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Morphology of the mandibles and the first maxillae in the family Lithobiidae (Myriapoda, Chilopoda), with remarks on their phylogenetic significance

Abstract The mandibles and the first maxillae of 37 species of the family Lithobiidae (Myriapoda, Chilopoda) were investigated and compared to provide a structural overview and evaluate their significance for the systematics of the family. The species sampling focused on the genus Lithobius, examining 33 species of four subgenera (Lithobius, Monotarsobius, Sigibius, Ezembius), as this genus represents about half of the known diversity of Lithobiidae, including more than 500 assigned species and subspecies. The microstructures on the mandibular gnathal edge and the first maxillary telopodites and coxal projections were studied using scanning electron microscopy. Although having a similar structural pattern, we demonstrate that the microstructures are variable within and between species of adult specimens and commonly show intergradation. To check for intraspecific variability of microstructures and character stability, specimen sampling was extended for the two common Austrian species Lithobius dentatus and Lithobius validus, for which seven specimens depicted no major differences in the mandibular gnathal edge and the first maxillae. Our data suggest the presence of three characters in the mandibular gnathal edge and the first maxillae useful for lithobiid phylogeny. These characters were tested in a phylogenetic analysis together with previously described and novel morphological characters. Subgenera of Lithobius are mostly non‐monophyletic, and several other genera of Lithobiinae as well as other subfamilies group with particular species or clades of Lithobius. The results corroborate a close relationship between Disphaerobius loricatus and Lithobius (Ezembius) giganteus, strengthening the hypothesis that Pterygoterginae is nested within Lithobiinae and specifically within Lithobius, allied to L. (Ezembius) and Hessebius.

the first maxillary telopodites and coxal projections were studied using scanning electron microscopy.
Although having a similar structural pattern, we demonstrate that the microstructures are variable within and between species of adult specimens and commonly show intergradation. To check for intraspecific variability of microstructures and character stability, specimen sampling was extended for the two common Austrian species Lithobius dentatus and Lithobius validus, for which seven specimens depicted no major differences in the mandibular gnathal edge and the first maxillae. Our data suggest the presence of three characters in the mandibular gnathal edge and the first maxillae useful for lithobiid phylogeny. These characters were tested in a phylogenetic analysis together with previously described and novel morphological characters. Subgenera of Lithobius are mostly non-monophyletic, and several other genera of Lithobiinae as well as other subfamilies group with particular species or clades of Lithobius. The results corroborate a close relationship between Disphaerobius loricatus and Lithobius (Ezembius) giganteus, strengthening the hypothesis that Pterygoterginae is nested within Lithobiinae and specifically within Lithobius, allied to L. (Ezembius) and Hessebius.

K E Y W O R D S
first maxillae, Lithobiidae, mandibles, morphology, phylogeny

| INTRODUCTION
The mandibles are a key character underpinning the Mandibulata hypothesis for arthropod interrelationships, unifying myriapods, hexapods, and crustaceans as a monophyletic group (Snodgrass, 1950).
This monophyly is additionally supported by homologous structures on the most distal part of the mandible, the mandibular gnathal edge (Edgecombe, Richter, & Wilson, 2003). The gnathal edge mainly consists of the pars incisiva and the pars molaris, and variably by the lacinia mobilis, each of which bears several microstructures . These microstructures provide phylogenetic information at finer taxon levels within the mandibulate subphyla (see Edgecombe et al., 2003 and references therein). For example, for representatives of the myriapod class Chilopoda, the microstructures of the mandibular gnathal edge have proven to be important in several studies addressing the morphology, taxonomy, systematics, and phylogeny of different orders (e.g., Edgecombe, 2004a;Edgecombe & Giribet, 2004;Edgecombe, Giribet, & Wheeler, 2002;Edgecombe & Hollington, 2002;Hollington & Edgecombe, 2004;Koch & Edgecombe, 2012;Koch, Edgecombe, & Shelley, 2010). In the order Lithobiomorpha, as in the three other chilopod orders that comprise the Pleurostigmophora, the mandibular gnathal edge consists principally of the pars incisiva, which bears aciculae (pectinate lamellae), the mandibular teeth (dentate lamina) and branching bristles. A well-developed lobe equipped with trichomes dorsally viz. the pulvillus (sensu Crabill, 1960) or Haarpolster (sensu Verhoeff, 1918), was considered as the pars molaris . However, the pulvillus has been reinterpreted in recent studies as the dorsalmost part of the pars incisiva whereas the pars molaris is confined to a sclerite that bears the mandibular condyle, the lamina condylifera (Koch & Edgecombe, 2012).
In addition to the mandibles, the first maxillae of Lithobiomorpha have also been documented in some systematic works (e.g., Edgecombe, 2004a;Edgecombe & Giribet, 2004;Edgecombe & Hollington, 2002;Hollington & Edgecombe, 2004). The first maxillae consist of paired coxosternites and twosegmented telopodites, each with a basal and a distal article, and a coxal projection. The presence of plumose bristles along the inner margin of the distal article of the maxillary telopodite is autapomorphic for Lithobiomorpha (Edgecombe, 2004b).
The mandibles and the first maxillae have provided characters that were used in combination with molecular data for phylogenetic analyses on the lithobiomorph family Henicopidae (e.g., Edgecombe & Giribet, 2003;Koch & Edgecombe, 2008).
To date, only a few representatives (13 species and eight genera) of the family Lithobiidae were investigated in detail for their mandibular and first maxillary microstructures but mainly as an outgroup in analyses of henicopids, and the character coding was invariant Edgecombe & Giribet, 2004;Koch & Edgecombe, 2008). Therefore, it has remained unclear if the mandibles and the first maxillae of the Lithobiidae bear useful characters for systematic and phylogenetic analyses. This is especially the case for the species-rich genus Lithobius Leach, 1814 known to accommodate more than 500 species/subspecies (see Bonato et al., 2016;Zapparoli & Edgecombe, 2011). Thus, any new morphological or molecular information is of potential value to help solve some of the unclear species-interrelationships within the genus, its phylogeny and evolutionary history. The genus Lithobius was hitherto subject to one morphological phylogenetic analysis based on 40 characters resolving it as non-monophyletic, with a few species being more closely related to the genera Australobius Chamberlin, 1920, Hessebius Verhoeff, 1941 and Pleurolithobius Verhoeff, 1899 (Koch & Edgecombe, 2008). A more recent study on the peristomatic structures (epipharynx and hypopharynx) of 32 species of the same genus highlighted eight new characters, which could also be systematically useful .
In this study, we focus on the mandibles and maxillae of the family Lithobiidae. Therefore, we examined and compared the mandibular gnathal edge and the first maxillae of 37 species using scanning electron microscopy. Among these, 33 species were from the genus Lithobius, including four subgenera: Lithobius Leach, 1814; Monotarsobius Verhoeff, 1905;Sigibius Chamberlin, 1913 andEzembius Chamberlin, 1919, as well as four species from allied lithobiid genera, viz. Eupolybothrus Verhoeff, 1907;Disphaerobius Attems, 1926 andNeolithobius Stuxberg, 1875. The aim of this work is twofold: First, we provide a descriptive and comparative overview on the morphology of the mandibular gnathal edge and the first maxillae of the studied species. Second, we evaluate the phylogenetic importance of the investigated microstructures together with other morphological characters for the systematics of Lithobiidae, especially within the genus Lithobius. Intraspecific variability and plasticity of the structures were checked with an extended specimen sampling for two Lithobius species. Phylogenetic analyses using maximum parsimony and maximum likelihood of the newly described characters together with characters from other morphological systems were performed, including characters extracted from other Lithobiidae, Henicopidae, and two representatives of Scutigeromorpha as outgroup. We further compared the mandibular gnathal edge of Disphaerobius loricatus (Sseliwanoff, 1881) and Lithobius (Ezembius) giganteus Sseliwanoff, 1881 and, together with the phylogenetic results, verified that it supports previous suggestions on their relationships based on external morphological and peristomatic characters (Farzalieva, Nefediev, & Tuf, 2017;Ganske et al., 2018). This is of particular systematic importance because Disphaerobius is usually classified separately from Lithobius, in the subfamilies Pterygoterginae and Lithobiinae, respectively.

| Material
The studied material consists of 62 specimens belonging to 37 species preserved in 70 or 95% EtOH (Table 1)

| Sample preparation
The mandibles and/or first maxillae were detached from the head with forceps and dissecting needles in one to seven adult male or female individuals per species (see Table 1; Supporting Information 1). Manual multifocus images of the sclerotized parts of the mandibles and first maxillae were obtained with a Nikon Eclipse Ni compound microscope (LM) equipped with a Nikon DS-Ri2 camera using NIS-Elements Microscopic Imaging Software. For scanning electron microscopy (SEM), the specimens were: (a) cleaned in an ultrasonic bath (50-60 Hz) for 5 to 10 s (maximum); (b) dehydrated in an ascending alcohol series (70%, 80%, 90%, 96% EtOH, 2 × 10 min each); (c) covered with HMDS (hexamethyldisilazane) and air dried overnight. The mouthparts were mounted on sticky aluminium tape fixed on aluminium stubs, glued with conductive silver and coated with platinum (Leica EM SCD500). Then, they were studied and photographed with a JEOL JSM 6610-LV at an accelerating voltage of 10-15 kV. LM and some SEM images were stacked and processed using Zerene Stacker

| Morphological characters and coding
The data matrix in Table 2 codes for 62 morphological characters described below under "Morphological characters and observations." The character matrix (Table 2) was compiled using Mesquite version 3.40 (Maddison & Maddison, 2018) and is available as RTF-file (Supporting Information 2).
Information on the peristomatic structures of the species included in the phylogenetic analysis was obtained from Koch and Edgecombe (2008) and Ganske et al. (2018). Other morphological information was acquired from Koch and Edgecombe (2008), new observations, or from literature resources.

| Phylogenetic analysis
Cladograms were rooted with Scutigeromorpha as an outgroup to Lithobiomorpha. Parsimony analyses under equal weights were conducted using TNT version 1.1 (Goloboff, Farris, & Nixon, 2003) with heuristic searches involving 1,000 random addition sequences and TBR branch swapping saving up to 100 trees per replicate. Nodal support was measured using Jackknife resampling (Farris, 1997) and Bremer support (Bremer, 1994), both with TNT. Jackknifing used 1,000 replicates with 36% deletion, each replicate involving a heuristic search. Bremer support was calculated from collections of suboptimal trees obtained by heuristic searches. Implied weighting implemented in TNT used the same heuristic search parameters as for equal weights, across concavity constants k = 2, 3, 4, and 5. Multistate characters 17 and 18 were ordered (additive), the others unordered (nonadditive). Character optimisations were examined using ASADO version 1.61 (Nixon, 2004 3 | RESULTS

| Mandibles
In all examined species, the mandibular gnathal edge ( Figure 1) agrees with the previous descriptions provided for the same family .
a. Aciculae (aci, aco) and pinnules (pi) The aciculae are present on the ventral part of the gnathal edge ( Figure 1) and occur as an inner and an outer row (Figures 1 and 2a,b).

| First maxillae
In all examined species, the first maxillae ( Figure accessory denticles on the mandibular teeth are separated by a distinct median ridge (arrow) and pass abruptly into the trichomes of the pulvillus, the two dorsalmost teeth are bicuspid, (b) details from Figure 4a with accessory denticles of different scale types and a fringe of "scale-like" branching bristles; (c) Lithobius (Lithobius) piceus, multifurcate and structured or smooth accessory denticles divided by a less distinct ridge (arrow), right mandible, right is dorsal; (d) Lithobius (Monotarsobius) aeruginosus, multifurcate and structured accessory dentilces, right mandible, right is ventral; (e) Lithobius (Lithobius) pyrenaicus, multifurcate or triangular and structured or smooth accessory denticles, right mandible, right is ventral; (f) Lithobius (Lithobius) castaneus, multifurcate or triangular and structured or smooth accessory denticles, right mandible, right is ventral. Abbreviations: ad-Accessory denticles on mandibular teeth; fr-Fringe of branching bristles on mandibular gnathal edge; pu-Pulvillus on mandible; tpu-Trichomes on pulvillus trichodea and microtrichodea (Figure 17b-d). In D. loricatus, the coxal projection bears multiple rows of stout plumose bristles and scattered sensilla trichodea and microtrichodea (Figure 17a). The sensilla trichodea expand more posteriorly along the coxal projection than the other types of trichomes and setae (Figures 15a,b,e, 16a,c,d, and 17a-c).
State (0) is shared by species of the subgenera Sigibius, Monotarsobius (except for L. holstii [Eason, 1973, Figure 52] and L. crassipes [Koren, 1992, FIGURE 5 Details of mandibular gnathal edge of Lithobiidae (SEM). (a, b) triangular and smooth accessory denticles that pass continuously into the trichomes of the pulvillus; the trichomes of the pulvillus transition continuously from branched to simple ones; the branching bristles of the fringe have a widened base, branch on the upper two-thirds and are not in contact with the trichomes of the pulvillus, right mandible, top is ventral, (a) Lithobius (Ezembius) giganteus, (b) Disphaerobius loricatus; (c) Lithobius (Lithobius) nodulipes, the trichomes of the pulvillus transition from branching to simple ones with a break (arrow), right mandible, top is ventral; (d) Lithobius (Lithobius) dentatus, the transition of accessory denticles to the trichomes of the pulvillus is continuous, left mandible, right is dorsal; (e) Lithobius (Lithobius) fagei, the branching bristles are in contact with the trichomes of the pulvillus (arrow) and there is a trichome-free strip between accessory denticles and trichomes of the pulvillus, left mandible, right is dorsal; (f) Lithobius (Lithobius) cyrtopus, the branching bristles of the fringe are not in contact with the trichomes of the pulvillus and the accessory denticles transition abruptly into the trichomes of the pulvillus, right mandible, top is ventral. Abbreviations: ad-Accessory denticles on mandibular teeth; fr-Fringe of branching bristles on mandibular gnathal edge; pu-Pulvillus on mandible; tpu-Trichomes on pulvillus Coding is inapplicable for Scutigeromorpha and Henicopidae.
Information is missing for some species (see Table 1).
pontis (Chamberlin, 1911, p. 34). Representatives of the subgenera Monotarsobius and Sigibius as well as Scutigeromorpha possess rounded tergites without projections (state (0)), and this is also the case of several species of the subgenus Lithobius and most Henicopidae.
62. Dense concentration of short setae on posterior part of sternite and coxa 15 of male: (0) absent; (1) present. (see Koch & Edgecombe, 2008, char. 26) A dense concentration of short setae on the posterior part of the sternite and the 15th coxa of males was coded present (state (1)) for L. giganteus and H. plumatus in Koch and Edgecombe (2008). We observed the presence of those setae in L. electus (Silvestri, 1935, Figure 13 and new observation) as well. Interestingly, these setae are absent in males of D. loricatus, but were described for its congener Disphaerobius svenhedini (Verhoeff, 1934) (Farzalieva et al., 2017, Figure 8).

| Phylogenetic analyses
One-hundred and thirty-eight shortest cladograms of 303 steps were obtained under equal weights (consistency index 0.265, retention index 0.541) using the matrix with 62 morphological characters ( Table 2). The strict consensus tree is shown in Figure 18 and all characters are optimized on one of the 138 minimal-length trees (unambiguous optimizations; Figure 19). Under implied weights, the same two shortest trees were retrieved for k = 4 and 5 (see consensus tree of k = 4 in Figure 20 with a fit of 21.2880). The consensus tree constructed from 1,000 bootstrap trees under maximum likelihood analysis with a log-likelihood of −1,437.394991 is illustrated in Figure 21.
Although Lithobiidae and Henicopidae are rendered monophyletic in the ML analysis (Ultrafast Bootstrap 100%; Figure 21) and under Based on implied weighting Ethopolyinae is monophyletic, but this is not the case under equal weights or ML (Figures 18, 20-21

| Mandibles
In contrast to the family Henicopidae, which displays either a single row or two rows of aciculae (Edgecombe, 2004a [char. 50]; Edgecombe & Giribet, 2004 [char. 151]), the examined species of the family Lithobiidae showed a consistent number of two rows of aciculae (see also Edgecombe & Giribet, 2004;Edgecombe & Hollington, 2002 (Figure 5a,b) as was described and illustrated for the henicopid Zygethobius pontis Chamberlin, 1911(Figure 6c in Edgecombe et al., 2002. Edgecombe and Giribet (2004 [char. 153]) stated that accessory denticles are ubiquitous in Lithobiomorpha, though also present in the scolopendromorph family Cryptopidae. Drawing on the fairly large number of examined lithobiid species and specimens in this study, we provide additional information on the differences in shape (triangular/ multifurcating, simple/structured), including intergrading forms.
Accessory denticles as multifurcating scales were only described for the genus Lamyctes (Figure 7b in  while Edgecombe & Giribet (2004) coded these denticles (see char. 156) as simple and triangular only. Furthermore, the transition of multifurcating scales to simple scales was not described in detail before, although it was depicted in Lamyctes emarginatus (Newport, 1844) for example ( Figure 7b in . If accessory denticles are structured, they can be equipped with tubercles or spines. Edgecombe (2004a) described "tuberculate accessory denticles" in character 51 (state (2)) for the henicopid subgenus Haasiella as well. However, the mandible of a studied moulting specimen of L. lapidicola showed tuberculate denticles on the old part and spinous multifurcating scales on the freshly moulted mandibles, indicating that tuberculate accessory denticles are perhaps the result of attrition (e.g., due to chewing).
It could be worth cross-checking individuals of different stadia to verify the state of this character and possible environmental influence.
The grooved ridge on the ventral edge of a mandibular tooth interrupts the accessory denticles in all examined lithobiid species, consistent with the findings of Edgecombe et al. (2002 [char. 27]) for the family. There are however slight differences in the expansion of the ridge along the tooth.
For the Lithobiidae, we revised the description of character 28 in , which describes the connection of the accessory denticles and the trichomes of the pulvillus. It now appears subjective to define the elongation as abrupt or progressive according to Edgecombe & Giribet (2004 [char. 155]). We now specify if there is a contact between the denticles and the trichomes of the pulvillus or not in character 32: continuous transition of accessory denticles into trichomes of pulvillus: (0) absent (trichome-free strip); (1) present (either gradual or abrupt). This character conflicts with monophyly of the subgenera Lithobius and Sigibius ( Figure 19).
As a new character for lithobiid systematics, we suggest the occurrence of an internal spinulation of the mandibular gnathal edge (Figures 8 and 9), as follows (char. 31): spinulation on internal side of mandibular gnathal edge posterior to margin of ventralmost mandibular tooth: (0) absent; (1) present (simple spines or branching bristles).
The presence of an internal spinulation was hitherto illustrated in L.
However, the internal spinulation appears to be absent on illustrations  Sensilla microtrichodea occur as a cluster or row between the coxal projection and the telopodite (Figure 14b-f) and vary in number between species, conspecifics and within an individual. This high variation makes it impossible to define a character. A cluster of sensilla microtrichodea was described by Edgecombe and Hollington (2002) for Australobius scabrior as well and, being present in Henicopidae as well as Lithobiidae, was proposed as an autapomorphic character for Lithobiomorpha (Edgecombe, 2004b).

| Comments on the variation of microstructures
Ecological influence may affect the expression of characters within a single species due to food availability, habitat choice or environmental conditions during development (e.g., Tobias, 1969). For example, intraspecific variation of the aciculae was described for the scolopendromorph Ectonocryptoides quadrimeropus Shelley & Mercurio, 2005 by Koch et al. (2010;p. 56). However, the described mandibular and first maxillary characters are invariant between conspecifics of the genus Henicops Newport, 1845 from different populations (Edgecombe, Colgan, & Sharkey, 2006 [char. 10-13]). Our study of seven individuals of the species L. validus and L. dentatus from different locations in Austria showed a low intraspecific variation of microstructures. There are mainly differences in the number of bristles on the internal side of the mandibular gnathal edge and the number of aciculae, reported above in "Results". Besides examining several individuals of the same species, the investigation of different stadia per species is necessary to check for structural variability and, in a more systematic context, character stability as already suggested by Tobias (1969).
There are also differences in number of internal spines/bristles and aciculae of left and right mandibles or sensilla microtrichodea (between the coxal projection and telopodite) of left and right first maxillae of a single individual. The same was observed for the number of aciculae and even number of mandibular teeth in Henicops washpoolensis (Edgecombe & Hollington, 2005).

| Phylogenetic implications
As revealed by their cladistic analyses based on 40 morphological characters earlier, Koch and Edgecombe (2008) showed that the genus Lithobius is non-monophyletic. The present phylogenetic results with an extended set of taxa of the genus Lithobius and more charac- shared characters from external morphology identified in a recent taxonomic work (Farzalieva et al., 2017). Here, we further compared the mandibular gnathal edge of Disphaerobius loricatus, Hessebius plumatus (Zalesskaja, 1978) and Lithobius (Ezembius) giganteus and noted strong similarities, especially in the presence of simple spines on the internal side; the low number of simple, triangular accessory denticles; and the similar pattern of the fringing bristles, these branching distally only and showing broad bases. These characters and the phylogenetic results, which depict strong support for the node grouping D. loricatus and L. (E.) giganteus, provide additional evidence in favour of the inference made by Ganske et al., (2018) that Pterygoterginae is an invalid subfamily because its recognition renders Lithobiinae paraphyletic.
Nevertheless, our data are sensitive to character weights, resulting in unstable groupings, and most nodes receive low support values. This is likely a consequence of the low number of characters in relation to the number of species, and a rather high degree of homoplasy, as measured by the Consistency Index (CI 0.265).

| Conclusion/outlook
The mandibles and the first maxillae of Lithobius and more generally the Lithobiidae might be useful as a source for new morphological characters, but do not provide as much information as their homologous structures in Henicopidae (see . However, new information on microstructures, for example, from the mandibles, the first maxillae and the peristomatic structures (Ganske et al., 2018) might be useful for taxonomists using SEM data as a data source to delimit cryptic species (e.g., Pilz, Melzer, & Spelda, 2008).
As stated by Edgecombe (2007, p. 337), "… the taxonomic sampling in microanatomical studies is limited, generally involving detailed descriptions of single or few species within each of the major chilopod groups and as such these data are informative for questions involving relationships between chilopod orders but are generally neutral on taxonomic problems at finer levels, even between families." The expanded taxon sampling in this study mainly based on the genus Lithobius showed that there are differences suggesting at least three new characters. The considerable degree of sensitivity of our results to character weights and optimality criteria (i.e., parsimony versus likelihood) and weak support for most deep nodes within Lithobiidae suggest that molecular data will be especially valuable for advancing lithobiid phylogeny. The morphological characters compiled in this study will be combined with sequence data in a forthcoming phylogenetic analysis focusing on the genus Lithobius.