A stereotyped cell division pattern is found at the posterior border of the mandibular segment
Like all malacostracan crustaceans studied in this respect (for a recent review see Dohle et al. [42]), amphipods show a stereotyped cell divisions pattern in the post-naupliar region during growth, differentiation and segmentation of the germ band [36,43,44]. In contrast to this, the naupliar region does not exhibit an obvious stereotyped cell division pattern [37,42]. Only Scholtz [36] suggested that there might be a certain regularity in the divisions and arrangements of the posterior cells of the developing mandibular segment in the amphipod Gammarus pulex but with the methods then at hand the details were not resolvable With the technique of 4D-microscopy we have been able to provide the first evidence for an invariant cell division pattern in the naupliar region of another amphipod species, the freshwater beach hopper Orchestia cavimana. At least in the posterior part of the region of E(0) we recognized a relatively strict cell division pattern. This pattern has only superficial similarities to the post-naupliar cell division pattern of malacostracan crustaceans but it is not as elaborated in terms of timing of mitoses, cell size, and the spatial arrangement of the resulting cells. In E(0) the sequence of the individual divisions is not as strict and the direction of the mitotic spindles is more or less longitudinally oriented. It appears that the posterior border of the mandibular segment forms some kind of transition between the more irregular divisions and cell arrangements of the anterior naupliar region and the highly complex stereotyped post-naupliar patterns.
It is not clear whether our findings of a regular pattern in the posterior part of the mandibular segment holds true for other malacostracans as well, although some data from the isopod Porcellio scaber hint to that possibility [45]. However, the cellular events in the corresponding region in Porcellio are much more irregular when compared with those in Orchestia. It has been even shown for Porcellio that some cells of the most anterior row of the post-naupliar segments can migrate into the posterior area of the mandible segment [45], a phenomenon that does not occur in Orchestia.
Interestingly enough, our results reveal that the posterior segmental boundary of the mandibular segment corresponds to the genealogical border between rows E(0) and E(1), i.e. E(1) does not contribute to the posterior part of the mandibular segment. This stands in contrast to all more posterior post-naupliar segmental boundaries which are formed within the descendants of one ectoderm row and thus do not match the genealogical borders (see Fig. 2), [42]. Row E(1) forms a kind of transition between these segmentation modes because its posterior region follows the typical post-naupliar pattern in that it contributes to the anterior portion of the segment of the first maxillae whose posterior part is formed by anterior descendants of the next adjacent row E(2) [36,37]. These differences indicate that the parasegmental organization (i.e. a frame-shift between the early metameric anlagen and the resulting morphological segments) of the post-naupliar germ band (see [42]) is not found in the naupliar region with a transition in the first maxillary segment.
Cell lineage data and clonal analyses reveal that paragnaths are not limbs but outgrowths of the sternal region of the mandibular segment
Based on our knowledge of the cell division pattern of the early developing mandibular region (see above) we were able to look at its morphogenesis at a very high level of resolution. By means of single cell labeling with the fluorescent dye DiI we were able to reconstruct and analyze the clonal composition of the mandibular region from the beginning of ectodermal proliferation up to the differentiation of the mouthparts. The cell labeling reveals that the paragnaths have their origin in the area I and area II which comprises columns 1 to 3 of region E(0). The mandibles originate from cells of the areas II and III (columns 2 to 4). Areas I and II contribute also to the sternal region and the mandibular ganglia whereas area III forms parts of the tergites as well. In more posterior segments, columns 1 and 2 mainly contribute to the formation of segmental ganglia and probably sternites, and columns 3 to 5 mainly give rise to limbs [36,46]. Hence, when compared with clonal composition of the post-naupliar segments it is evident that the columns that form the ganglia and sternites in these segments correspond to those that give rise to the paragnaths in the mandibular segment. In addition, the mandibular buds are formed in a comparable position to the other limbs. This clearly reveals that paragnaths of Orchestia are processes of the mandibular sternal region.
Further evidence for the claim that crustacean paragnaths belong to the mandibular segment is based on gene expression data. For instance, the segment polarity gene engrailed is expressed in a regular stripe in the posterior region of the mandibular segment, as in all other segments, comprising cells that form the posterior part of the paragnaths in amphipods, isopods and decapods [14,18,37]. Moreover, the expression of the Hox-gene Deformed (Dfd) is mainly found in the mandible segment of hexapods [47,48], myriapods [49,50], and crustaceans [14,16], and in the latter case expression of Dfd comprises the buds of the paragnaths [14,16].
All these data reveal that paragnaths are part of the mandibular segment and that they are not limb derivatives as has been suggested by several authors. This confirms previous ideas on the origin and nature of paragnaths based on embryological and larval evidence [17]. With our clonal analysis we can definitely rule out the possibility that paragnaths indicate the existence of an additional head segment as has been claimed for example by Casanova [29], Chaudonneret [26], Denis [34], and Hansen [28]. Furthermore, the limb-bud like early appearance of the paragnaths in Orchestia and other crustaceans is no indication for a limb-related nature of these structures but is only a superficial similarity that does not represent a genealogical relation. Our results show the power of cell lineage and clonal analyses for inferences on the nature, origin and thus homology of morphological structures. With this kind of investigation morphological and gene expression data can be complemented.
In many cases the origin of the paragnaths during crustacean embryonic and larval development has either not been specified (e.g. Ref. [51-53]) or it has been suggested that paragnaths develop from mandibular and/or maxillary segments (e.g. Ref. [54]). A look at the corresponding figures in these articles with our results in mind reveals that it is possible in almost all examples to relate the paragnathal structures to the mandibular segment (e.g. Manton [51], plate 24, fig. 27; Manton [52], plate 25, fig. 23; Moeller [53], figs. 2, 7). Even the median lobe of the so-called lower lip of the raptorial cladoceran Leptodora kindtii might represent fused paragnaths [55]. Accordingly, we tentatively conclude that the paragnaths lobes are homologous throughout Crustacea. Stein et al. [56] suggest that a pair of paragnaths humps is an apomorphy for a crustacean subgroup comprising Eucrustacea and Phosphatocopina (Labrophora). However, these authors did not consider similar structures in myriapods and hexapods which indicate a much more widespread occurrence among euarthropods. (see next chapter).
Are the crustacean paragnaths homologous to the superlinguae in hexapods and myriapods?
There are several reports of post-oral paired bud-like anlagen in myriapods (e.g. Chilopoda: Heymons [57], Progoneata: Tiegs [19]) and Hexapoda (e.g. Ref. [24,25,58-60]). In dicondylian hexapods the situation is somewhat ambiguous. Larink [61] and Scholl [62] report for Lepisma and Carausius an undivided early hypopharynx anlage whereas Rohrschneider [24] for Periplaneta and Ibrahim [63] for Tachycines describe two separated buds. Whether these are factual differences is not clear. However, the presence of paired buds in a number of pterygotes as well as collembolans, diplurans, and archaeognathans allows the conclusion that these paired buds were present in the hexapod stem species. Nevertheless, all these buds have in common that they originate from the sternum of the mandible segment directly ventral to the mandibular ganglion anlagen and between the mandibular limb buds, after these have formed. This very much resembles the early stages of crustacean paragnaths (see above, [14,17,18,51,52]). These sternal buds of the mandibular segment give either rise to the superlinguae, paired lateral lobes of the hypopharynx, in Symphyla [19], Collembola [58,59], Diplura, Archaeognatha [25], or to (another part of) the hypopharynx [22,24,57] when superlinguae are not present as for instance is the case in many Pterygota and all Chilopoda [22,64]. However, the contribution of these mandibular sternal buds to the hypopharynx body (linguae) is interpreted controversially. The hypopharynx of Hexapoda and Progoneata is thought to be a composite structure formed by protrusions of the sternites of different numbers of gnathal segments (e.g. Ref. [19,22,25,61,62]). In contrast to this, Haget [65] and Wada [66] suggest, based on their experimental teratological studies that the entire hypopharynx originates from the mandibular sternites. According to Heymons [57], this is also the case in Chilopoda. The situation is further complicated by the fact that buds appearing in the intercalary segment of hexapods are called "hypopharyngeal lobes" (Hypopharynxhöcker) and have been suggested to form (part of) the hypopharynx, an obvious misinterpretation (see Ref. [23,63,67]). Reading the numerous articles dealing with this problem it is evident that investigations focusing on the differentiation of the hypopharynx in myriapods and hexapods using modern approaches are badly needed to solve this issue.
Authors such as Crampton [27], Snodgrass [68], and Bitsch and Bitsch [5] suggest that the paragnaths and the superlinguae are homologous. However, based on our data and what we find in the above discussed literature about the development of the hexapod and myriapod hypopharyngeal complex, we think that the conclusion of these authors is simplifying the matters because paragnaths cannot be homologous to superlinguae alone if the major part of the hypopharynx of myriapods and hexapods originates from the mandibular sternites as well. Accordingly, we modify the homology statement concerning crustacean paragnaths and the myriapod/hexapod superlinguae/hypopharynx by suggesting that the early anlagen of these structures are homologous, taking into account that the homology of early developmental stages does not necessarily mean that more advanced stages are also homologous [69]. Since a comparable structure is absent in the corresponding segment of Chelicerata and Onychophora (first walking leg, see Ref. [1,2,70-72]) it is likely that a pair of sternal buds in the mandibular segment is a shared apomorphy of Crustacea, Myriapoda, and Hexapoda. Accordingly these structures provide further support for the Mandibulata hypothesis (see also Ref. [2,73-75]) which has been disputed based on molecular data (e.g. Ref. [76-78]).
Acknowledgements
We thank Greg Edgecombe for invaluable comments on the manuscript and for improving the English. The support by the Deutsche Forschungsgemeinschaft (DFG) (Scho 442/5-3) is gratefully acknowledged.
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