-eleodes clade.” Among these, we recover very strong support for the monophyly of the core-eleodes clade, which contains the type species of Eleodes and the bulk of the species-level diversity currently placed within the genus, and for the embaphion clade, which strongly supports the monophyly of the genera Embaphion and Neobaphion. The monophyly of the blapylis clade and the metablapylis group is more equivocal and the relationships between all four of these clades are not supported and vary based on partitioning scheme (see published dataset Johnston 2022). In particular, some members of the metablapylis group were recovered as early diverging lineages in a grade leading to the rest of the tribe; however, since neither of the topologies are supported for the placement of these taxa we refer to them as a group instead of a clade in recognition that their monophyly is dubious. Table 1 summarizes the topologies recovered by each of the analyses.

Figure 1.

Recovered phylogeny for the Monophyly of Amphidorini dataset. Topology shown corresponds to the maximum likelihood reconstruction highlighting the two unrelated clades of Amphidorini and the three other Blaptinae tribes sampled. Branches marked with a red dot were recovered in both Bayesian inference and maximum likelihood analyses with at least .99 posterior probabilities and 90 bootstrap values, respectively.

Out of the 19 non-monotypic genus-group taxa, as defined by Bousquet et al. (2018), only seven were recovered as monophyletic: the genera Embaphion and Trogloderus and the Eleodes subgenera Blapylis, Discogenia, Melaneleodes, Pseudeleodes, and Steneleodes. Relationships and composition of the constituent genera and subgenera are discussed in more detail below.

Placement of Nycterinus

Nycterinus is hereby placed as incertae sedis within the subfamily Tenebrioninae. Taxon sampling across Tenebrionidae was not dense enough to draw major conclusions about extralimital tribal and sub-familial composition, but several relationships are significant to point out within the scotobiine clade. First, the representatives of Tenebrionini did not form a monophyletic lineage (Fig. 1), and indeed the widespread Western Hemisphere genera Rhinandrus LeConte, 1866 and Zophobas Dejean, 1834 show affinities to Nycterinus along with the Western Hemisphere tribes Cerenopini, Eulabini, and Scotobiini. This is somewhat unsurprising given ambiguous definitions of many Western Hemisphere lineages that have not been reviewed recently (Johnston et al. 2020) and because Nycterinus was recovered near a composite ‘Tenebrionini’ taxon based on morphological cladistic and phenetic analyses (Doyen and Tschinkel 1982).

Figure 2.

Among-Amphidorini Phylogeny. Topology shown corresponds to the maximum likelihood reconstruction showing the four major clades recovered within Amphidorini. Branches marked with a red dot were recovered in both Bayesian inference and maximum likelihood analyses with at least .99 posterior probabilities and 90 bootstrap values, respectively.

The clade containing Nycterinus can generally be recognized in the Western Hemisphere externally by the generally elongate head capsule and compound antennal sensoria and internally by possessing pleated defensive glands and lacking a spherical spermatheca (Berry 1973, Doyen and Tschinkel 1982, Silvestro et al. 2015, Johnston et al. 2020). This group has historically been associated with the Eastern-Hemisphere Scaurini Billberg, 1820 or the global Tenebrionini. More data are needed to understand the exact nature of the relationships within this group, but the defensive glands and antennal sensoria clearly exclude them from Blaptinae and place them near more derived groups of the Tenebrioninae and Stenochiinae Kirby, 1837 (Kanda 2017, Johnston et al. 2020).

Genus-group composition

The lack of branch support at the base of Amphidorini complicates the delimitation of genera based on molecular data alone. The current broad concept of Eleodes spans three of the four groups defined above (Fig. 2), with the embaphion clade being the only exception, and if maintained would require the inclusion of the genera Trogloderus, Lariversius, and Eleodimorpha. However, there is no unequivocal support for the sister relationship of the embaphion clade to the remainder of the Amphidorini. This has strong nomenclatural ramifications because Embaphion is the oldest available genus-group name within the tribe and would take priority over Eleodes were an even broader concept of that genus to be adopted. It is clear that the currently recognized six genera do not sufficiently convey the evolutionary history of the group, though taxonomic changes will require significant morphological investigation (Johnston and Smith, in prep.). Each of the four main groups are further discussed with regards to their main subcomponents.

Table 1.

Summary of major topological elements by analysis. Major groups are annotated as monophyletic or not and the earliest diverging clade sister to the rest of the tribe is indicated.

The embaphion clade (Fig. 2) comprises two well-supported lineages. The monophyletic Embaphion is consistent with the historically recognized group which is diagnosable by its upturned pronotal margins and ovipositor morphology (Blaisdell 1909, Johnston et al. 2015). Sister to the latter genus is Neobaphion, which was erected for species with ovipositor morphology similar to Embaphion but the body habitus resembling Eleodes (Blaisdell 1925, Triplehorn and Aalbu 1985, Johnston et al. 2015). Notably, the species Eleodes wynnei Aalbu, Smith and Triplehorn, 2012 is recovered in this clade with strong support. The latter species was described in the subgenus Caverneleodes which is herein recovered as polyphyletic and discussed in more detail below. Museum sequencing of Neobaphion alleni, currently known only from the five specimens of the type series, confirmed the generic placement of this rare species and provides insight for future revisionary studies on a genus where most species are relatively uncommon in collections.

The blapylis clade (Fig. 2) comprises five lineages which range from the Rocky mountains to the Pacific coast, not ranging into the Great Plains or arid reaches of continental Mexico. Trogloderus, Lariverisus, and Eleodes barbata Wickham, 1918 are all strongly associated with sandy substrates and dunes in the Intermountain Region (Johnston 2019). The latter species was recently placed as incertae sedis within Eleodes due to a lack of morphological similarity with any other known subgenera (Johnston 2016). The subgenera Blapylis and Discogenia are each recovered as well-supported monophyletic lineages which are consistent with their historical recognition (Blaisdell 1909, Somerby 1972, Bousquet et al. 2018). This clade was recovered in every analysis though with variable statistical support. Female ovipositor morphology and integument sculpturing seem to further unite these taxa morphologically.

The heterogeneous metablapylis group (Fig. 3) consists of six well-supported lineages which span most of the geographic range of the tribe, though its monophyly is not well supported and different topologies are found across different analyses (see dataset in Johnston 2022). This group is in part morphologically supported by most members bearing a wide and evenly tapered elytral epipleuron which is not found in any of the other clades defined here. The largest component lineage corresponds to the Eleodes subgenus Steneleodes which is well established based on female ovipositor morphology and is particularly diverse in central Mexico (Blaisdell 1909, Bousquet et al. 2018). The Eleodes subgenus Metablapylis is recovered in a strongly supported clade intermixed with most sampled members of the subgenus Caverneleodes. Museum sequencing of the latter's type species, Eleodes easterlai Triplehorn, crucially places it within this clade; hence, a detailed morphological reexamination and characterization is recommended for these taxonomic groups. Museum sequencing further confirmed the distinct monotypic lineage Eleodimorpha, which was recovered sister to a monotypic lineage of the type species of the current Eleodes subgenus and tribal basionym Amphidora, both of which are restricted to California. Placement of the monotypic Eleodes subgenus Tricheleodes was unstable across all analyses and its inclusion in the metablapylis group is dubious. However, the morphological circumscription by Johnston (2016) is supported. The final lineage in this group consists of the Eleodes subgenus Cratidus, including part of the current subgenus Amphidora as a sister lineage to two current members of the subgenus Caverneleodes, including the museum-sequenced E. (Caverneleodes) sprousei along with E. (Caverneleodes) thomasi Aalbu, Smith & Triplehorn 2012. This clade is well supported in the analyses but has not been recognized in previous taxonomic treatments within Amphidorini and warrants further examination to potentially identify morphological synapomorphies shared between the species.

Figure 3.

Among-Amphidorini phylogeny continued. Topology shown is the same as Fig. 2 with the metablapylis group expanded. Branches marked with a red dot were recovered in both Bayesian inference and maximum likelihood analyses with at least .99 posterior probabilities and 90 bootstrap values, respectively.

The core-eleodes clade (Fig. 4) encompasses the bulk of what is currently treated within the genus Eleodes, including all sampled members of nine subgenera. The monophyly of this clade is very strongly supported, as are many of its constituent component clades, yet we again see a lack of support for many of the relationships between these components. In addition to the monotypic subgenera Ardeleodes and Heteropromus, the recently reviewed subgenera Pseudeleodes (see Johnston 2016) and Melaneleodes (see Triplehorn and Thomas 2012) were the only previously recognized groups recovered as monophyletic. The nominate subgenus Eleodes was recovered as polyphyletic, with the California-restricted type species Eleodes dentipes Eschscholtz, 1829 recovered in a small clade with several species which range southward into Baja California. The remainder of the nominate subgenus was recovered as a paraphyletic grade which includes components of the subgenera Promus and Litheleodes. The latter subgenera were recovered as polyphyletic, with Litheleodes, as historically defined and recently revised by Triplehorn and Thomas (2015), largely nested within Promus except for its most atypical member Eleodes arcuata Casey, 1884 (Triplehorn and Thomas 2015) representing an early-diverging lineage within the core-eleodes clade. Promus was defined for a number of species distributed in the United States (Blaisdell 1909, Johnston 2015) and was expanded to include a large number of species from Mexico in a worldwide catalog for the family (Gebien 1938). The species from the United States, including the type Eleodes opaca (Say, 1824) were recovered with the bulk of Litheleodes, whereas a large portion of the later included Mexican species were recovered in a strongly supported clade that also includes the currently monotypic Omegeleodes.

Evolutionary insights

The phylogeny presented here may provide some insights to the origins and biogeographic trends within Amphidorini, though phylogenetic dating and historical biogeographic analyses are complicated by the lack of reliable fossil calibrations. The exclusion of Nycterinus and sister relationship between the western North American Amphidorini and Palearctic Blaptini suggests the origin of Amphidorini was due to either a vicariance event across Laurasia or a subsequent dispersal event to the Nearctic, perhaps through a land bridge. A broader phylogenetic study within the family is necessary in order to infer lineage ages required for testing either of these hypotheses. In addition, Kamiński et al. (2021) recovered Amphidorini sister to (Blaptini + Platyscelidini Lacordaire, 1859), the latter being a Palearctic lineage not included in our analyses which would be critical to include for understanding the origins of these groups. A dispersal event from the Palearctic may be further supported by most of Amphidorini's lineages with a center of diversity found in the far western regions of the United States from the California Floristic province and Intermountain Region, i.e. embaphion clade: Neobaphion, Embaphion; blapylis clade: Trogloderus, Lariversius, E. barbata, Blapylis; metablapylis clade: Tricheleodes, Eleodimorpha, Amphidora, Cratidus, Metablapylis; core-eleodes clade: Litheleodes, Eleodes (Eleodes) (Bousquet et al. 2018, Johnston 2019). The lineages that inhabit the eastern and southern range of Amphidorini are primarily more derived groups within the core-eleodes clade that also have some species present in the more western regions, i.e. the Eleodes subgenera Melaneleodes, Omegeleodes, and Promus.

Morphological character systems historically used to classify Amphidorini groups need to be reevaluated in the context of this phylogeny. The primary system for generic and subgeneric placement used by Blaisdell (1909) and carried on through recent works is the female ovipositor. Species placements based on ovipositor morphology were largely supported across our phylogeny, but placements based upon external characters (e.g., cavernicoly and long legs in Caverneleodes see Triplehorn 1975 and Aalbu et al. 2012) and via cataloguing efforts instead of revisionary works (e.g., Promus see Johnston 2015) were not. Previously used external diagnostic characters may yet provide some utility in identifications, but are nearly all homoplasious across the tree, including males with profemoral spines (found in e.g., Cratidus, Trogloderus, Eleodes (Eleodes), some Eleodes (Promus)), all femora with spines (found in e.g., E. armata LeConte, 1851 and E. loretensis Blaisdell, 1923), and dense protarsal setal pads (found in e.g., Eleodes subgenera Amphidora, Blapylis, and Promus).

Figure 4.

Among-Amphidorini phylogeny continued. Topology shown is the same as Fig. 2 with the core-eleodes clade expanded. Branches marked with a red dot were recovered in both Bayesian inference and maximum likelihood analyses with at least .99 posterior probabilities and 90 bootstrap values, respectively.

The phylogenetic hypotheses presented in this paper are strongly divergent from current taxonomy. To illuminate this phylogeny/classification gap (Franz 2005), Table 2 summarizes the current taxonomic classification and the likely changes needed to address the inferences discussed above. We look forward to future studies that reconcile this molecular phylogenetic scaffold with the rich legacy of Amphidorini morphology to robustly circum-scribe and diagnose composite genera and subgenera.

Table 2.

Summary of current Amphidorini classification following the catalog of Bousquet et al. (2018) according to the molecular phylogenetic reconstruction presented here.