New mid-Cretaceous cryptic slime mold beetles and the early evolution of Sphindidae (Coleoptera: Cucujoidea)

The cryptic slime mold beetles, Sphindidae, are a moderately diverse cucujoid beetle family, whose members are obligately tied to slime molds throughout their life. The fossil record of sphindid beetles is sparse; stem-sphindids and crown-group members of uncertain systematic placement have been reported from Cretaceous ambers. Here we review the Mesozoic fossil record of Sphindidae and report a new sphindid genus and species, Trematosphindus newtoni gen. et sp. nov., from Albian/Cenomanian amber from northern Myanmar (ca. 99 Ma). Trematosphindus is set apart from all other sphindids by the presence of distinct lateral cavities on the anterior pronotal angles. Our phylogenetic analysis identifies Trematosphindus as an early-diverging genus within Sphindidae, sister to the remainder of the family except Protosphindus, or Protosphindus and Odontosphindus. The new fossils provide evidence that basal crown slime mold beetles begun to diversify by the mid-Cretaceous, providing a valuable calibration point for understanding timescale of sphindid co-evolution with slime molds.


Introduction
The family Sphindidae, cryptic slime mold beetles, is a group of widespread beetles belonging to the diverse and, as currently conceived, paraphyletic polyphagan su-perfamily "Cucujoidea" (Forrester and McHugh 2010). Sphindidae are represented in the Recent fauna by only nine genera and approximately 66 valid extant species (J.V. McHugh, personal communication), though much of their biodiversity remains undocumented. For example, Forrester and McHugh (2007) mentioned that they have identified more than a hundred undescribed species in a single sphindid genus, Aspidiphorus Latreille. As their common name suggests, sphindid beetles feed exclusively on slime molds (myxomycetes) in both larval and adult stages, while most other aspects of their ecology remain elusive (Lawrence and Newton 1980;Burakowski and Ślipiński 1987). Sphindidae appears to be closely related to Protocucujidae, which is supported by multiple lines of morphological and molecular evidence (e.g., Leschen et al. 2005;Robertson et al. 2015;McKenna et al. 2019). Four extant subfamilies have been proposed within Sphindidae based on a morphological phylogenetic analysis, i.e., Protosphindinae, Odontosphindinae, Sphindiphorinae, and Sphindinae (McHugh 1993).
The fossil record of Sphindidae is very sparse. All putative pre-Quaternary sphindid fossils were reported from amber deposits. Kirejtshuk et al. (2015) described five species of Libanopsis Kirejtshuk from the Early Cretaceous Lebanese amber, and assigned them to a new subfamily, Libanopsinae. Later, Kirejtshuk et al. (2019) described the genus Burmops Kirejtshuk from the mid-Cretaceous Burmese amber, and moved the previous described genus Pleuroceratos Poinar & Kirejtshuk from Silvanidae to Sphindidae, treating both as members of the extant subfamily Protosphindinae. However, Pleuroceratos has externally open procoxal cavities , which is discordant with a placement in Protosphindinae. Tihelka et al. (2020) further found that the morphology of Pleuroceratos is actually characteristic of Phloeostichidae, and confirmed its position in Phloeostichidae with a formal phylogenetic analysis.
Here, we report a new sphindid genus and species from mid-Cretaceous Burmese amber, Trematosphindus newtoni gen. et sp. nov., adding to our knowledge on the Mesozoic diversity of this family.

Materials
The Burmese amber specimens studied herein (Figs 1-6) originated from amber mines near Noije Bum (26°20' N, 96°36' E), Hukawng Valley, Kachin State, northern Myanmar. The specimens are deposited in the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing, China. The amber pieces were trimmed with a small table saw, ground with emery papers of different grit sizes, and finally polished with polishing powder.

Fossil imaging
Photographs under incident light were taken with a Zeiss Discovery V20 stereo microscope. Widefield fluores-cence images were captured with a Zeiss Axio Imager 2 light microscope combined with a fluorescence imaging system. Confocal images were obtained with a Zeiss LSM710 confocal laser scanning microscope, using the 488 nm Argon laser excitation line. Images under incident light and widefield fluorescence were stacked in Helicon Focus 7.0.2 or Zerene Stacker 1.04. Confocal images were stacked with colour coding for depth in ZEN 2.3 (Blue Edition), or without colour coding in Helicon Focus 7.0.2. Microtomographic data were obtained with a Zeiss Xradia 520 Versa 3D X-ray microscope at the micro-CT laboratory of NIGP and analyzed in VGStudio MAX 3.0. Scanning parameters were as follows: NIGP175114 [isotropic voxel size, 3.2569 μm; power, 4 W; acceleration voltage, 50 kV; exposure time, 1 s; projections, 2501]; NIGP175115 [isotropic voxel size, 2.3931 μm; power, 3 W; acceleration voltage, 40 kV; exposure time, 3 s; projections, 3001]. Images were further processed in Adobe Photoshop CC to enhance contrast.

Morphological phylogenetic analysis
To evaluate the systematic placement of the new species, a morphological phylogenetic analysis was performed using both parsimony and Bayesian inference. The data matrix was mainly derived from a previously published dataset (McHugh 1993) (File 1, 2). Ericmodes sylvaticus (Philippi) (Protocucujidae) was selected as the outgroup. Character 4 in McHugh (1993) was originally coded for Protosphindus chilensis Sen Gupta & Crowson as "clypeus deeply embedded in head with one-third length or less projecting beyond anterior margin of head". In fact, the clypeus of Protosphindus Sen Gupta & Crowson is similar to other sphindids in having more than half of its length projecting beyond the anterior margin of the head ( fig. 1 in Sen Gupta and Crowson 1979), and hence the coding of the character was amended accordingly. Parsimony analysis was performed under implied weights using the program TNT 1.5 (Goloboff et al. 2008(Goloboff et al. , 2016. Parsimony analyses achieve highest accuracy under a moderate weighting scheme (i.e., when concavity constants, K, are between 5 and 20) (Goloboff et al. 2018;Smith 2019). Therefore, the concavity constant was set to 12 here, as suggested by Goloboff et al. (2018). Most parameters were set as default in the "new technology search", while the value for "find min. length" was changed from 1 to 100. A strict consensus tree was calculated, and standard bootstrap analysis was implemented by 10,000 pseudoreplicates, where the support values were shown as frequency differences (Goloboff et al. 2003).
A Bayesian inference for morphological traits was conducted using MrBayes 3.2.6 (Ronquist et al. 2012). Two MCMC analyses were run simultaneously, each with one heated chain and three cold chains. Trees were sampled every 10,000 generations. Analyses were stopped when the average standard deviation of split frequencies remained below 0.01. The first 25% of sampled trees were discarded as burn-in, and the remains were used to build a majority-rule consensus tree.
Trees were drawn with the online tool iTOL 5.7 (Letunic and Bork 2019) and graphically edited with Adobe Illustrator CC 2017.

Abbreviations
The following abbreviation of institution is used: NIGP -Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences. The following abbreviations of morphological characters are used: BL -apparent body length in dorsal view; BW -body width; EL -elytral length; HL -head length; HW -head width; PL -pronotal length; PW -pronotal width. Etymology. The generic name is composed of the Greek "trema", hole, and generic name Sphindus, in reference to the cavity at each anterior pronotal angle. The name is masculine in gender.

Remarks.
Trematosphindus is somewhat similar to several families in (or formerly in) the broadly defined Cucujoidea (e.g., Biphyllidae, Cryptophagidae, Boganiidae, and Protocucujidae) in the general habitus, shape of antennal club, or the presence of cavities/glandular pores. Boganiidae and Cryptophagidae can be easily ruled out as potential relatives of Trematosphindus, based on their externally open procoxal cavities (procoxal cavities externally closed in Trematosphindus and Sphindidae). Biphyllidae (now in Cleroidea) and also Cryptophagidae can be distingushed from Trematosphindus by their laterally closed mesocoxal cavities (mesocoxal cavities laterally open in Trematosphindus and Sphindidae). As the sister taxon of Sphindidae, the monogeneric family Protocucujidae shares many features with Trematosphindus (and other Sphindidae, Ślipiński 1998). However, Protocucujidae lacks the distinct elytral striae and the distinct basal row of depressions on ventrites 2-5 (elytral striae distinct and basal row of depressions on ventrites 2-5 present in Trematosphindus and at least most Sphindidae). Thus, we think Trematosphindus can be quite confidently assigned to Sphindidae, despite the presence of some characters unusual for a crown-group sphindid (e.g., supraocular grooves absent, anterior pronotal angles with cavities/glandular pores, scale-like setae; see also Discussion).

Results
The parsimony analysis under implied weights yielded two most parsimonious trees, where the placement of Notosphindus McHugh & Wheel differed (Fig. 7). The results are consistent with the parsimony analysis under equal weights by McHugh (1993). The Bayesian inference recovered the position of Notosphindus as sister to (Carinisphindus McHugh + Sphindus Chevrolat), though with only a low posterior probability (Fig. 7). In both analyses, the fossil Trematosphindus newtoni, was recovered in a relatively basal position within Sphindidae. In the implied-weighted parsimony analysis, Trematosphindus was recovered as sister to all extant sphindids except Protosphindus, while in the Bayesian inference it appeared to be sister to all extant sphindids except Protosphindus and Odontosphindus LeConte. Kirejtshuk et al. (2015)   represents a crown-group sphindid. Our phylogenetic analysis placed it as the sister taxon of all other extant sphindids except Protosphindus under parsimony, and as sister to all other sphindids excluding Protosphindus and Odontosphindus in the Bayesian analysis. Trematosphindus possesses a combination of apomorphic and plesiomorphic characters. It shares some plesiomorphic characters with Protosphindus, the earliest branching lineage in crown-group Sphindidae, including the absence of supraocular antennal grooves, the non-emarginate lateral margin of the clypeus, and the 11-segmented an-tenna (also shared with Odontosphindus and Sphindiphorus Sen Gupta & Crowson). However, Trematosphindus differs from Protosphindus in abdominal ventrites 2-4 with a distinct basal band of depressions (Fig. 2E), and the absence of raised carinae on elytra. It differs from Odontosphindus and Sphindiphorus, two other basal sphindid genera, as well as Protosphindus in having a pygidium with a longitudinal median groove (Fig. 3E). This well-defined median groove on pygidium was previously known in Aspidiphorus and Sphindiphorus (Forrester 2003). In other sphindids, the pygidium either en-  an,antenna;cl,clypeus;el,elytron;fr,frons;lbp,labial palp;md,mandible;mtf,metafemur;mttb,metatibia;mtts,metatarsus;mtv,metaventrite;mxp,maxillary palp;pc,procoxa;pf,profemur;pn,pronotum;ps,prosternum;ptb,protibia;sc,scutellum; tirely lacks any depressions or grooves, or has only an indistinctly defined median depression. The mandibles of Trematosphindus are probably flattened at apex, while in Protosphindus and Odontosphindus the mandibles are broad at apex (McHugh 1993).

Discussion
A notable character distinguishing Trematosphindus from all extant and fossil members of Sphindidae is the presence of a large oval cavity at each anterior pronotal angle (Figs 2F, 3C). External exoskeletal cavities have been widely reported in Coleoptera, and some of them have been suggested as a storage place for fungal transport (Grebennikov and Leschen 2010). Sphindidae are known to feed on myxomycetes (slime molds). Though these large pronotal cavities are absent in extant sphin-dids, the surface punctures of sphindids have been associated with slime mold spores, and therefore likely play a role in transporting slime molds (McHugh 1990(McHugh , 1993. As such, the large pronotal cavities in Trematosphindus may have fulfilled a similar function. The presence of surface punctures is a plesiomorphic for the family. Trematosphindus possibly evolved large lateral pronotal cavities later to further increase the efficiency of fungal transport. Alternatively, those cavities may be interpreted as glandular pores on callosity. A somewhat similar glandular callosity at anterior pronotal angles can be found in some Boganiidae (Crowson 1990;Escalona et al. 2015; Cai and Huang 2019) and some Cryptophagidae (Bousquet 1989; Otero and Johnson 2013; Otero and Pereira 2019). Historically, Sphindidae, Boganiidae and Cryptophagidae are all placed in the superfamily Cucujoidea. However, recent phylogenetic analyses recovered that this Cucujoidea sensu lato is paraphyletic and contains three separate clades (McKenna et al. 2019;Cai et al., 2021). Each of the three families mentioned above is placed in a different clade, and are therefore only distantly related to each other. Therefore, the openings at the anterior pronotal angles probably evolved independently in these families.
Slime molds are ubiquitous in humid substrates such as most wood, soil, and dung worldwide (Stephenson et al. 2008). While the spores and spore-bearing structures of slime molds provide food for a diverse range of bee-tles, sphindids stand out as the only family in which all species appear to be obligately associated with this food source (Lawrence and Newton 1980). The fossil record of slime molds is exceedingly scare, owing to the fragile nature of their fruiting bodies. Nonetheless, recent discoveries of exceptionally preserved slime molds in Burmese amber (Poinar and Vega 2019;Rikkinen et al. 2019) suggest that the group was diverse in saproxylic habitats by the mid-Cretaceous. The growing fossil record of Cretaceous sphindids provides corroborating evidence that slime molds were important players in terrestrial ecosystems in the Mesozoic. Future discoveries of fossils sphindids can shed further light on the co-evolution between slime molds and beetles. Because some sphindids display   a degree of host specificity (Lawrence and Newton 1980), the fossil record of cryptic slime mold beetles can provide valuable indirect calibration points for inferring the timescale of the slime mold tree of life.

Competing interests
The authors have declared that no competing interests exist.

Acknowledgements
We are grateful to Richard A. B. Leschen and Steven L. Stephenson for the help discussion, and Joseph V. McHugh and one anonymous reviewer for the detailed comments on the earlier version of this paper.
We also thank Su-Ping Wu for technical help in micro-CT reconstruction, and Yan Fang for technical help in confocal imaging.  Supplementary material 1