The peristomatic structures as a source of systematic characters in the genus Lithobius Leach, 1814 (Myriapoda, Chilopoda)

Abstract Morphological characters have been widely used in centipede systematics. Here, we aim to obtain morphological information from the preoral chamber and peristomatic structures of lithobiomorph centipedes, with taxonomic sampling focused on the species-rich genus Lithobius Leach, 1814. Towards this goal, we (i) examined the epipharynx and hypopharynx of 32 species belonging to four subgenera of the genus Lithobius, viz. Lithobius Leach, 1814, Monotarsobius Verhoeff, 1905, Sigibius Chamberlin, 1913 and Ezembius Chamberlin, 1919 using light and scanning electron microscopy, (ii) searched for phylogenetically informative characters and (iii) described interspecific variation. Three species of the lithobiid genera Eupolybothrus Verhoeff, 1907, Disphaerobius Attems, 1926 and Neolithobius Stuxberg, 1875 were additionally examined and considered as likely outgroups. New characters and character states are proposed as additions to current phylogenetic datasets. Similarities in the peristomatic structures ally Disphaerobius with Lithobius (Ezembius), suggesting that the subfamily Pterygoterginae is nested within Lithobiinae and Lithobius.


Introduction
The peristomatic structures -the epipharynx and hypopharynx -of Chilopoda have hitherto been studied in the orders Scutigeromorpha, Lithobiomorpha, Geophilomorpha (Koch and Edgecombe 2006, 2012, and Scolopendromorpha (Edgecombe andKoch 2008, 2009) revealing numerous characters bearing phylogenetically useful information (see also Koch et al. 2010, Vahtera et al. 2013. Two characters of the peristomatic structures, viz. 'bottle-shaped' epidermal glandular shafts on the epipharynx and a characteristic shape of the hypopharynx, support the monophyly of the order Lithobiomorpha, whereas paired oblique rows of spines on the clypeal part of the epipharynx are thought to be apomorphic for the family Lithobiidae . Until now, Lithobius, the most diverse genus in Chilopoda, with more than 500 described species Edgecombe 2011, Bonato et al. 2016), is resolved as non-monophyletic on the basis of morphological data. Particular species were recovered in cladistic analysis as most closely related to the genera Australobius Chamberlin, 1920, Hessebius Verhoeff, 1941, and Pleurolithobius Verhoeff, 1899, and this likely applies to other genera of Lithobiinae as well, if not even some of other five subfamilies of Lithobiidae (for current classification of this family see Zapparoli and Edgecombe 2011). However, broad information on species-interrelationships is still missing and the monophyly of subgenera remains questionable, being based on combinations of the same set of characters (Edgecombe 2007). Aiming to obtain further morphological information from the peristomatic structures of Lithobius to evaluate whether those might be useful for identifying clades within this very large genus, we study the epipharynx and hypopharynx of 32 species of Lithobius, including the subgenera Lithobius (23 spp.), Sigibius (3 spp.), Monotarsobius (5 spp.), and Ezembius (1 sp.) using light and scanning electron microscopy. We describe the variation of the microstructures between species and propose new characters for which patterns of variability suggest a potential for phylogenetic analyses. Additionally, we examine species of the lithobiid genera Neolithobius Stuxberg, 1875 (Lithobiinae), Eupolybothrus Verhoeff, 1907 (Ethopolyinae), andDisphaerobius Attems, 1926 (Pterygoterginae), for comparison with Lithobius.

Material
The studied material consists of 61 specimens belonging to 35 species preserved in 70% or 95% EtOH (Table 1), deposited at the Natural History Museum Vienna (NHMW), the Natural History Museum London (BM/NHMUK) and the Hungarian Natural History Museum Budapest (HNHMB). All material was examined with light and scanning electron microscopy.

Sample preparation
The epipharynx and hypopharynx were dissected from the preoral chamber as described in Koch and Edgecombe (2008) in one to four adult male or female individuals per species. Multifocus images of the sclerotized parts of the epipharynx and hypopharynx were obtained with a Nikon SMZ25 stereomicroscope equipped with a Nikon DS-F2.5 camera using NIS-Elements Microscope Imaging Software with an Extended Depth of Focus (EDF) patch. For scanning electron microscopy (SEM), the specimens were: (1) cleaned in an ultrasonic bath (50-60 Hz) for 5 to 10 seconds (maximum), occasionally in a solution of 15% hydrogen peroxide for 2 hours; (2) dehydrated in an ascending alcohol series (70%, 80%, 90%, 96% EtOH, 2 × 10-15 min each); (3) air dried overnight (or covered with HMDS) or critical point dried (Leica 300 CPD).
Specimens were mounted on aluminium stubs equipped with a sticky aluminium tape, glued with conductive silver, coated with platinum (Leica EM SCD500) and studied with a JEOL JSM 6610-LV at an accelerating voltage of 15 kV. Figures were processed with Adobe Photoshop CS6 and assembled in Adobe InDesign CS6. Terminology follows Koch and Edgecombe (2008).
ally with up to two or more rows on the lateral sides (Figs 2C, 3A, 4A, 5B), or consistently two to more rows (Figs 2A, 3B). The number of glandular shafts varies from 19 in L. microps to more than 80 in L. validus and is generally higher in larger species. The number of glandular shafts can also differ between individuals of the same species, e.g. 20-22 in L. aeruginosus or 42-48 in L. pyrenaicus. Figure 2. Details of transverse bulge, 'bottle-shaped' epidermal glandular shafts and median spine field of the epipharynx of Lithobiidae. A Disphaerobius loricatus; no transverse bulge; consistently two rows of 'bottle-shaped' epidermal glandular shafts; narrow and slightly medially widening median spine field B Lithobius (Lithobius) pyrenaicus; parallel aligned margins of a single transverse bulge; one row of 'bottle-shaped' epidermal glandular shafts; rhomboid and medially widening median spine field C Lithobius (Lithobius) fagei; single transverse bulge with parallel margins; more than one row of 'bottle-shaped' epidermal glandular shafts laterally; laterally widening median spine field D Lithobius (Sigibius) microps; single transverse bulge with non-parallel margins; subequal width of median spine field E Lithobius (Lithobius) mutabilis; single transverse bulge with non-parallel margins; one row of 'bottle-shaped' epidermal glandular shafts; subequal width of median spine field F Lithobius (Monotarsobius) aeruginosus; single transverse bulge with non-parallel margins; one row of 'bottle-shaped' epidermal glandular shafts; subequal width of median spine field. bu -transverse bulge, gl -'bottle-shaped' epidermal glandular shafts, msp -median spine field.  Fig. 4A) and non-parallel aligned margins; one row of 'bottle-shaped' epidermal glandular shafts medially with a transition to two rows laterally (see Fig. 4A); weak transverse furrow distally to the transverse bulge (arrow); laterally widening median spine field B Lithobius (Lithobius) piceus; weak transverse furrow (arrow) distally to the single transverse bulge (non-parallel margins); irregular two rows of 'bottle-shaped' epidermal glandular shafts; subequal width medially and laterally of median spine field C Lithobius (Lithobius) lucifugus; distal and proximal transverse bulges with surface striation (see Fig. 4B-C) D Eupolybothrus (Eupolybothrus) grossipes; distal and proximal transverse bulges; medially widening median spine field. butransverse bulge, bud -distal transverse bulge, bup -proximal transverse bulge, gl -'bottle-shaped' epidermal glandular shafts, msp -median spine field.
Proximal to the 'bottle-shaped' epidermal glandular shafts is a median spine field arranged as a wide or a narrow band with a subequal width, medially or laterally widened and consisting of a variable number of branching spines (Figs 1A, C, 2, 3A-B, D: msp, 5D, 6A, D). The spines are always directed towards the labral part of the epipharynx but differ in shape, size and texture. The shape can be scaly, apically furcated or not (Figs 5D, 6).
Paired labral bristle bands occur on the distal bars on each side of the tooth plate ( Fig. 1C: bdb). The bristle bands consist of long, simple bristles medially with a gradual transition to branching bristles laterally (Fig. 7). The branching bristles occur with a few or several outer rows, more or less covering the distal bar ( Fig. 7A-B). The bristles point dorsomediad towards the transverse bulge. The branching bristles on the distal bar of the outer rows are generally 'hassock-like' (Fig. 8A-C), but they can also be 'palmleaf- like' as for L. validus (Fig. 8D) or 'comb-like' in L. trebinjanus (Fig. 8E). The base of the branching bristles ranges from narrow to wide, with intermediate forms (Fig. 8).
The labral branching bristles on the distal bar expand towards the proximal part in a continuous manner (  On the lateral borders of the distal bar, ovoid tubercles are observed in nearly all investigated species (Figs 1C: tub, 10H).
The median sensilla cluster ( Fig. 1C: msc) on the clypeal part is always transversely aligned. It displays a highly variable interspecific arrangement of the sensilla. These sensilla can be arranged in line (Fig. 10A inset), in an offset-pattern (Fig. 10A, C-D) or symmetrical (Fig. 10B). The number of sensilla in the studied species varies between five in L. aeruginosus to 65 in E. grossipes (Fig. 10D). Variation of the arrangement and number of sensilla is also recorded in individuals of the same species (e.g. L. tenebrosus and L. aeruginosus).
Proximal to the clypeal part pairwise lateral spine fields are present bordering the median sensilla cluster except for N. aztecus (Fig. 10C), D. loricatus and E. grossipes (Fig. 10D) in which the sensilla overlap with the spine fields ( Fig. 1C: lsp). The lateral spine fields are arranged in one oblique row or more than one row (Fig. 10A-G). If there is more than one row there is a tendency for spines to cluster or form small groups (Fig. 10E). These spines are surrounded by pores (Fig. 10E) and vary in number from two per side in L. peregrinus to approximately 17 in L. crassipes. They always point proximomediad towards the mouth opening and show a dissimilarity in number and distribution per side within a single individual. The spines are mainly long and tapering, with shorter ones in between ( Fig. 10A-G). In some other species, they can be bi-or trifurcate (Fig. 10B, E).
Lateral to the pharyngeal plate, hypopharyngeal spines are always present (Figs 1D, 11A, 12A: hsp). They are arranged in clusters of five to 37 spines unilaterally ( Fig. 13A-C) and they are surrounded with single or clustered pores (up to six) from apparently epidermal glands ( Fig. 13A-B, D, F). The spines mainly taper (Figs 11A, 13A-D, F), sometimes with ridges along the lateral side of the spine shaft (Fig. 13E) or are apically furcate (Fig. 13A). They can be long or short, sometimes with a more flattened appearance (Figs 11A, 13). The hypopharyngeal spines may occur with a continuous transition distomedially to the tuft area (Fig. 13A) or with a distinct break (Fig. 13C).
'Button-shaped' sensilla are arranged in continuous clusters on the lips of the median crest medially up to the ventrolateral bars within the branching bristles and are present in all examined species (Figs 1D: bsc, 14, 16B-D). The median crest is flanked by intergrading rows of branching bristles (Fig. 1D: smc), which can be stout and short (Fig. 15D) or slender and long (Fig. 15E). In several species, we observed a transition from branching bristles to flattened spines on the outermost rows (Figs 14A, 15A-C, F). The flattened spines show a structured surface (Fig. 15C).
The trichomes on the paired lips forming the median crest exhibit an intergrading transition from the tuft area proximal to distal up to the tips of the ventrolateral bars and medially to the proximoventral parts of the hypopharynx (Fig. 1D). At the border to the tuft area, there are generally 'fan-shaped' or plumose branching bristles, which mostly shorten in length, transitioning to 'brush-', 'tuft-', 'feather-like' or simple bristles (Figs 14A, 15A, E-F, 16A-C, E-F, 17A-D, F). On the proximoventral part, the bristles change over into clearly separated brush-tufts that are intermingled by 'button-shaped' sensilla (Fig. 16D). The shape of trichomes varies greatly between species. In D. loricatus, for example, there are scales on the distal tips of the lips bordered by the margin of the ventrolateral bar (Fig. 17E) in comparison to other species showing bristles in this area ( Fig. 17A-D, F).

Peristomatic characters with phylogenetic significance
In the following, eight peristomatic characters are proposed for the genus Lithobius, three of which are newly described (see char. 4, 6, 7). Additionally, we verified the consistency of two characters (see char. 2, 8) and adjusted three (see char. 1, 3, 5) from those indicated by Koch and Edgecombe (2008). Codings are provided in Appendix 1.
Lateral expansion of median sensilla cluster of epipharynx: (0) isolated from the lateral spine fields; (1) partly overlapping with the lateral spine fields. In all Lithobius species we examined (except for L. tricuspis and L. nodulipes for which the samples were damaged), the median sensilla cluster is bordered laterally by fields of spines (state (0); Fig. 10A-B). The sensilla in D. loricatus and N. aztecus slightly overlap with the lateral spine fields medially (state (1); e.g. Fig. 10C). In E. grossipes the sensilla of the median sensilla cluster strongly overlap with the lateral spine fields proximolaterally (state (1); Fig. 10D).  Fig. 15B). The Ezembius species L. electus studied here displays state (1), which differs from Lithobius (Ezembius) giganteus Sseliwanoff, 1881, stated by Koch and Edgecombe (2008). State (1) (Fig. 15A-C, F) is more common throughout the other subgenera of Lithobius and species of the other examined genera compared to state (0) (Fig. 15D-E).

Discussion
Studies on the external morphology and microanatomy of the peristomatic structures of centipedes have hitherto unveiled phylogenetically useful information (Koch and Edgecombe 2006, 2012, 2009). The 'bottleshaped' epidermal glandular shafts of the epipharynx and the discrete shape of the hypopharynx support the monophyly of the order Lithobiomorpha and paired oblique rows of lateral spines on the clypeal part of the epipharynx is, for example, considered as an apomorphic character for the family Lithobiidae . The inclusion of characters from these structures in a morphological dataset that also included other (mostly external) parts of the body further revealed the genus Lithobius as a non-monophyletic taxon . Within the genus Lithobius, five out of eleven described characters of the peristomatic structures display different states , which might give hints on speciesinterrelationships within the genus. These data from the peristomatic structures are presented as a set of coded characters (Appendix 1) that will be analysed cladistically with characters from other character systems in a later study.

Phylogenetic significance of the peristomatic structures of Lithobiidae
While studying the peristomatic structures of Lithobiomorpha and Scutigeromorpha, Koch and Edgecombe (2008) compared the presence of the 'bottle-shaped' epidermal glandular shafts between the labral and clypeal part of the epipharynx. These glandular shafts were reported to be constantly present in Lithobiomorpha  and absent in other chilopods (Koch and Edgecombe 2006, 2012. We confirmed the presence of glandular shafts in the specimens we examined in the lithobiid genera Lithobius, Neolithobius, Eupolybothrus and Disphaerobius and further recorded differences in number and regularity of rows (character 1). The same authors  described the presence of a transverse bulge dividing the labral and clypeal part on the epipharynx for all Lithobiomorpha except for Hessebius plumatus Zalesskaja, 1978 and L. (Ezembius) giganteus displaying no bulge at all. This study confirms the absence of the bulge in the species D. loricatus ( Fig. 2A) and for the first time the presence of a second bulge (distal transverse bulge) as recorded for the species L. calcaratus, L. lucifugus, L. tenebrosus and E. grossipes as well as E. fasciatus (Newport, 1845) (specimens used by Koch and Edgecombe 2008). The alignment of the bulges is further described and proposed as an additional character state (character 4).
The examination of additional taxa within Lithobiidae revealed more variation in the shape of the median spine field than previously described and having surveyed more species we include additional character states to those already described by Koch and Edgecombe (2008) (character 5).
Although differences in shape of the bristles on the labral flap were briefly mentioned by Koch and Edgecombe (2008), our study unveiled four consistent states in the shape of bristles and transition of those from laterally to medially, which serves as a new multistate character for Lithobiidae (character 6). A transition of bristles from plumose to 'fan-shaped' was described for Pleurolithobius patriarchalis (Berlese, 1894) , as in the majority of the investigated species in the present study. In contrast, only 'fan-shaped' bristles are observed in the lithobiid Harpolithobius anodus (Latzel, 1880) and the henicopid Lamyctes (Lamyctes) emarginatus (Newport, 1844). On the other hand, the interpretation that Lithobius (Monotarsobius) holstii (Pocock, 1895) possesses only 'fan-shaped' bristles ( Fig. 6E in Koch and Edgecombe 2008) seems erroneous as their figure reveals a pattern in accordance with the other examined Monotarsobius-species, which exhibit a transition from plumose to 'fan-shaped' bristles (e.g. L. aeruginosus, Fig. 9C).
Generally, the median sensilla cluster borders or overlaps marginally with the lateral field of spines in Lithobiomorpha . However, we observed a median sensilla cluster considerably expanding along the length of the lateral spine fields on the epipharynx in E. grossipes for Lithobiomorpha (Fig. 10D). This was also verified in E. fasciatus (specimens used by Koch and Edgecombe 2008), which also displays a large but partial overlap.
As mentioned in the introduction, the hypopharynx as a short outgrowth with a median crest is an apomorphic character for Lithobiomorpha. This is verified in all examined lithobiid species. Moreover, the median crest margin of all studied species of the subgenus Monotarsobius displays flattened spines (character 8) as previously described for L. holstii .

Variability of the peristomatic structures in Lithobiidae
Besides the well-defined characters listed in the previous paragraph, our investigation also yielded several structures with high variability in appearance and/or intermediate forms between and even within species. For example, the branching bristles of (i) the labral bristle band on the distal bar, (ii) the spines of the median spine field of the epipharynx and (iii) the branching bristles as a tuft on the hypopharynx occur with several non-definable forms. Koch and Edgecombe (2008) described a smooth transverse bulge for Lithobiidae, which we confirmed for most of the examined species. However, we also observed a longitudinal striation of the whole bulge surface or at least on the lateral parts of the bulge for some species (Figs 3C, 4D). A similar description of the latter state was observed for the henicopid Lamyctes emarginatus, where more defined longitudinal grooves occur .
The paired oblique rows of elongated lateral spines on the clypeal part of the epipharynx were also considered as an apomorphic character for Lithobiidae . This is also confirmed in all examined lithobiid species we studied. However, the proposed character states, i.e. (2): oblique rows of single spines and (3): a few small groups of branching spines for the lateral field of spines on Lithobiidae were not consistent across the species we examined and showed many intermediate states. On this basis we excluded the character for conclusions on the systematics in Lithobiidae, especially Lithobius, in our study. Koch and Edgecombe (2008) recorded groups of lateral fields of spines in the subgenus Monotarsobius in contrast to pairs of oblique rows in the rest of Lithobiidae (character 32, state (3)). These spines seem to be arranged in oblique rows as in the rest of Lithobiidae in the species L. (Monotarsobius) aeruginosus and L. (Monotarsobius) curtipes (Fig. 10F-G).
A correlation between the number of 'bottle-shaped' epidermal glandular shafts of Lithobiomorpha and body size was also mentioned by Koch and Edgecombe (2008), implying that larger species tend to have higher numbers. Here, we suggest the same for the number of glandular shafts, sensilla in the median sensilla cluster and the 'nipple-shaped' sensilla cluster, lateral spines, and the hypopharyngeal spines. This size correlation needs to be confirmed by morphometrics and statistical analysis but the phylogenetic significance of these characters is cast into doubt.

Assumptions on the relationship of Disphaerobius with (sub)genera Lithobius and Ezembius
The peristomatic structures of H. plumatus and L. (Ezembius) giganteus described by Koch and Edgecombe (2008) and D. loricatus examined in this study, i.e. a missing transverse bulge (character 3), simple bristles on the labral flap of the epipharynx (character 6) and scales on the distal tips of the lips of the hypopharynx (Fig. 17E), differ from all other studied species of Lithobius, including L. (Ezembius) electus, even if the latter is correctly placed in the subgenus Ezembius. Several taxa in Central Asia, also species of the giganteus-group of Lithobius (Eason 1983(Eason , 1986) and of the genus Hessebius Verhoeff, 1941 share some morphological characters with the genus Disphaerobius Attems, 1926, as mentioned by Farzalieva et al. (2017): "… functionally biarticulated tarsi of leg 1-13, the antennae composed of 20 antennomeres, the rounded posterior angles of all tergites, the 1-segmented male gonopods, and Tömösváry's organ being equal in size to the nearest ocellus or smaller." In contrast to the three other species of the giganteus-group of Lithobius, L. (Ezembius) giganteus displays secondary sexual modifications of the tergites in males similar to Disphaerobius (Farzalieva et al. 2017). Here, we assume that the epipharyngeal and hypopharyngeal structures may confirm a closer relationship of L. (Ezembius) giganteus to D. loricatus than to L. (Ezembius) electus. This relationship is inconsistent with the classification of Disphaerobius as a separate subfamily, Pterygoterginae Verhoeff, 1933, because that classification would render Lithobiinae, as well as Lithobius and L. (Ezembius) as paraphyletic groups.