Introduction

Philonthus Stephens, 1829 is the second largest genus in the diverse beetle family Staphylinidae, containing over 1,250 species (Hromádka 2008; Chani-Posse 2010). Many Philonthus species are very similar, their phylogenetic relationships are still poorly known (Hromádka 2008; Chani-Posse 2010; Schillhammer 2012), and often they can be distinguished from each other only by subtle differences in the shape of the male genitalia or minute external characters. At the same time, many Philonthus display significant intraspecific morphological variation; in particular, the same species may have different color morphs, which is especially true for the species with a metallic coloration. Such clusters of closely related species would benefit from a rigorous study by means of integrative taxonomy; these, amongst others, include the rotundicollis, picimanus, and formaneki groups (Schillhammer 2003) as well as the varians, longicornis, micans, and other groups (Schillhammer 2009, 2012; Hromádka 2012).

Integration of DNA data with morphology allows researchers to overcome some of the previous challenges faced by traditional taxonomy, and can help reveal cryptic species overlooked in the past (Vitecek et al. 2017; Brunke et al. 2020; Hansen & Jenkins Shaw 2023). Integrative taxonomy, as a tool for species delimitation, also involves a rigorous assessment of the distribution and bionomic data that were always valued by careful taxonomists. In Staphylinidae, methods of integrative taxonomy are gaining momentum, as summarized in Gebremeskel et al. (2023) and as exemplified by Park et al.'s (2024) recent study on the morphologically variable and widespread Philonthus nudus Sharp. Philonthus splendens (Fabricius, 1793) and its reported intraspecific form, sometimes formally recognized as subspecies P. s. sideropterus Kolenati, 1846, are a noteworthy case suitable for careful integrative taxonomic examination (Fig. 1).

Fig. 1.

Habitus of P. splendens (A) and P. sideropterus (B), showing differences in elytral coloration.

Philonthus splendens is a large predatory rove beetle between 12.0 and 16.5 mm in length, commonly collected and well-represented in museum collections. It typically has a shiny black body and its pronotum lacks the characteristic dorsal rows of punctures present in most Philonthus species. The elytra are often with a brass/bronze metallic luster. Philonthus splendens is the type species of the genus Philonthus and is distributed across Europe and large parts of Asia, with some records from North Africa (Schillhammer 2000). Philonthus splendens var. sideropterus is much less widespread, known only from the Caucasus region. It was first described from Transcaucasia by Kolenati (1846) as having “Elytris cyaneis”, i.e., a cyan-blue metallic luster on the elytra in contrast to the brass/bronze of P. splendens, while otherwise being very similar to the latter. The seemingly non-overlapping geographical distributions of both P. splendens and P. sideropterus and their subtle morphological differences urged staphylinid taxonomists to consider them as varieties or subspecies (examples of habitats suitable for P. splendens and P. sideropterus are shown in Fig. 2). The ambiguity of the subspecies concept and lack of data showing a transition between both subspecies has further made the true categorization of P. sideropterus difficult. The blue-colored Transcaucasian form seems, as Kolenati (1846) and several other authors (Hochhuth 1849; Coiffait 1967, 1974; Schillhammer 2000) noticed, endemic to the Caucasus. Caucasian variants of otherwise widespread species have often shown signs of speciation in various organismal groups, such as spiders (Chaladze et al. 2014), plants (Gagnidze et al. 2002), and beetles (Gebremeskel et al. 2023; Justesen et al. 2023), to mention just a few. The unique geological and climatic conditions of the Caucasus, with its diverse landscapes ranging from mountains and high plateaus to semi-arid lowlands, function as a southern refugium for several temperate species (Milne & Abbott 2002; Beridze et al. 2023). Species previously with widespread populations were fragmented and/or isolated in the Caucasus due to the gradual uplift and cooling of the region in the Miocene and Pliocene (23.3–2.58 Ma) or the more recent climatic fluctuations in the Quaternary (2.58 Ma–present) (Burke et al. 2018; Hrivniak et al. 2020; Beridze et al. 2023; Hansen et al. 2023). Many of these isolated, remnant populations have presumably diverged enough from the originally widespread populations to become distinct species, making the Caucasus a biodiversity hotspot with high numbers of endemics (Chaladze et al. 2014; Victorovich & Pavlovich 2018).

To investigate the outlined case of P. splendens var. sideropterus, we here aim to study morphologically and molecularly a wide pool of material that covers the entire distribution of this species complex. For the molecular work, we targeted the widely accepted barcode fragment of the mitochondrial COI gene and the fragment of the nuclear Wingless gene. The COI barcode is widely used to delimit closely related species across most animal phyla (Ratnasingham & Hebert 2007; Shearer & Coffroth 2008; Gebremeskel et al. 2023; Gorring & Cognato 2023). The nuclear gene Wingless (Wg) was chosen as a supplement to COI due to its similar usage in other studies (Toussaint et al. 2015; muÑoz-Tobar & Caterino 2020). To not rely solely on genetic evidence, we also performed a morphological assessment of targeted individuals as well as a careful mapping of the distribution of both color forms, an integrative approach.

Fig. 2.

Habitats of of P. splendens and P. sideropterus. A. Bakhmaro, Georgia; P. sideropterus collected in cattle dung at 1900–2500m. B. Nekselø, Denmark; P. splendens collected in cattle dung near sea level. C. Altai, Russia; P. splendens collected in cattle dung at 1500m. D. Lessinia, Italy; P. splendens collected in nest debris near marmot burrow entrance at 1600 m.

Taxonomic history of Philonthus splendens var. sideropterus

Philonthus splendens var. sideropterus Kolenati, 1846 was described as a variant of P. splendens Fabricius, 1793 with cyan-colored elytra. No data on the type material is mentioned in Kolenati (1846), except that it originated from “Transcaucasia”. It is noteworthy that Kolenati (1846) mentioned the possibility of sideropterus being a separate, new species. Hochhuth (1849) brought the first elaborate description of P. splendens var. sideropterus and reported one specimen collected from Tiflis (now Tbilisi, Georgia) and three from Helenendorf (now Goygol, Azerbaijan). The first mention of a specimen that can be considered a syntype of P. s. var. sideropterus from the Caucasus was located at some “der Königl. Sammlung” by Kraatz (1857). Ganglbauer (1895) listed sideropterus as a synonym of P. splendens without any mention of its previous classification as a variant. This was repeated in the catalog of Bernhauer & Schubert (1914), who also listed it as a synonym. Contrary to this, the catalog by Scheerpeltz (1933) returned to the taxonomic differentiation between P. splendens and P. sideropterus by classifying the latter as an aberration of the former. Dissected specimens of P. sideropterus were first reported in Smetana (1955), who did not see any difference in the structure of the aedeagus between sideropterus and splendens but suggested subspecies status for sideropterus based on its allopatric distribution. Smetana (1955) mistakenly assumed that Kolenati (1846) described P. sideropterus as a species of its own. Smetana (1958, 1959), consistent with his earlier decision, listed sideropterus as a subspecies of P. splendens. Coiffait (1967) also included sideropterus as a subspecies of P. splendens, but in a later work he (Coiffait, 1974) again listed sideropterus as a variety of P. splendens. Schillhammer (2000) acknowledged the volatile status of sideropterus and, similarly to Smetana (1955, 1958, 1959), classified it as a subspecies.

Material and methods

DNA extraction and PCR

DNA extractions were done using the EZNA DNA Tissue kit (Omega Bio-tek, Norcross, GA, USA), following the product protocol for tissue with a prolonged lysis time (24 hours). For non-destructive extraction, the entire specimen was soaked in a lysis buffer and immediately transferred to 96% ethanol after lysis. After DNA extraction, the physical vouchers were glued on card and mounted on pins, with the terminalia segments and aedeagus fixed with water-soluble glue on the same card. See Supplementary File 1 for specimens included in the figures.

For COI, the PCR reaction consisted of 1 µl of DNA extract, 4 µl of 5× HOT FIREPol Blend Master Mix Ready to Load With 10 mM MgCl2 (Solis BioDyne), 1 µl of each primer at 0.5 µM and 18 µl distilled water. Primers and the specific PCR cycles were as follows:

Two different strategies were used to obtain DNA sequences. These were Sanger sequencing and Nanopore sequencing using a Flongle flow cell on the MinION, respectively.

Forward and reverse Sanger sequencing and purification of target COI amplicons was done by Macrogen (Amsterdam, The Netherlands). The forward and reverse raw sequences were aligned to a reference P. splendens barcode (GenBank Accession: KJ966992) in Geneious Prime (v2023.1) ( https://www.geneious.com) and edited for obvious alignment errors.

MinION-based sequencing of the PCR amplified extractions containing the short COI (sCOI) loci (see Supplementary File 1 for specimen list) was done by Flongle Sequencing (Srivathsan et al. 2021). During the previous PCR step, tagged primers were used on samples intended for Flongle Sequencing, enabling differentiation between samples. All included material from the sCOI PCR was pooled together, followed by a thorough Clean-up, DNA repair, and end-prep step. This was designed to remove unwanted primer sequences and to ensure all DNA fragments were of the same length and free of any artifacts introduced by the polymerase during PCR. The Flongle flow cell was placed in the Flongle adapter connected to the MinION, and then loaded with the pooled and cleaned DNA material. After running for 24 hours, the output data were made available after transfer from the MinION to a computer with Filezilla ( https://filezilla-project.org/, v3.65.0-rc1). Basecalling was done using Guppy ( https://timkahlke.github.io/LongRead_tutorials/BS_G. html) on the extracted file output to interpret the ionic signal into base pairs. The basecalled output sequences were then demultiplexed using ONTbarcoder (v0.1.9b, and v0.1.11c), sorting out the sequences based on the combination of tagged primers (Srivathsan et al. 2021). The final output file was uploaded to Geneious Prime (v2023.1) for alignment and reference barcode mapping using the same reference as above.

The procedure for the nested PCR of Wg was similar to that for COI, except that two runs that were done with the second nested Wg using 1 µl of DNA product from the first initial Wg. Primers and specific PCR cycles for the initial Wg and nested Wg as follows:

If this initial PCR failed to show a band on the gel electrophoresis, another reaction was set up using the initial PCR product as DNA template with a new set of internally nested primers using the following conditions:

Forward and reverse Sanger sequencing and purification of target Wg amplicons was done by Macrogen (Amsterdam, The Netherlands). The forward and reverse raw sequences were aligned to a reference P. splendens barcode (GenBank Accession: GU377489).

Results

Geographical distribution and bionomics

As clearly shown in Fig. 3, the wide distribution range of P. splendens extends from Western Europe to Central Siberia. The range of P. sideropterus is much more restricted, confined to the Caucasus and Northern Turkey, and well separated from the range of P. splendens by the drier Western Turkey in the East and by the Ciscaucasian open plains in the North. An outlier record from the southern part of Turkey (Gaziantep, Dîlok) is based on a single old specimen (no date, but from the collection of EDUARD EPPELSHEIM [1837–1896]), which we consider a mislabeling as we have yet to see other material from the area, which usually houses another fauna. The distribution of P. splendens occurs at lower altitudes in more northern areas, while it is mostly found in mountainous areas in the southern part of its range, for instance in the European Alps and Atlas Mountains of North Africa. Here, also a few outlier records were found in the literature, with mentions of findings in Middle Asia (Kadyrov et al. 2014), North Korea (Schillhammer 2000), and Northeast China (Li & Chen 1993). Neither of these records could be confirmed by us, and we believe they could represent misidentifica-tions, mislabelings, or sporadic introductions. Philonthus splendens seems to be confined to the nemoral forests of Eurasia, occurring mainly in or around dung. Noteworthy is the absence of records from the plains of West Siberia, but the notable presence of the species in the more montane south-eastern part of that region. Based on our personal collecting experience and records with sufficient bionomic data, P. sideropterus is always found in the humid elevated mountain areas, mainly in or around dung, in the zones with temperate forest or in the subalpine belt with bushy vegetation. Interestingly, in the extensive summary by Salnitska et al. (2022) of the faunistic literature for the open plains of South European Russia, P. splendens is also reported as absent there, meaning that the gap between the distributions of P. splendens and P. sideropterus in the Ciscaucasian plain is real and not an artifact of poor sampling.

Fig. 3.

Distribution map of P. sideropterus (blue) and P. splendens (brown). Localities based on examined specimens are shown by circles; localities based on records in GBIF or the literature are shown by stars.

Morphology

As repeatedly noted by earlier authors, P. splendens and P. sideropterus differ from each other in the coloration of the elytra. All specimens from the distribution area of Philonthus splendens have a metallic elytra coloration ranging from bright brass to a deep dark brown, while all Caucasian specimens, i.e., P. sideropterus, have metallic-colored elytra with a blue hue. The blue hue ranges from bright ocean blue to a deep, almost blackish blue. The only exception was a single specimen of P. sideropterus from Gümüşhane (NE Turkey) with reddish non-metallic brown elytra (Fig. 4A).

Schillhammer (2000) showed a slight difference between P. splendens and P. sideropterus in the structure of the aedeagus: in the shape of the median lobe and position of the sensory peg setae on the paramere (line drawings in Fig. 4C). However, this difference does not hold when more specimens are compared (photos in Fig. 4C). Investigation of the female 10th tergite also showed no difference between the investigated taxa (Fig. 4B).

Phylogenetic and species delimitation analyses

ML and BI trees support P. sideropterus and P. splendens as sister clades with respective support values of UFB=99.2/87.9, SH-aLRT=100/89, and PP=1/0.86 and support for the monophyletic ingroup as high as UFB=99.8, SH-aLRT=100, and PP=1 (Fig. 5).

Fig. 4.

Morphological variation in P. splendens and P. sideropterus. A. Variation in elytra coloration ranging from dark to clear metallic colors for P. sideropterus (blue) and P. splendens (brass); includes rare reddish brown color morphs found in both taxa. B. Morphological variation in 10th tergite of female P. sideropterus (top four) and P. splendens (bottom four). C. Morphological variation in the median lobe and paramere of the aedeagus in P. splendens and P. sideropterus (localities and other information about every illustrated specimen can be found in the supplementary dataset for the material examined, where illustrated specimens are flagged in the “Figure” column, available in Supplementary File 1. Line drawings of median lobe (in lateral view) and paramere (in dorsal view, i.e., underside facing median lobe) provided by HARALD Schillhammer show previously hypothesized shape of the aedeagus for both taxa.

All species delimitation methods, ASAP, ABGD, and bPTP, showed congruent results supporting species-level differentiation between P. splendens and P. sideropterus (Fig. 5).

The haplotype network analysis (Fig. 5) shows the significant molecular distance between P. splendens and P. sideropterus. Nucleotide differences between P. splendens and P. sideropterus ranged from 16 to 19. A much lower level of intraspecific divergence was observed for both P. splendens and P. sideropterus: 1–4 and 1–2 nucleotides, respectively.

For Wg, we experienced poor quality of sequencing, resulting in aligned forward and reverse reads of very variable lengths (204–438bp instead of the targeted 464). The low amplification success of this marker could be related to many of our extracts being from museum specimens, from which nuclear genes are generally more difficult to recover. Another potential issue could be low specificity of the used primers for our target taxa, as these were general insect primers and not modified to fit our target. We found no difference in Wg between the two species in both the BI and ML analyses (Supplementary Fig. 1).

Discussion

Historical biogeography

Distributional and ecological evidence supports P. sideropterus and P. splendens as separate species that likely originated as a result of the boreo-montane disjunction (VukojiČiĆ et al. 2014) of the ancestral species' range. One hypothesis would be that the cooler climate during the Pliocene (5.30–2.58 Ma) (Burke et al. 2018; Beridze et al. 2023; Hansen et al. 2023) enabled populations of a common ancestor of P. splendens and P. sideropterus to expand their range south to Asia Minor and the Caucasus. Presumably, this expansion occurred via the more forested areas of Eastern Europe, especially along the north-south oriented, large forested river valleys or via “mountain hopping” through the Alps, Balkans, and mountainous areas of Turkey, finally reaching the Greater Caucasus via the Lesser Caucasus. Regardless of the initial southwards expansion routes of a temperate species during the cooler period, subsequent rises in temperatures and increasing continentality possibly led to the isolation of the south-easternmost populations at the cooler and more humid higher elevations of the Caucasus and Northern Turkey, where they evolved into a separate species, P. sideropterus. Presumably, the original range of the lineage which is now P. splendens was also fractured between the more northern populations and the more southern one(s) retracting to higher elevations in the central and southern European mountains or even North Africa, but their isolation apparently was not as significant. More recent Quaternary climate fluctuations may also have enabled populations from North Africa, the Alps, and the Balkans to mix with more northern Euro-Siberian populations.

Fig. 5.

An integer neighbor-joining haplotype network (center) and consensus phylogenetic tree based on Bayesian and Maximum Likelihood analyses of the COI barcode, with support values. Support values at each node are shown in the following order: UFB, SH-aLRT, PP. Bars with gaps on the right indicate the most probable clustering of three species delimitation methods. The size of the circles in the haplotype network is proportional to the number of specimens in the clusters; vertical bars between the clusters represent nucleotide differences.

Conclusion and taxonomy

We used an integrative taxonomic approach, namely three lines of evidence such as molecular species delimitation, morphology, and biogeography, to resolve the ambiguous taxonomic status of P. sideropterus, previously treated as either a variety or a subspecies of P. splendens. Molecularly, P. sideropterus and P. splendens are clearly supported as separate species by COI barcode data but not by the nuclear Wg marker, which gave an inconclusive answer to the same question largely due to a shortage of data related to practical difficulties in obtaining this marker from museum specimens. The earlier noted external morphological difference in the coloration of elytra between P. sideropterus and P. splendens is here confirmed as stable and easy to see in the great majority of specimens. The earlier stated subtle genitalic differences between males of both taxa are here not confirmed because of the newly revealed intermediate forms that, however, do not show any geographic pattern. The distributions of both taxa are clarified and shown to be confined to areas of sufficient humidity; they are clearly allopatric and well separated from each other by a large drier area without suitable habitats for wither taxon. The molecular distance (clear in COI), morphological differences (clear in the elytral coloration), and wide geographic gap between P. splendens and P. sideropterus indicate that they are well established and well diagnosable allopatric sister lineages that lack gene flow. Even without visible differences between their male genitalia, they do not match the definition of subspecies as emerging allopatric lineages that did not completely lose genetic connectivity (Braby et al. 2012). Although, recently, Dufresnes et al. (2023, 2024) and Lukhtanov (2024) removed the demand of genetic connectivity as a criterion for subspecies and encouraged to use this category in taxonomy to accommodate closely related phylogeographic lineages, here we explicitly abandon the previous very ambiguous treatment of P. sideropterus as a subspecies (Smetana 1955, 1958, 1959; Coiffait 1967; Schillhammer 2000) or a variety (Kolenati 1846; Hochhuth 1849; Kraatz 1857; Coiffait 1974) of P. splendens. Instead, we raise the status of Philonthus sideropterus Kolenati, 1846, sp. propr. to species level, which in fact was already suspected by Kolenati (1846) nearly two centuries ago.