The pattern of segment formation,as revealed by engrailed expression,in a centipede with a variable number of segments

Arthropods vary enormously in segment number, from less than 20 to more than 200. This between-species variation must have originated, in evolution, through divergent selection operating in ancestral arthropod species with variable segment numbers. Although most present-day arthropod species are invariant in this respect, some are variable and so can serve as model systems. Here, we describe a study based on one such species, the coastal geophilomorph centipede Strigamia maritima. We investigate the way in which segments are formed using in situ hybridization to demonstrate the expression pattern of the engrailed gene during embryogenesis. We also analyze segment number data in mother-offspring broods and thereby demonstrate a significant heritable component of the variation. We consider how natural selection might act on this intraspecific developmental variation, and we discuss the similarities and differences in segment formation between the geophilomorphs and their phylogenetic sister-group.     

Latitudinal cline in segment number in an arthropod species, Strigamia maritima

Arthropods vary more than 30-fold in segment number. The evolutionary origins of differences in segment number among species must ultimately lie in intraspecific variation. Yet paradoxically, in most groups of arthropods, the number of segments is fixed for each species and shows no intra- or interpopulation variation at all. Geophilomorph centipedes are an exception to this general rule, and exhibit intraspecific variation in segment number, with differences between individuals being determined during embryonic development and hence independent of population age structure. Significant differences in segment number between different geographical populations of the same species have been previously reported, but insufficient sampling has been conducted to reveal any particular geographical pattern. Here, we reveal a latitudinal cline in segment number in the geophilomorph species Strigamia maritima: segment number in British populations decreases with distance north. This is the first such cline to be reported for any centipede species; indeed as far as we are aware it is the first such cline reported for any arthropod species. In vertebrates, fish are known to exhibit a latitudinal cline in segment number, but interestingly, this is in the opposite direction; fish add segments with increasing latitude, centipedes subtract them.     

Temperature-dependent plasticity of segment number in an arthropod species: the centipede Strigamia maritima

SUMMARY The evolution of arthropod segment number provides us with a paradox, because, whereas there is more than 20‐fold variation in this character overall, most classes and orders of arthropods are composed of species that lack any variation in the number of segments. So, what is the origin of the higher‐level variation? The centipede order Geophilomorpha is unusual because, with the exception of one of its families, all species exhibit intraspecific variation in segment number. Hence it provides an opportunity to investigate how segment number may change in a microevolutionary context. Here, we show that segment number can be directly altered by an environmental factor (temperature)—this is the first such demonstration for any arthropod. The direction of the effect is such that higher temperature during embryogenesis produces more segments. This potentially explains an intraspecific cline in the species concerned, Strigamia maritima, but it does not explain how such a cline is translated into the parallel interspecific pattern of lower‐latitude species having more segments. Given the plastic nature of the intraspecific variation, its link with interspecific differences may lie in selection acting on developmental reaction norms.     

Demonstration of a heritable component of the variation in segment number in the centipede Strigamia maritima

SUMMARY Here we address the question of how arthropod segment number may evolve by reporting the results of further work on the model system Strigamia maritima . Recently, we showed that there was a plastic component of the variation in segment number within this species; now we demonstrate that there is also a heritable component. This is important because it enables a connection to be made between the known latitudinal trend among species of geophilomorph centipedes (more segments at lower latitudes) and the parallel trend within them. This latter trend is best documented in S. maritima but is also known in several other species. However, while a general connection between the inter- and intraspecific trends can now be made, deciding upon a specific hypothesis of the nature of the selection involved is still problematic. We provide two alternative hypotheses, one based on the temperature-related plasticity in segment number being adaptive, the other based on it being nonadaptive.     

A double segment periodicity underlies segment generation in centipede development

The number of leg-bearing segments in centipedes varies extensively, between 15 and 191, and yet it is always odd. This suggests that segment generation in centipedes involves a stage with double segment periodicity and that evolutionary variation in segment number reflects the generation of these double segmental units. However, previous studies have revealed no trace of this. Here we report the expression of two genes, an odd-skipped related gene (odr1) and a caudal homolog, that serve as markers for early steps of segment formation in the geophilomorph centipede, Strigamia maritima. Dynamic expression of odr1 around the proctodaeum resolves into a series of concentric rings, revealing a pattern of double segment periodicity in overtly unsegmented tissue. Initially, the expression of the caudal homolog mirrors this double segment periodicity, but shortly before engrailed expression and overt segmentation, the intercalation of additional stripes generates a repeat with single segment periodicity. Our results provide the first clues about the causality of the unique and fascinating "all-odd" pattern of variation in centipede segment numbers and have implications for the evolution of the mechanisms of arthropod segmentation.     

Decoupling elongation and segmentation: Notch involvement in anostracan crustacean segmentation

Repeated body segments are a key feature of arthropods. The formation of body segments occurs via distinct developmental pathways within different arthropod clades. Although some species form their segments simultaneously without any accompanying measurable growth, most arthropods add segments sequentially from the posterior of the growing embryo or larva. The use of Notch signaling is increasingly emerging as a common feature of sequential segmentation throughout the Bilateria, as inferred from both the expression of proteins required for Notch signaling and the genetic or pharmacological disruption of Notch signaling. In this study, we demonstrate that blocking Notch signaling by blocking γ‐secretase activity causes a specific, repeatable effect on segmentation in two different anostracan crustaceans, Artemia franciscana and Thamnocephalus platyurus. We observe that segmentation posterior to the third or fourth trunk segment is arrested. Despite this marked effect on segment addition, other aspects of segmentation are unaffected. In the segments that develop, segment size and boundaries between segments appear normal, engrailed stripes are normal in size and alignment, and overall growth is unaffected. By demonstrating Notch involvement in crustacean segmentation, our findings expand the evidence that Notch plays a crucial role in sequential segmentation in arthropods. At the same time, our observations contribute to an emerging picture that loss‐of‐function Notch phenotypes differ significantly between arthropods suggesting variability in the role of Notch in the regulation of sequential segmentation. This variability in the function of Notch in arthropod segmentation confounds inferences of homology with vertebrates and lophotrochozoans.     

Arthropod-like expression patterns of engrailed and wingless in the annelid Platynereis dumerilii suggest a role in segment formation

The origin of animal segmentation, the periodic repetition of anatomical structures along the anteroposterior axis, is a long-standing issue that has been recently revived by comparative developmental genetics. In particular, a similar extensive morphological segmentation (or metamerism) is commonly recognized in annelids and arthropods. Mostly based on this supposedly homologous segmentation, these phyla have been united for a long time into the clade Articulata. However, recent phylogenetic analysis dismissed the Articulata and thus challenged the segmentation homology hypothesis. Here, we report the expression patterns of genes orthologous to the arthropod segmentation genes engrailed and wingless in the annelid Platynereis dumerilii. In Platynereis, engrailed and wingless are expressed in continuous ectodermal stripes on either side of the segmental boundary before, during, and after its formation; this expression pattern suggests that these genes are involved in segment formation. The striking similarities of engrailed and wingless expressions in Platynereis and arthropods may be due to evolutionary convergence or common heritage. In agreement with similarities in segment ontogeny and morphological organization in arthropods and annelids, we interpret our results as molecular evidence of a segmented ancestor of protostomes.     

Notch/Delta signalling is not required for segment generation in the basally branching insect Gryllus bimaculatus

Arthropods and vertebrates display a segmental body organisation along all or part of the anterior-posterior axis. Whether this reflects a shared, ancestral developmental genetic mechanism for segmentation is uncertain. In vertebrates, segments are formed sequentially by a segmentation 'clock' of oscillating gene expression involving Notch pathway components. Recent studies in spiders and basal insects have suggested that segmentation in these arthropods also involves Notch-based signalling. These observations have been interpreted as evidence for a shared, ancestral gene network for insect, arthropod and bilaterian segmentation. However, because this pathway can play multiple roles in development, elucidating the specific requirements for Notch signalling is important for understanding the ancestry of segmentation. Here we show that Delta, a ligand of the Notch pathway, is not required for segment formation in the cricket Gryllus bimaculatus, which retains ancestral characteristics of arthropod embryogenesis. Segment patterning genes are expressed before Delta in abdominal segments, and Delta expression does not oscillate in the pre-segmental region or in formed segments. Instead, Delta is required for neuroectoderm and mesectoderm formation; embryos missing these tissues are developmentally delayed and show defects in segment morphology but normal segment number. Thus, what initially appear to be 'segmentation phenotypes' can in fact be due to developmental delays and cell specification errors. Our data do not support an essential or ancestral role of Notch signalling in segment generation across the arthropods, and show that the pleiotropy of the Notch pathway can confound speculation on possible segmentation mechanisms in the last common bilaterian ancestor.     

Variation in body segment number within and between populations of the centipede Strigamia maritima (Chilopoda: Geophilomorpha)

Although most arthropod species have a fixed number of body segments, one order of centipedes – the Geophilomorpha – provides an unusual opportunity to study the variation and microevolution of segment number. This is because all species in all but one family exhibit variation in the number of leg‐bearing segments (LBS) within and between natural populations. One species in particular, the coastal geophilomorph Strigamia maritima, has become a ‘model system’ for these studies, because of its high population densities and the consequent ease of collecting large samples. Previous studies on this species have examined various aspects of segment number variation. However, most studies have characterized each population by an LBS distribution and a mean LBS number that are based on data from all life‐stages. Here, we dissect the variation within as well as between populations and show that different cohorts within a population often have significantly different LBS number distributions. This is almost certainly due to developmental plasticity, probably related to the prevailing microhabitat temperature within brood chambers, but possibly related to other environmental factors too. Although we found no evidence of selection, the fact that different species of geophilomorphs have different LBS distributions suggests that, in the long term, selection may act on the developmental reaction norm of LBS number. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107 , 678–685.     

The segmentation cascade in the centipede Strigamia maritima: involvement of the Notch pathway and pair-rule gene homologues

The centipede Strigamia maritima forms all of its segments during embryogenesis. Trunk segments form sequentially from an apparently undifferentiated disk of cells at the posterior of the germ band. We have previously described periodic patterns of gene expression in this posterior disc that precede overt differentiation of segments, and suggested that a segmentation oscillator may be operating in the posterior disc. We now show that genes of the Notch signalling pathway, including the ligand Delta, and homologues of the Drosophila pair-rule genes even-skipped and hairy, show periodic expression in the posterior disc, consistent with their involvement in, or regulation by, such an oscillator. These genes are expressed in a pattern of apparently expanding concentric rings around the proctodeum, which become stripes at the base of the germ band where segments are emerging. In this transition zone, these primary stripes define a double segment periodicity: segmental stripes of engrailed expression, which mark the posterior of each segment, arise at two different phases of the primary pattern. Delta and even-skipped are also activated in secondary stripes that intercalate between primary stripes in this region, further defining the single segment repeat. These data, together with observations that Notch mediated signalling is required for segment pattern formation in other arthropods, suggest that the ancestral arthropod segmentation cascade may have involved a segmentation oscillator that utilised Notch signalling.     

Expression of Pax group III genes suggests a single-segmental periodicity for opisthosomal segment patterning in the spider Cupiennius salei

Pair-rule patterning forms a key step for segmentation in insects. The expression patterns of pair-rule gene orthologs in representatives of other arthropod groups imply that these genes were segmentation genes in the last common ancestor of the various arthropod groups, but almost nothing is known about the underlying mechanism in noninsect arthropods. Here, we cloned and analyzed members of the Pax group III genes from the spider Cupiennius salei. Pax group III genes comprise genes like the Drosophila genes paired, gooseberry, and gooseberry-neuro, as well as the vertebrate Pax 3 and Pax 7 genes. We recovered three Pax group III genes from the spider C. salei, Cs-pairberry-1, Cs-pairberry-2, and Cs-pairberry-3, and show that the combined expression of the three spider genes mimics the patterns in insects, suggesting an ancestral role for Pax group III genes in segmentation, neurogenesis, and appendage formation in arthropods. One of the genes, pairberry-3, is expressed in a segmental periodicity before overt morphological segmentation is visible, suggesting a single segmental periodicity for opisthosomal segment pattering in the spider. Comparisons among arthropods suggest that the underlying mechanisms for pair-rule gene orthologs are more diverged than the ones for the segment-polarity genes. We argue that there may be a correlation between the lower variation in patterns of segment-polarity genes and the phylotypic stage. The segment-polarity genes are required to define the segment borders of the embryo at the germ-band stage, the arthropod phylotypic stage. Pair-rule gene orthologs act more upstream and may display more variation in their action.     

An early temperature‐sensitive period for the plasticity of segment number in the centipede Strigamia maritima

Geophilomorph centipedes show variation in segment number (a) between closely related species and (b) within and between populations of the same species. We have previously shown for a Scottish population of the coastal centipede Strigamia maritima that the temperature of embryonic development is one of the factors that affects the segment number of hatchlings, and hence of adults, as these animals grow epimorphically—that is, without postembryonic addition of segments. Here, we show, using temperature‐shift experiments, that the main developmental period during which embryos are sensitive to environmental temperature is surprisingly early, during blastoderm formation and before, or very shortly after, the onset of segmentation.     

A three-phase model of arthropod segmentation

Molecular and morphological evidence (expression patterns of pair-rule genes and segmental position of the genital openings and other segmental markers) suggest that the segmental units of the arthropod body are specified, in early ontogeny, by three spatially and/or temporally distinct mechanisms and do not appear in a strict antero-posterior sequence. A first anterior set of indivisible segments (naupliar segments, possibly three in all arthropods) is followed by a set of more caudal (post-naupliar) primary units (eosegments, possibly ten in all arthropods) which then undergo a process of secondary segmentation, thus giving rise to a higher number of definitive segments (merosegments). The number of merosegments deriving from each eosegment is characteristic of the different arthropod clades and is mostly stable at the level of the traditional arthropodan classes or subclasses. All their segmentation patterns, however, including those found in the segmental organisation of highly segmented forms (such as centipedes and millipedes, notostracan, lipostracan and anostracan crustaceans, and trilobites) are reducible to the basic groundplan with three naupliar and ten postnaupliar segments. These basic units of arthropod segmentation may also have an equivalent in other Ecdysozoa, despite the lack of any segmentation (nematodes) or, at least, of an overt segmentation (kinorhynchs).     

Exploring developmental modes in a fossil arthropod: growth and trunk segmentation of the trilobite Aulacopleura konincki

Trilobites offer the opportunity to explore postembryonic development within the fossil record of arthropod evolution. In contrast to most trilobites, the Silurian proetid Aulacopleura konincki from the Czech Republic exhibits marked variation in the mature number of thoracic segments, with five morphs with 18-22 thoracic segments. The combination of abundant articulated specimens available from a narrow stratigraphic interval and segmental intraspecific variation makes this trilobite singularly useful for studying postembryonic growth and segmentation. Trunk segmentation followed a hemianamorphic pattern, as seen in other arthropods and as characteristic of the Trilobita; during a first anamorphic phase, segments were accreted, while in the subsequent epimorphic phase, segmentation did not proceed further despite continued growth. Size increment during the anamorphic phase was targeted and followed Dyar's rule, a geometric progression typical of many arthropods. We consider alternative hypotheses for the control of the switch from anamorphic to epimorphic phases of development. Our analysis favors a scenario in which the mature number of thoracic segments was determined quite early in development rather than at a late stage in association with a critical size threshold. This study demonstrates that hypotheses concerning developmental pattern and control can be tested in organisms belonging to an extinct clade.     

How does arthropod segment number evolve?—some clues from centipedes

Summary Studies of intraspecific variation in the number of trunk segments of geophilomorph centipedes provide clues as to how different species of arthropods, and whole clades in some cases, come to be characterized by different segment numbers. However, although previous work in this area has revealed an interesting geographical pattern—a latitudinal cline in which segment number decreases with increasing latitude—the causality of the cline remains obscure. Is it because of selection on genetically based variation, or is it a result of a form of phenotypic plasticity in which the segmentation process is directly affected by a latitude‐correlated factor such as temperature? Here, we provide some indirect evidence for plasticity. If the cline is indeed a plastic one, a paradox arises, because the cline mirrors interspecific variation—geophilomorph species with more northern ranges typically have fewer segments than those from further south—but interspecific differences cannot arise from nonheritable variation. We propose a resolution of this apparent paradox via a model in which genetic and environmental factors interact through selection acting on developmental reaction norms.     

Gene expression suggests conserved mechanisms patterning the heads of insects and myriapods

Segmentation, i.e. the subdivision of the body into serially homologous units, is one of the hallmarks of the arthropods. Arthropod segmentation is best understood in the fly Drosophila melanogaster. But different from the situation in most arthropods in this species all segments are formed from the early blastoderm (so called long-germ developmental mode). In most other arthropods only the anterior segments are formed in a similar way (so called short-germ developmental mode). Posterior segments are added one at a time or in pairs of two from a posterior segment addition zone. The segmentation mechanisms are not universally conserved among arthropods and only little is known about the genetic patterning of the anterior segments. Here we present the expression patterns of the insect head patterning gene orthologs hunchback (hb), orthodenticle (otd), buttonhead-like (btdl), collier (col), cap-n-collar (cnc) and crocodile (croc), and the trunk gap gene Krüppel (Kr) in the myriapod Glomeris marginata. Conserved expression of these genes in insects and a myriapod suggests that the anterior segmentation system may be conserved in at least these two classes of arthropods. This finding implies that the anterior patterning mechanism already existed in the last common ancestor of insects and myriapods.     

Evolution of the pair rule gene network: Insights from a centipede

Comparative studies have examined the expression and function of homologues of the Drosophila melanogaster pair rule and segment polarity genes in a range of arthropods. The segment polarity gene homologues have a conserved role in the specification of the parasegment boundary, but the degree of conservation of the upstream patterning genes has proved more variable. Using genomic resources we identify a complete set of pair rule gene homologues from the centipede Strigamia maritima, and document a detailed time series of expression during trunk segmentation. We find supportive evidence for a conserved hierarchical organisation of the pair rule genes, with a division into early- and late-activated genes which parallels the functional division into primary and secondary pair rule genes described in insects. We confirm that the relative expression of sloppy-paired and paired with respect to wingless and engrailed at the parasegment boundary is conserved between myriapods and insects; suggesting that functional interactions between these genes might be an ancient feature of arthropod segment patterning. However, we find that the relative expression of a number of the primary pair rule genes is divergent between myriapods and insects. This corroborates suggestions that the evolution of upper tiers in the segmentation gene network is more flexible. Finally, we find that the expression of the Strigamia pair rule genes in periodic patterns is restricted to the ectoderm. This suggests that any direct role of these genes in segmentation is restricted to this germ layer, and that mesoderm segmentation is either dependent on the ectoderm, or occurs through an independent mechanism.