On some surface structures of potential taxonomic importance in families of the suborders Polydesmidea and Dalodesmidea (Polydesmida, Diplopoda)

Abstract Surface structures have rarely been the subject of a comprehensive study in Polydesmida despite their tremendous variety within this order. A number of these peripheral structures are here studied in most families of the suborders Polydesmidea and Dalodesmidea (sensu Hoffman 1980), using scanning electron microscopy. An illustrated description of the surface sculpture of the prozonite, the limbus and the intercalary cuticular micro-scutes on the metazonite is given for the first time for the studied families, together with an account of some other poorly known surface structures. Taken together, these characters allow us to recognize two main groupings of families. The families Ammodesmidae, Cryptodesmidae, Cyrtodesmidae, Haplodesmidae, Oniscodesmidae and Pyrgodesmidae have knobs on the posterior part of the prozonites, a toothed to lobed limbus, and no micro-scutes on the metazonites, wheras the families Fuhrmannodesmidae, Polydesmidae, Dalodesmidae, Macrosternodesmidae, Nearctodesmidae, Opisotretidae and Trichopolydesmidae have no knobs on the posterior part of the prozonites, a spiky or reduced limbus, and intercalary micro-scutes on the metazonites. The results are complemented with literature records and compared with current taxonomic and phylogenetic interpretations of the group.

introduction Whereas the gonopods have hitherto been acknowledged to be the most reliable source of characters for millipede identification, the details of external morphology have in most cases remained under-prospected. This is also true for the order Polydesmida although it is by far the most diverse millipede order in terms of non-gonopodal morphology. The taxonomy of several polydesmidan families, notably Fuhrmannodesmidae and Pyrgodesmidae, is in a deplorable state, and new taxonomic characters are badly needed. Rowe and Sierwald (2006) drew attention to the fact that the external morphology has been studied in only a few cases in millipedes, and gave an overview of the major works which dealt with this topic.
Scanning electron microscopy has, in many contemporary works, significantly helped to illustrate fine surface structures in millipedes. Mesibov (2009a) studied and described the several shapes of spiracles in species of the families Paradoxosomatidae and Dalodesmidae, demonstrated the great variation in the shape, location and density of the sphaerotrichomes on male legs in Dalodesmidae and noted the presence of different patterns in the integument sculpture of the metatergal tuberculation in two genera of the same family (see also Mesibov 2008). Moreover, the different arrangements and structures of the spinnerets in 16 families of Polydesmida were studied by Shear (2008), who delineated a notable variation of these structures within the studied taxa (see also Mesibov 2009a). The structure of the spinnerets was furthermore studied by Mesibov (2008Mesibov ( , 2009a who suggested a possible synapomorphy for some of the 10 studied dalodesmid genera even if recognizing that the use of spinneret structure in taxonomy of Polydesmida requires more sampling (see Mesibov 2009a).
The limbus, or posterior margin of the metazonites, was investigated by Schmidt (1962) who systematically described and compared the variation of shapes within numerous families, using only light microscopy.
Some polydesmidans of the families Cryptodesmidae, Haplodesmidae, and Pyrgodesmidae are earth-incrusted, i.e., adult specimens bear a coat of dirt. Shear (1973Shear ( , 1977 described and illustrated special "boxes" and "branched tree-like setae" (Shear 1977) which supposedly keep the dirt on the cuticle.
During the study of a new pyrgodesmid species from Tunisia using scanning electron microscopy (Akkari and Enghoff 2011), we found a number of cuticular structures which have not hitherto been described. In order to assess their significance, we made a comprehensive survey of 22 species belonging to all the families of the suborders Polydesmidea and Dalodesmidea (see the list below and Table 1) except for the Dorsoporidae (Polydesmidea) and Vaalogonopodidae (Dalodesmidea) of which material was inaccessible for study.
Our study was mainly focused on three sets of characters: a) surface sculpture of the prozonite (anterior, cylindrical part of body ring); b) the limbus (posterior margin of body rings); c) intercalary micro-scutes on the surface of the metazonite. A few additional structures such as the cuticular outgrowths in earth-incrusted species and the ozopores are briefly presented below and compared. Moreover, recent literature ac-counts of 29 species from relevant families (see Table 2), including SEM illustrations or descriptions were checked and compared with our results.

Materials and methods
The studied material is preserved in 70 % ethanol and deposited in the Natural History Museum of Denmark (Zoological Museum, University of Copenhagen, ZMUC). Earth-incrusted specimens were cleaned for scanning electron microscopy by soaking in a solution of commercial detergent (©Biotex) and/or by ultrasound then air dried. SEM pictures were made with a JEOL JSM-6335F scanning electron microscope, then processed and assembled with Adobe Photoshop CS5 software.

Results
Fine sculpture of the prozonite Figs 1-9 The prozonite of the studied species is divided into two main parts separated by a transverse ridge. While the anterior part is rather uniform, showing a scaly aspect, the posterior surface displays varied patterns within the studied families. In the examined species of Ammodesmidae, Cryptodesmidae, Cyrtodesmidae, Haplodesmidae, Oniscodesmidae and Pyrgodesmidae the anterior part of the prozonite (a) displays a covering of small scaly units, juxtaposed in series of irregular transverse rows, becoming elongated in the posteriormost row to form a transverse ridge (r) marking the border of this part (Figs 1-9). In front of the ridge, the general aspect is quite uniform in all the above cited families, with lozenge-shaped units. These units could sometimes be star-like, furrowed, and marginally jagged (cyrtodesmid sp.) (Fig. 3). In Amphitomeus attemsi (Oniscodesmidae), the units are more elongated and strongly prominent, interconnected with parallel cuticular ridges (Fig. 5). On the other hand, the microsculpture of the anterior part of the prozonite in Prosopodesmus jacobsoni (Haplodesmidae) takes the shape of hollow chambers separated by walls of "microvilli-like" structures (Fig. 4). The posterior surface of the prozonite (b) is, in all examined species of these six families, characterized by a regular covering of subspherical knobs (s) placed on a smooth to microgranulated background. The cover of knobs is regularly dense in most cases (Figs 1, 3-9) though fairly dispersed in Elythesmus enghoffi (Cryptodesmidae) (Fig. 2). These knobs are generally uniformly smooth (Figs 1-3, 6-9) but sometimes exhibit special configurations: lobed in P. jacobsoni and spiky in A. attemsi (Figs 4, 5).
The examined species of the families Fuhrmannodesmidae, Polydesmidae, Dalodesmidae, Macrosternodesmidae, Nearctodesmidae, Opisotretidae and Trichopolydesmidae show an anterior surface of the prozonite with polygonal units serrated marginally. However, these units are much less conspicuous and prominent than in the species of the first set of families (Figs 10-17). The posterior border of the anterior part of the prozonite is similarly marked by a transverse ridge but its units are nevertheless only slightly modified. The posterior surface of the prozonite remarkably differs by the complete absence of the sub-spherical knobs described above; instead the surface is smooth to scaly (Figs 10-17).

A peculiar structure of the metazonites: intercalary cuticular micro-scutes Figs 35-41
The studied species of the families Fuhrmannodesmidae, Polydesmidae, Dalodesmidae, Macrosternodesmidae, Nearctodesmidae, Opisotretidae and Trichopolydesmidae (cf. Table 1) present a peculiar structure on the metazonital surface: between the normal polygonal cuticular scutes which cover the metazonital surface there are rows of small ovoid "intercalary scutes". The placement of these structures is unlikely to be accidental or indicating any kind of bacterial infection as they seem well arranged in a regular pattern, appearing like spaced nodes or pearls aligned on strings crossing the surface of the metazonites (Figs 35-41). These structures have never been documented. However, they are visible on an illustration in Mesibov [2003, fig. 3, (right)], for Procophorella innupta Mesibov, 2003 (Dalodesmidea).

Some other poorly known surface structures Figs 42-49
The surface structure of most earth-incrusted species is characterized by the presence of papilla-like cuticular outgrowths which are particularly abundant in Ammodesmidae, Cyrtodesmidae and Pyrgodesmidae. These papillae are boletiform (mushroom-shaped)  and are presumed to keep the cuticular secretions and adhering soil particles in place. Displaying variation in length, shape of the apex, and density on the surface, the papillae are short, with a rounded apex in Rharodesmus tabarkensis (Fig 42) and Cynedesmus sp., elongate and slender in Elassystremma sp. (Fig. 43) and in cyrtodesmid sp. The same structures have been mentioned in previous works, generally quoted as "papillis" (Silvestri 1925(Silvestri , 1947, "Papillen" (Attems 1940), or "microvilli" in most of Golovatch's works -illustrations can be found in Golovatch et al. (2009a, figs 24A, B, E, F). Shear (1977) was the first to provide SEM illustrations of such structures and to comment on their possible function.

Discussion
Polydesmida is the most speciose millipede order, and despite the fact that it has remained quite stable in terms of number of families (Shelley 2003), its taxonomy is far from being satisfactory. In his attempt to classify the suborders Polydesmidea and Dalodesmidea, Hoffman (1980: 146) expressed his dissatisfaction and pessimism: "The groupings set forth in the following pages are to a large extent exercises in futility, but may have some reference value in a bibliographic sense". Nevertheless, Hoffman's (1980) main classification scheme still stands and has been adopted by most subsequent authors. Simonsen (1990), studying the phylogeny of Polydesmida, made a number of changes and synonymies. Notably, Simonsen (1990) placed Dalodesmidae + Vaalogonpodidae as sister-group of Polydesmidae, i.e., the suborder Dalodesmidea nested within the suborder Polydesmidea. However, most of Simonsen's conclusions were soon after criticized by several authors because they were based on insufficient data and bold assumptions (e.g. Golovatch 1996, Shelley 2003. Taken together, the fine sculpture of the prozonite, the structure of the limbus and the presence/absence of metazonital micro-scutes indicate two main groupings of families within the suborders Polydesmidea and Dalodesmidea. The first group (A) comprises the families Ammodesmidae, Cryptodesmidae, Cyrtodesmidae, Haplodesmidae, Oniscodesmidae and Pyrgodesmidae. These six families have a cover of subspherical knobs on the posterior surface of the prozonites (Figs 1-9) and a toothed limbus constituted of a series of lobes which may or not be surmounted by spikes (Figs  18-26).
The second group (B) encompasses the families Fuhrmannodesmidae, Polydesmidae, Dalodesmidae Macrosternodesmidae, Nearctodesmidae, Opisotretidae and Trichopolydesmidae, and is characterized by 1) an absence of knobs on the posterior surface of the prozonite which is smooth to scaly (Figs 10-17), 2) an absence of lobes on the limbus which has instead a jagged margin or spikes with varied complexity (Figs 27-34), and 3) intercalary micro-scutes on the surface of the metazonites (Figs  35-41), absent in the first set of families.
The fine structure of the prozonite in both species of the Tasmanian dalodesmid genus Noteremus, N. summus and N. infimus (Mesibov 2009c, fig. 3) perfectly agrees with what we recorded in the dalodesmid Icosidesmus sp. (Fig. 13). The genus Asphalidesmus, in contrast, exhibits a pyrgodesmid-like pattern with a conspicuous cover of sub-spherical knobs on the posterior surface of the prozonite. In his description of A. golovatchi, Mesibov (2009c) wrote "prozonites with narrow band of longitudinal ridges just anterior to suture, elsewhere uniformly covered with very small protuberances with blunt, rounded tips directed slightly posteriorly". Mesibov (2011) did later describe several new species of the same genus on which the posterior prozonite protuberances were visible, especially on A. bellendenkerensis Mesibov, 2011(Mesibov 2011 . 4A).
Although assigned to Dalodesmidea (Mesibov 2009c), the similarity of the genus Asphalidesmus to the first set of families (Ammodesmidae, Cryptodesmidae, Cyrtodesmidae, Haplodesmidae, Oniscodesmidae and Pyrgodesmidae) might not be a surprise knowing that the taxonomic position of Asphalidesmus has been matter of controversy. In fact, the genus was originally described in Dalodesmidae then subsequently placed in Fontariidae, Vanhoeffeniidae, and Haplodesmidae, listed in Xystodesmidae, and assigned to Cryptodesmidae and Oniscodesmidae (see Mesibov 2009c). Despite lacking sphaerotrichomes and showing a few further peripheral structures resembling more Pyrgodesmidae than Dalodesmidae and Vaalogonopodidae, Asphalidesmus was informally placed in the suborder Dalodesmidea (see Golovatch 2003). This was subsequently confirmed by Mesibov (2009c) although both authors agree in not assigning the genus to any family (Golovatch 2003, Mesibov 2009c. Several literature records clearly support the patterns described above for the limbus. For example, the limbus shows a series of lobes with or without additional spikes in the pyrgodesmid species L. vanuatu (Golovatch et al. 2008, fig. 4D) and Myrmecodesmus hastatus (Bergholz et al. 2004, figs 5, 6), the ammodesmids Elassystremma prolaeve and E. laeve (VandenSpeigel and Golovatch 2003, figs 7, 18), and the haplodesmids Agathodesmus steeli (Mesibov 2009b, fig. 6C), Cylindrodesmus hirsutus (Golovatch et al. 2001, fig. 11) and Eutrichodesmus similis (Golovatch et al. 2009a, fig. 19E). The limbus is, on the other hand, composed of a number of ramified spikes in, for example, the dalodesmid Ginglymodesmus tasmanianus (Mesibov 2005, fig. 4A). Schmidt (1962, figs 22-29) (Fuhrmannodesmidae). In all these species, the limbus is strikingly similar to what we observed in the studied species belonging to the same families.
Though we are aware that the present data alone do not warrant a strict cladistic analysis, we have compared our findings (Fig 51) with the only existing phylogenetic work on the order Polydesmida (Simonsen 1990). The two main groups (A) and (B) mentioned above agree remarkably well with the basal dichotomy in Simonsen' cladogram (Fig 50), except for the families Haplodesmidae and Cryptodesmidae which belong to our group (A) but which according to Simonsen are in the clade which otherwise includes our group (B). However, the general knowledge about these two families and their diversity is still incomplete, and no evidence has hitherto been provided in support of their monophyly. In his revision of the Haplodesmidae, Golovatch (2009a) recognized two major 'grades', the haplodesmid and doratodesmid grades, according to their somatic (non-sexual) characters and capacity for "volvation" (coiling into a sphere). In the present study, only the haplodesmid genus Prosopodesmus was studied. Golovatch et al. (2009a) characterised Prosopodesmus as a "pyrgodesmid-like haplodesmid" and considered it as basal in the Haplodesmidae (together with Rhipidopeltis Miyosi, 1958).
The Cryptodesmidae studied by us alos present a complication as Elythesmus has knobs on the posterior part of the prozonites (Fig. 2) and a lobed limbus (Fig. 26) as in families group A, whereas Aporodesmus has a posterior prozonite surface free of knobs (Fig. 52) and dentate leaf-shaped (le) elements and spikes (sp) on the limbus (Fig. 53).
As stated above, the second set of families (B) have no lobes on the limbus. Instead, ramified spikes and "icicles" (Figs 27-30) can be observed in the families Dalodesmidae, Macrosternodesmidae and Polydesmidae, which clearly separates  Simonsen (1990). Haplodesmidae here corresponds to Haplodesmidae + Doratodesmidae on Simonsen's original cladogram; families not studied here are marked with asterisks 51 Branching diagrams (not cladograms) based on Fig. 50 but modified to illustrate the distribution of the different states of the three studied characters: A. presence of knobs on the anterior part of the prozonite, B. shape of the limbus, C. presence of intercalary micro-scutes on the metazonites (see Appendix for character states). them from the Fuhrmannodesmidae, Trichopolydesmidae, Nearctodesmidae and Opisotretidae in which the limbus is hardly developed and bears but few scattered bulges (Figs 31-34). However, this separation fits neither with Hoffman's (1980) classification in which the Macrosternodesmidae, Nearctodesmidae, Trichopolydesmidae and Fuhrmannodesmidae form the superfamily Trichopolydesmoidea (see also Shelley 2003), nor with the phylogenetic analysis of Simonsen (1990) in which the Macrosternodesmidae and Nearctodesmidae appear in the same clade while the Opisotretidae, a separate clade, is grouped with a different set of families (Fig. 50). The simple limbus could well be a plesiomorphic character state expressed in the Fuhrmannodesmidae, Trichopolydesmidae, Nearctodesmidae and Opisotretidae (see also Simonsen 1990).
In the present work, we do not pretend to offer a new subordinal classification of Polydesmida, or to solve any of the taxonomic problems related to families -a colossal task which definitely will require a lot more sampling and character scoring, including the gonopod structures which haven't been considered here. However, documenting these remarkable surface structures and trying to compare them between the different families will perhaps contribute to bringing new insights, leading towards a better understanding of polydesmidean and dalodesmidean relationships.