A review and phylogeny of Scarabaeine dung beetle fossils (Coleoptera: Scarabaeidae: Scarabaeinae), with the description of two Canthochilum species from Dominican amber

Despite the increasing rate of systematic research on scarabaeine dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae), their fossil record has remained largely unrevised. In this paper, we review all 33 named scarabaeine fossils and describe two new species from Dominican amber (Canthochilum alleni sp.n., Canthochilum philipsivieorum sp.n.). We provide a catalogue of all fossil Scarabaeinae and evaluate their assignment to this subfamily, based primarily on the original descriptions but also, where possible, by examining the type specimens. We suggest that only 21 fossil taxa can be reliably assigned to the Scarabaeinae, while the remaining 14 should be treated as doubtful Scarabaeinae. The doubtful scarabaeines include the two oldest dung beetle fossils known from the Cretaceous and we suggest excluding them from any assessments of the minimum age of scarabaeine dung beetles. The earliest reliably described scarabaeine fossil appears to be Lobateuchus parisii, known from Oise amber (France), which shifts the minimum age of the Scarabaeinae to the Eocene (53 Ma). We scored the best-preserved fossils, namely Lobateuchus and the two Canthochilum species described herein, into the character matrix used in a recent morphology-based study of dung beetles, and then inferred their phylogenetic relationships with Bayesian and parsimony methods. All analyses yielded consistent phylogenies where the two fossil Canthochilum are placed in a clade with the extant species of Canthochilum, and Lobateuchus is recovered in a clade with the extant genera Ateuchus and Aphengium. Additionally, we evaluated the distribution of dung beetle fossils in the light of current global dung beetle phylogenetic hypotheses, geological time and biogeography. The presence of only extant genera in the late Oligocene and all later records suggests that the main present-day dung beetle lineages had already been established by the late Oligocene–mid Miocene.


INTRODUCTION
Scarabaeine dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae) are a primarily dungfeeding subfamily comprising ∼6,200 species and ∼267 genera worldwide (Tarasov & Génier, 2015). Recently, this subfamily was the subject of 15 key studies using molecular and morphological data (summarized in Scholtz (2009b) and Tarasov & Génier (2015)) that aimed at constructing a robust phylogeny and/or at facilitating comparative evolutionary studies in dung beetles. Further development of scarabaeine systematics and evolutionary research would benefit greatly from analytical approaches that integrate fossils with morphology and molecules for combined phylogenetic inference (Ronquist et al., 2012b;Warnock et al., 2015). While our knowledge of molecular and morphological evolution of dung beetles has grown considerably over the last decade, their fossil record has remained almost unstudied. Nevertheless, fossils represent an essential data source for resolving relationships, understanding morphological character evolution and assessing the tempo and mode of diversification. Ignoring the fossil record makes tree calibration procedures less robust methodologically (Ronquist et al., 2012a;Ronquist et al., 2012b). Currently, inference of a time-annotated evolutionary history of dung beetles is hampered by the lack of a detailed investigation of their known fossils. In the present study we aim to address this issue by providing a critical overview of the fossil record of the group.
Fossil Scarabaeinae are rare in collections, with just 33 described species prior to this study. Here, we describe two additional species of the genus Canthochilum from Dominican amber (16 Ma), bringing the total number of fossil species to 35. Such a scarce fossil record is likely the result of taphonomic biases, rather than a lack of paleontological studies on this group. Herein, we review the scarabaeine fossil record and provide a catalogue of all described species, which is updated from previous works (Krell, 2000a;Krell, 2007) and supplemented with notes on the credibility of the fossils' taxonomic placement. Due to the poor preservation of fossilized specimens important characters are often missing, which often leads to misidentifications. In this paper, we examine the original descriptions and illustrations of all described scarabaeine fossil species and evaluate the potential of misidentifications in the original taxonomic placements. Because proper taxonomic placement needs an investigation of type specimens and we could not obtain the types of all 33 fossil taxa known prior to this study, the notes on potential issues with taxa for which the types were not examined do not represent formal taxonomic decisions. However, they raise flags where caution and further taxonomic work is necessary. The preserved or documented characters of 14 of the fossils currently described as scarabaeine do not allow their unambiguous placement in Scarabaeinae. Available evidence does support the placement of the remaining 21 fossils species (including the two new species described herein) in Scarabaeinae. We discuss the distribution of these 21 species across the scarabaeine phylogeny and through geological time. While most reliable fossil scarabaeines belong to extant genera, one of them, Lobateuchus parisii from the Eocene (53 Ma), is a member of an extinct genus and is also the oldest reliably identified scarabaeine fossil. We argue that the minimal age for the Scarabaeinae should be aligned with the age of Lobateuchus and not with the ages of the oldest recorded doubtful "scarabaeines" known from the Cretaceous (i.e., Prinocephale Lin, 1980 andCretonitis Nikolajev, 2007).
We selected the best-preserved dung beetle fossils, the Canthochilum species described herein and the oldest scarabaeine fossil Lobateuchus, all of which are known from amber, and added them to the most recent morphological character matrix of Scarabaeinae (Tarasov & Génier, 2015). Then, we analysed the resulting dataset (including both fossil and extant species) using parsimony and Bayesian methods of phylogenetic inference.

Examination of fossils
The Dominican amber pieces containing the fossilized remains of the two new species described herein were examined dry using a Leica MZ16 stereomicroscope. Photos were taken with a Canon EOS 500D digital camera attached to a Leica MZ16 stereomicroscope and with a Canon EOS 1DS Mark III attached to an Infinity K2/SC system. Several fossils described by Heer (1862) were studied from detailed photos kindly provided by the curators of the Geological-Paleontological Collection at Eidgenössische Technische Hochschule Zürich in Switzerland. The remaining non-amber fossils were examined using only literature sources, i.e. original descriptions, other relevant works and illustrations when available.

Dating of fossils
The age of the fossils is derived from the latest publications discussing in depth the relevant deposits (Table 1). If applicable, absolute ages were adjusted to the current dating of epochs or stages as summarized by Cohen, Finney & Gibbard (2015) and Gradstein et al. (2012).

Character matrix
We scored the examined scarabaeine fossils (Lobateuchus parisii, Canthochilum philipsivieorum and C. alleni) into the character matrix previously developed for extant Scarabaeinae (Tarasov & Génier, 2015). The remaining described fossils, including the ones from La Brea, are too incomplete (preserving information allowing to score just one or two characters at best) to integrate into existing character matrices, preventing us from determining their phylogenetic affinities. The character matrix was constructed using Mesquite ver. 3.03  and includes 114 taxa and 205 characters; it can be downloaded as Supplemental Information 1 or from MorphoBank Table 1 List of the described fossil Scarabaeinae and the confidence of their placement in Scarabaeinae. The table puts the scarabaeine fossils in two categories-those which can be confidently assigned to Scarabaeinae and those whose assignment is doubtful. The column Stat. (Status) summarizes our confidence for treating a fossil as a member of Scarabaeinae and classifies them in the following categories: (S) true scarabaeine with correct generic placement; (?S) true scarabaeine but its generic placement needs a further investigation; (DS) doubtful scarabaeine, the description lacks any evidence for assigning of the fossil to Scarabaeinae; (L) the fossil specimen is presumed lost. The justification for placing a fossil in any of these four categories is given in the Catalogue section. The column Age provides the fossil age data (derived from the age of the strata or amber that contain the fossils) for those fossils that can be confidently placed in Scarabaeinae. The age information was retrieved from references listed in the Dating source column.

Epoch
Age ( (http://www.morphobank.org project 2184). Since we did not add any new characters to the matrix of Tarasov & Génier (2015) and did not modify its general structure, the character report for the present matrix is the same as in Tarasov & Génier (2015) and we refer the reader to that paper for details.

Phylogenetic analyses
For the phylogenetic inference we applied Bayesian inference in addition to the parsimony approach that is traditionally used in morphology. Bayesian analysis samples topologies from their posterior probability (PP), thus explicitly assigning a probability score to every sampled split of lineages. Comparison of probability scores between alternative splits allows straightforward evaluation of alternative positions for taxa. Parsimony analysis may also infer numerous trees, but in contrast to Bayesian inference, the split frequency in parsimony does not bear any explicit statistical explanation. Even if numerous trees are inferred in a parsimony analysis, the alternative positions of splits are not evaluated and usually one specific position for a split tends to be shown-either by using a consensus tree or by showing a "preferred" topology. The evaluation of alternative placements using the Bayesian approach is especially interesting for fossil taxa as they usually have many missing entries in the matrix, which tends to generate numerous alternative placements.
In this paper, we ran both Bayesian and parsimony analyses with two versions of the data matrix: one including all characters and one with ambiguous characters (characters #122, 71, 73, 74, 161, 204) excluded as suggested in Tarasov & Génier (2015).

Parsimony (MP)
The parsimony analysis was conducted in TNT ver. 1.1 (Goloboff, Farris & Nixon, 2008) under equal weights using the following TNT settings: traditional search with 3,000 replications and up to 200 trees saved per replication, tree buffer set to store 1 M trees, TBR, trees automatically condensed after search, the default collapsing rule was used.
To assign support values onto branches of the consensus trees, we calculated Bremer support values (BSV) by searching suboptimal trees up to 10 steps longer than the shortest one using TBR swapping on the shortest trees.
To summarize sampled trees and frequencies after burnin we used the sumt command with minpartfreq = 0.01 in order to include rare splits in the posterior sample. We used the resulting .tstat and .parts files to extract and analyse information about splits and their probabilities. The alternative splits for fossil species are shown in Fig. 2. Due to illustrational constraints confining visualization to 2D space, we demonstrate only those alternatives that are not nested within hierarchically higher splits. We call such splits elementary. We choose this illustration approach over other methods (e.g., networks or density trees) as it improves readability of the results in the present case and provides a good summary of the alternative relationships.

Nomenclatural acts
The electronic version of record of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the Official Registry of Zoological Nomenclature. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:D2719A53-A7C1-4D9D-BE94-FED25F17DC7C. The work is archived in PubMed Central and CLOCKSS. The genus Canthochilum is a Greater Antillean endemic comprising 23 extant species (Philips & Ivie, 2008) in addition to the two extinct species described here. The fossils of the two specimens described here were initially found and subsequently purchased on eBay. Because eBay is known to be extensively flooded with fake amber insects, we needed to critically examine the authenticity of our amber and specimens prior to species description. The proof of authenticity of amber requires sophisticated analytical techniques (Eriksson & Poinar, 2015). We ran simple tests which are usually sufficient to confirm authenticity as suggested in Eriksson & Poinar (2015). The results that alcohol drops did not affect amber pieces while burning of amber released a smell of pine resin align well with properties of authentic amber. Additionally, we consider the described Canthochilum fossils to be authentic because Canthochilum is a relatively rare, non-dungdwelling taxon that is difficult to collect. Most specimens are caught with flight intercept traps (Philips & Ivie, 2008). Furthermore, its distribution is restricted to the Greater Antilles. Thus, it would require an entomology expert to fake those pieces of amber, which does not seem plausible. Moreover, one fossil, C. philipsivieorum, is very distinct from all known extant species of Canthochilum, thus reinforcing its fossil nature. Tarasov  Material examined. Holotype, ♂, amber fossil, Dominican Republic, no additional data (CEMT).

Canthochilum alleni
Description. Body oblong, black, without visible colour differences between legs and remaining body surface.
Head: dorsal surface without any visible sculpturing, completely smooth (no visible punctures); clypeus with 4 teeth; mesal pair of teeth triangular, tips blunt; lateral pair of teeth obtuse, significantly wider than mesal teeth; clypeal margin angulate between lateral tooth and genal junction, not forming a tooth; clypeo-genal projection present and easily discernible; dorsal ocular area quite large, eyes separated by a distance equal to four times their transverse width.
Pronotum with surface covered by regular simple punctures, separated by about 3-7 puncture diameters; surface between punctures smooth.
Elytra with 8 striae, with finely shagreened surface and without visible punctures; 1st, 2nd and 8th elytral striae distinct, striae 3-7 not distinctly discernible; all elytral intervals flat except 1st which is very slightly convex; elytra with slight lateral carina that internally adjoins 7th stria; 7th stria appears to be formed by large punctures becoming distinct posteriorly. Proleg with trochanterofemoral pit; protibia with 3 teeth, tips of basal and medial teeth separated by 1.5 times the distance between tips of apical and medial teeth; protibial spur spatulate and blunt, reaching third tarsomere.
Metacoxal lateral margin not expanded.
Pygidial margins not discernable in examined specimen; pygidial disc moderately convex, shagreened, covered with scattered simple punctures.
Length: 3.9 mm. Observational note. The specimen is a male, indicated by the modified shape of the protibial apical spur.
Diagnosis. This species is most similar to other Canthochilum species occurring in Hispaniola. It can be separated from other Canthochilum by the following combination of characters: (1) elytra with slightly expressed lateral carina, (2) body uniformly black, (3) clypeal margin with 4 teeth, (4) clypeo-genal projections present, (5) clypeal margin angulate between lateral tooth and genal junction, (6) eyes separated by a distance equal to four times their transverse width, (7) proleg with trochanterofemoral pit.
In Philips & Ivie's (2008) key to Canthochilum from Hispaniola, this new species falls into couplet 6 and can be distinguished from the other two species in this couplet (C. magnum Philips &Ivie, 2008 andC. darlingtoni Matthews, 1969) by the clypeal margin being angulate between the lateral tooth and genal junction.
Etymology. This species is named after Albert Allen (Boise, Idaho, USA), who very kindly sent it to FVM as a donation.
Locality and age. The precise locality cannot be determined as the specimen was bought on eBay. The age of Dominican amber has been somewhat controversial. While older ages had been suggested previously (34-38 Ma: Dilcher, Herendeen & Hueber, 1992;23-30 Ma: Grimaldi, 1995), Iturralde-Vinent (2001) restricted the age of fossiliferous Dominican amber to 15-20 Ma, most likely close to 16 Ma (Miocene). This restriction seems to have been largely accepted (Grimaldi & Engel, 2005;Penney, 2010).
Description. Body elongate with subparallel sides, brown with metallic sheen, colouration not differ between legs and remaining body except antennal clubs yellow.
Head: dorsal surface punctate, punctures simple, visible only on frons between eyes; clypeal margin with 4 teeth, mesal pair of teeth long, acute, subparallel, lateral pair of teeth triangular, acute and almost twice as short as mesal one; clypeal margin slightly angulate between lateral tooth and genal junction, without forming a tooth; clypeo-genal projection forms small acute tooth; eyes separated by a distance equal to two times their transverse width. It is noteworthy that the frons is elevated over the rest of the head and that the clypeo-frontal area forms a distinct carina; however, we tend to treat these two structures as likely to be artefacts of deformation.
Pronotum quadrate, anterior angles straight, surface covered by simple punctures, posterior fourth of midline with trace of depression.
Meso-metasternal suture obtusely angled; metasternum grooved posteriorly. Proleg with trochanterofemoral pit; protibia with 3 teeth, tips of basal and medial teeth separated by 1.5 times the distance between tips of apical and medial teeth; lateral outer margin of tibia denticulate; protibial spur spatulate with acute tip with a downward hook; spur reaches third tarsomere. Metacoxal lateral margin not expanded. Metatibia strongly curved in the middle. Length: 3.8 mm.
Observational note. The sex of this specimen cannot be identified. Although the apical spur of its protibia is spatulate and hooked downward, which might be considered a male trait, the degree of spur modification is not sufficiently extreme to rule out the female sex.
The holotype described here was subjected to taphonomic deformations that caused numerous longitudinal carinae across the entire body by squeezing the surface of the exoskeleton. These deformations are sometimes difficult to distinguish from the actual beetle morphology. In order to filter the deformations out, we used the bilateral symmetry of beetles and treated structures as artefacts if they were not symmetrical. However, in some cases the unequivocal identification of symmetry was doubtful, as mentioned in the description.
Despite the presence of strong elytral deformations that complicate observations, we assume that this species has a typical Canthochilum elytral morphology. Elytra in Canthochilum have a total of 8 striae. In some species, the external edge of the 7th stria adjoins either the longitudinal elytral carina or its trace or just the abruptly angulate edge. The presence of a carina in this species is supported by strong, bilaterally symmetrical (although deformed) elevations next to the 7th stria, while the presence of an 8th stria is suggested by a lateral row of setose punctures visible apically (the characteristic feature of many Canthochilum species). We conclude that this species has elytra with 8 striae and a strong carina that internally adjoins the 7th stria.
Diagnosis. This species can be unequivocally distinguished from all other Canthochilum species by its distinct elongate body shape and by the clypeal teeth forming a long, mesal pair of teeth and a shorter pair of lateral triangular teeth.
Etymology. This name derives from the combination of the last names of our colleagues Keith Philips and Michael Ivie, who worked extensively on the taxonomy of the genus Canthochilum.
Locality and age. See above under Canthochilum alleni.
Parsimony analysis of the full dataset yielded 66,400 trees of length 563, while analysis with ambiguous characters excluded yielded 29,400 of length 472. The convergence between runs in both Bayesian analyses was achieved after ∼2M generations and by the end of the runs the standard deviation of split frequencies was far below the acceptable limit of 0.01. Bayesian and parsimony analyses recovered similar phylogenetic placements of the fossil species. Lobateuchus parisii emerged within a clade formed by Ateuchus + Aphengium in both the analyses including all characters (BSV = 1, PP = 0.84) and the analyses with ambiguous characters excluded (BSV = 1, PP = 0.81) (Figs. 2E, 2F, 2H and 2I). The two fossil species of Canthochilum were recovered within a clade comprising two extant Canthochilum species, with the same support values in the analyses with and without ambiguous characters (BSV = 1, PP = 0.93) (Figs. 2C, 2D and 2G).
Alternative positions of fossil species in Bayesian analyses generally have significantly lower support than those revealed by the 50% majority consensus tree, nevertheless they are worth mentioning since they directly reflect the distribution of topological variability of the results. It is noteworthy that inclusion/exclusion of characters did not significantly change the results and mainly affected splits with low PP.
In the Bayesian analyses, Lobateuchus parisii is nested within the larger clade L (Figs. 2E and 2F), which also includes Ateuchus, Aphengium, Agamopus, Uroxys and Bdelyropsis. In turn the clade L forms a polytomy with many other dung beetle lineages and usually branches off early in the sampled trees. Most frequently, Lobateuchus forms a sister group with two Ateuchus species in both the analysis without ambiguous characters (PP = 0.53) and the analysis with ambiguous characters (PP = 0.47). Some splits support its sister position with taxa of the clade L, among which the most notable are: Ateuchus squalidus (PP with ambiguous = 0.19, PP without ambiguous = 0.30), Aphengium (PP with ambiguous = 0.15, PP without ambiguous = 0.1) and Agamopus (PP without ambiguous = 0.2). The remaining alternative splits illustrated in Figs. 2E and 2F have very low posterior probabilities.
The clade formed by Canthochilum species (including the two fossils) came up monophyletic with moderate posterior support (PP = 0.93). Interestingly the sample of the analysed splits does not contain any alternatives that support a non-monophyletic pattern for this clade due to the fact that such alternative splits have PP < 0.01 and therefore are not included in the summary statistics (sumt minpartfreq = 0.01) and are not discussed here. Since Canthochilum shows stable monophyly in our analyses, we focus our discussion on the alternative positions for this genus. The clade forming Canthochilum (clade C) makes up a polytomy with other scarabaeine clades, indicating the high uncertainty of the relationships between Canthochilum and other lineages. Among elementary splits the highest PP is found between Canthochilum and Bohepilissus (PP without ambiguous = 0.22) as well as Canthochilum and Paracanthon (PP without ambiguous = 0.12). All the remaining elementary splits are rare with posterior probabilities < 0.1. In addition to Canthochilum itself, these splits are formed by such taxa as Sinapisoma, Canthonella, Zonocopris, Arachnodes, and Apotolamprus.

Phylogenetic position of fossil Scarabaeinae from amber
The amber fossils Lobateuchus and Canthochilum are the best preserved scarabaeine fossils. Moreover, Lobateuchus represents the oldest reliable dung beetle fossil (see next section), which makes their phylogenetic assessment valuable for uncovering dung beetle evolutionary history.
The phylogenetic position of Lobateuchus (Figs. 1F-1I) in the same clade with Ateuchus and Aphengium is stable and supported in both the Bayesian and parsimony analyses. The authors who originally described Lobateuchus (Montreuil, Génier & Nel, 2010) probably noticed the body shape similarity between Ateuchus and Lobateuchus, thus deriving the name of the latter from the former and indirectly pointing out their similarity. However, in the discussion of the taxonomic position they suggest that Lobateuchus seems closely related to Haroldius. Our current morphological phylogeny lacks any representatives of Haroldius, thus precluding the test of this hypothesis.
However, the relatively high PP value supporting a nested position of Lobateuchus in the clade containing Ateuchus and Aphengium as inferred in present analyses must also be taken with caution because of data deficiency. Many important parts of Lobateuchus are covered with a layer of white impurities and hence hidden from direct observation. The piece of amber embedding the fossil is cracked and since it contains the holotype of Lobateuchus, it is preserved in a tightly closed glass capsule filled with Canada balsam for permanent storage. Capsule destruction and amber cleaning are needed to make the fossil specimen available for micro-CT investigation. These procedures along with potentially destructive power of x-rays significantly increase the risk of destroying the holotype specimen. Due to this reasons and MNHN policy the holotype was not available for tomography (decision of the curator of insect fossils at MNHN). It is unknown how much data would be available with tomography investigation (we doubt it would be much) but the investigation with stereomicroscope resulted in missing data for this taxon in the data matrix (72%, or 147 characters), which can bias the results of the analyses. To demonstrate this, let us evaluate the obtained results from the point of their character support. Clade Ateuchus + Aphengium is supported by one unique and three homoplasious synapomorphies (Tarasov & Génier, 2015). Two of the homoplasious synapomorphies are also preserved in Lobateuchus: epipleuron slightly protruded downward submedially and proleg lacking trochanterofemoral pit. In the analyses, these two synapomorphies appear to link Lobateuchus with Ateuchus + Aphengium, while the alternative placements are poorly supported due to extensive missing data. Thus, the clade formed by Lobateuchus, Ateuchus and Aphengium appears to be determined only by the characters that remain preserved in Lobateuchus. Although many phylogenetically important characters of internal body structures and male genitalia cannot be observed in Lobateuchus, the present evidence for the position of Lobateuchus is the best we could obtain. However, the limitations associated with the inference of the systematic placement of Lobateuchus must be kept in mind.
Unlike Lobateuchus, the placement of the two fossil Canthochilum species within the genus is well supported by both parsimony and Bayesian analyses, despite the extent of missing data (∼60% for each). The Canthochilum lineage is defined by two unique synapomorphies: the extremely reduced parameres and the highly modified shape of the aedeagal sclerite (Tarasov & Génier, 2015). None of these characters is observable in the fossil specimens, but in addition to these two synapomorphies Canthochilum species also share a unique set of diagnostic characters: (1) elytron with 8 distinctly visible striae; and (2) pro-, meso-, and metatarsus and apex of meso-and metatibia distinctly setose. In addition to these characters, some Canthochilum have the internal margin of the lateral elytral carina adjoining the 7th stria. The set of these three diagnostic characters is present in both described fossils, thus providing their well-corroborated position in Canthochilum by the phylogenetic analyses.
Notably, the body shape of the fossil C. alleni looks exactly like that of many extant Canthochilum species (e.g., C. magnum Philips &Ivie, 2008 andC. darlingtoni Matthews, 1969; see Philips & Ivie, 2008). Given an age for Dominican amber of 16 Ma, such close similarity points to a slow rate of morphological evolution in at least some Canthochilum lineages. The second fossil C. philipsivieorum differs from all other Canthochilum by its elongated body shape, but despite numerous deformations of its exoskeleton, all diagnostic Canthochilum characters are present in this species, which strongly supports its placement in Canthochilum.
The phylogenetic position of Canthochilum within Scarabaeinae, however, remains unresolved. Canthochilum forms a polytomy at the base of the scarabaeine tree in both parsimony and Bayesian consensus trees ( Figs. 2A and 2B). This polytomy is caused by the high uncertainty in sister-group relationships between Canthochilum and other main scarabaeine lineages. The alternative sister relationships for Canthochilum inferred by the Bayesian analyses (shown in Figs. 2C and 2D) suggest that this genus tends to cluster with genera such as Canthonella, Bohepilissus, Paracanthon and Zonocopris. This is consistent with the implied weight parsimony tree inferred by Tarasov & Génier (2015). Canthochilum species have not yet been included in any published molecular phylogeny, which makes such molecular analyses highly welcome in order to elucidate the placement of Canthochilum.
We believe that our character matrix provides a solid base for future studies of the phylogenetic relationships of extinct dung beetles. The updated catalogue of fossil Scarabaeinae and the detailed assessment of their placement should also be a useful tool for future studies of scarabaeine evolution and systematics.

Doubtful vs. Reliable Fossils
The fossil record of Scarabaeinae is poor, most likely due to their largely dung-associated lifestyle as living below the surface in terrestrial environments makes fossilization far less probable. Currently, the fossil record of Scarabaeinae comprises 35 described fossils (including the ones described in this study; see Catalogue section and Table 1). We also have evidence of "deltochiline-like" fossil(s) in Baltic amber which was unavailable for this study as we are in the process of locating the specimen. In the catalogue section below, we assess each fossil species based on its original description and illustrations and attempt to conclude whether its position in Scarabaeinae and its generic placement can be considered doubtful or reliable. Out of the 35 described fossil species, 14 cannot be confidently considered as scarabaeines due to the lack of any preserved character(s) that would unequivocally support their placement in Scarabaeinae; these taxa must therefore be treated as doubtful scarabaeines or even placed outside Scarabaeinae. Here we prefer to treat them as doubtful dung beetles whose placement in Scarabaeinae must be considered highly questionable. Contemporary revisions of old fossil descriptions usually tend to reduce the number of species described in a given taxon. For example, a recent revision of hydrophilid beetles described by Heer (1862) from the Ö hningen locality in Germany, known for its rich insect deposits (Selmeier, 1990), revealed numerous inaccurate family and genus attributions (Fikácek & Schmied, 2013). Heer (1862) Paulian, 1976) from the localities in Lake Victoria (Paulian, 1976) and Ö hningen, Germany (Heer, 1862) can be reliably considered as scarabaeines, but their generic placement will require a separate investigation of the specimens. Since this is beyond the scope of the present study, we adopt the generic placements used in their original descriptions or subsequent revisions.
Five extinct dung beetle genera have been described. We place four of them, namely, Ateuchites, Cretonitis, Prionocephale and Scelocopris, in the category of doubtful Scarabaeinae.

Distribution of fossils
The highest concentration of reliably identified scarabaeine fossils (Fig. 3) is observed in the Miocene (14 species; incl. 1 Mio-Pliocene), followed by the Pleistocene (5 fossil species). One fossil is known from the Oligocene (Rott, Gemany), and another (Lobateuchus), representing the oldest reliable dung beetle, dates back to the Eocene (53 Ma). The fossils are not uniformly distributed over the phylogenetic tree (e.g., that in Tarasov & Génier, 2015) and some taxa, namely Onthophagini + Oniticellini, Copris, and Phanaeus, have the largest number of fossil species (2-7 each). Fossil taxa tend to be concentrated in the large clade that includes Metacatharsius, Gymnopleurus, Anachalcos, Heliocopris and Onthophagini + Oniticellini likely due to the higher probability of fossilization in species-rich groups. The fossils described from the late Oligocene onwards can all be placed within extant genera, with many of them resembling extant species. This indicates that the main extant phylogenetic lineages of Scarabaeinae had at least evolved by the late Oligocene-mid Miocene (28-12 Ma).
Biogeographically, the majority of the fossils (16 species) are known from the Old World. However, the generally poor record of dung beetle fossils and the ambiguous taxonomic placement for several of them do not allow us to draw any biogeographic conclusions.

Scarabaeinae age based on fossil data
The oldest known fossils described as Scarabaeinae are Cretonitis copripes and Prionocephale deplanate, from the Lower and Upper Cretaceous, respectively. Fossilized dung beetle brood balls of the ichnogenus Coprinisphaera were recorded by Hasiotis (1999) and Hasiotis (2004) from the Upper Jurassic Morisson Formation in the Rocky Mountain Region of the U.S.A., but Bromley et al. (2007) questioned the interpretation of those fossils. A Coprinisphaera from the Lower Jurassic of So1tykó w, Poland, is neither described nor figured (Pie nkowski, 2004). Another Coprinisphaera specimen was recovered from the Upper Cretaceous Adamantina Formation in Brazil by Carvalho, Gracioso & Fernandes (2009). In addition, an ichnofossil representing a dinosaur coprolite with associated dung-filled tunnels similar to those made by dung beetles is known from the Cretaceous (Chin & Gill, 1996). These few inconclusive specimens are the only fossil record of putative dung beetles from the Mesozoic. As reliable identification and inference of the taxonomic position of Cretonitis and Prionocephale is not currently possible (see Catalogue section), the attribution of these Mesozoic fossils to Scarabaeinae is doubtful. The fossil brood balls need closer examination, but misinterpretation of spherical structures due to their scarcity of characters is easily possible. This means that currently "there are no reliable fossils or fossilized evidence which would support a Mesozoic origin for [scarabaeine] dung beetles" (Tarasov & Génier, 2015). The investigation of all described fossils conducted in this paper reveals that the oldest reliable scarabaeine fossil is Lobateuchus parisii, known from the Eocene (53 Ma). We suggest using this Eocene fossil in all relevant assessments of the minimum age for Scarabaeinae based on fossil data. Nevertheless, this Eocene fossil does not rule out the earlier origin of dung beetles. A recent comprehensive molecular phylogeny of Coleoptera (McKenna et al., 2015) that used calibration points from other beetle fossils dates the origin of Scarabaeinae at Upper Cretaceous which seems quite plausible. Contrary to that, the dating of dung beetle tree using published mutation rates (not fossil-based calibration points) yields relatively younger ages referring to Late Paleocene-Eocene: ∼56.5 Ma (Sole & Scholtz, 2010) and ∼45.3 Ma (Mlambo, Sole & Scholtz, 2015).

CATALOGUE OF FOSSIL SCARABAEINAE
This is an updated version of the catalogue of fossil Scarabaeinae, largely based on Krell (2000a) and Krell (2007). The fossil species are placed under two sections in alphabetic order. The first section lists species that we can confidently assign to Scarabaeinae, while the second lists those with doubtful assignment (see also Table 1). For every species we provide our justification explaining why it can or cannot be considered a scarabaeine, and we also express our concern if the current generic position of the species in Scarabaeinae needs to be separately investigated. Such species with doubtful generic position are marked with "?" before their genus names. The plausibility assessment for all species (except Lobateuchus and Canthochilum) is based on the literature data and illustrations provided in the original descriptions, and, for some of Heer's specimens, by photographs of the specimens.
Note. The species is described herein; see Systematic Paleontology section.
Note. The species is described herein; see Systematic Paleontology section.
Note. The well-illustrated original description (Pierce, 1946) and subsequent investigations (Matthews & Halffter, 1968;Miller, Gordon & Howden, 1981) strongly support the validity of this species and its placement in Copris.
Note. The well-preserved remains of elytra and pronotum support placement of this fossil in Eodrepanus (see Barbero, Palestrini & Roggero, 2009). Similar fragments possibly belonging to the same species were previously found by Gao et al. (2000) in Eemian deposits of the River Great Ouse in Cambridgeshire, UK.

?Gymnopleurus rotundatus Heer, 1862
Gymnopleurus rotundatus Heer, 1862: 73, pl. 6 (M Miocene, Ö hningen, Kesselstein, Germany). Heer, 1865: 378f;Heer, 1883: 404f;Scudder, 1891: 527;Handlirsch, 1906Handlirsch, -1908Meunier, 1921: 11;Krell, 2000a: 879. Note. The species is described from two specimens. The body shape and teeth of the anterior edge of the protibia of one specimen is mentioned in the original description (Heer, 1862). The photograph of the other specimen, which putatively belongs to the type series, shows the body shape (Fig. 4J). Those characters support the placement of this species within the Scarabaeinae. However, the poor preservation and lack of diagnostic characters at the generic level raise doubts about the generic placement of this species, and investigation of the type series is be needed to properly assess its generic placement and conspecificity.
Heliocopris antiquus Fujiyama, 1968 Heliocopris antiquus Fujiyama, 1968: 203 (L Miocene, Yanagida Formation, Noto, Japan). -Harusawa, 1994: 23;Krell, 2000a: 879;Krell, 2007: 21. Note. The description of this species is based on an incomplete fossil specimen consisting of elytra and part of the hind legs. The thorough original description and good illustrations provide solid evidence that this fossil is a true Heliocopris. This placement is supported by large elytron size, shape of tibiae and tarsi, and elytra with at least 7 striae (6 of which can be directly observed in the fossil, while traces of the 7th stria are observable on the internal margin of the lateral elytral carina).
Onthophagus mulleri. -Sphon, 1973: 52. Onthophagus statzi Krell, 1990: 187 (replacement name). -Krell, 2000a: 880;Krell, 2007: 22. Note. Based on the illustration of the body shape (Statz, 1952), this species should likely be assigned to Onthophagini. However, examination of the fossil specimen is required to clarify its taxonomic position within the tribe. The problems associated with placement of this fossil species in Onthophagus are the same as those discussed for O. bisontinus.
Note. This fossil is known by a well-preserved complete head, which strongly supports its placement in Phanaeus.

Ateuchites grandis Meunier, 1898
Ateuchites grandis Meunier, 1898: 114 (U Oligocene, Chattian, Armissan, Aude, France). -Handlirsch, 1906-Handlirsch, -1908Carpenter, 1992: 300;Krell, 2000a: 879;Krell, 2007: 19. Note. According to Martin Nose, curator at Bayerische Staatssammlung für Paläontologie und Geologie, München (where this species would likely be deposited), this fossil seems to be lost. The insufficient original description does not contain any characters which could shed light on the taxonomic position of this species or even support its placement within the superfamily Scarabaeoidea. We have to conclude that the taxonomic placement of this fossil remains a mystery and cannot support or refute its membership in Scarabaeinae.
Note. The limited original description (Horn, 1876) lacks any reasonable character that could unequivocally support the placement of this fossil in Scarabaeinae. Additionally, Horn (1876) seemed to hesitate assigning this species to Scarabaeinae, which likely indicates that poor preservation obscured the necessary diagnostic characters. Therefore, we doubt the assignment of this fossil to Scarabaeinae, but first-hand examination of the specimen is required to reach a substantiated conclusion.
Note. According to Janssens in Balthasar (1963: 79) this fossil rather represents a species of Zonitis Fabricius, 1775 from a different beetle family, Meloidae. Heyden may have confused two similar names, and erroneously assigned this fossil to the scarabaeine genus Onitis. The original description illustrates a body shape atypical for Scarabaeiodea. The slender tibiae without denticles also support this conclusion.
Note. The fossil is known from one elytron that indeed resembles those in the scarabaeine genus Onthophagus or in the tribe Onthophagini in general (Birket-Smith, 1977;Krell, 2010). However, the presence of only one elytron puts this fossil in a data deficient category where a reliable inference of the taxonomic position is impossible. Therefore, we mark this fossil as doubtful Scarabaeinae.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/ 10.7717/peerj.1988#supplemental-information.