Appendage patterning in the South American bird spider Acanthoscurria geniculata (Araneae: Mygalomorphae)

Matthias Pechmann 0 Nikola-Michael Prpic 0 0 Communicated by S. Roth 1 ) Johann-Friedrich-Blumenbach-Institut fr Zoologie und Anthropologie , Abteilung Entwicklungsbiologie, GZMB Ernst-Caspari-Haus, Georg-August-Universitt , Justus-von-Liebig-Weg 11, 37077 Gttingen, Germany Pattern formation by the genes dachshund (dac), Distal-less (Dll), extradenticle (exd) and homothorax (hth) in spider appendages has been studied previously only in members of the higher spiders (Araneomorphae). In order to study the diversity and conservation of pattern formation in spiders as a whole, we studied homologs of these genes in embryos of the bird spider Acanthoscurria geniculata, which belongs to the Mygalomorphae, a more primitive spider group. We show that the patterns of dac and Dll are largely conserved in all spiders studied so far. We find a duplication of hth and exd genes as previously identified in the higher spider Cupiennius salei. These data suggest that pattern formation shows little diversity in all spiders, including the duplication of hth and exd that likely occurred before the split of Mygalomorphae and Araneomorphae. We also find that the legs and pedipalps bear endites of which only the pedipalpal endite expresses Dll and is retained in the adult. Similarly, the limb buds of the posterior spinnerets express Dll and become segmented appendages in the adult, whereas the anterior spinnerets lack Dll expression and are absent in postembryonic stages. In both cases, the expression of Dll or the lack of it indicates structures which will be retained as adult traits or rudimentary structures that degenerate, respectively. The presence of embryonic rudiments of leg endites in Acanthoscurria and the leg-like pattern formation in the posterior spinnerets are interpreted as primitive traits that have been lost in the Araneomorphae. - The evolutionary success of the arthropods is, to a large part, founded on the diversity of their appendages which have been adapted to a large number of functions. A major goal of evolutionary developmental biology is to understand how evolutionary changes in developmental genetic mechanisms lead to novel morphological traits, e.g. new appendage types, that can adapt to new functions. Spiders have a number of different appendage types along their body axis. The opisthosoma (abdomen) bears four pairs of highly specialised appendages: the appendages on opisthosomal segments 2 and 3, after a short phase of outgrowth, invaginate and give rise to a complex respiratory system inside of the body. The appendages on opisthosomal segments 4 and 5 are the spinnerets that form a silk spinning and weaving apparatus. The appendages on the prosoma (head and trunk) are mainly used for feeding and locomotion, but can have additional functions as well. The most anterior appendage is the bilobed labrum that likely evolved from an anterior appendage pair by rotation and fusion (Kimm and Prpic 2006) and that serves as the upper lip during feeding. The following pair of appendages, the chelicerae, are used for prey capture and feeding and they inject the venom into prey animals. The next pair, the pedipalps, are a multifunctional appendage pair, which is used for sensory perception, feeding and, in males, sperm transfer during mating. The following four pairs of walking legs are mainly locomotory appendages, but are also equipped with organs for sensory perception and are also used for other functions such as prey capture. Previous studies in spiders have shown that the patterning of pedipalps and legs is very similar (Abzhanov and Kaufman 2000; Prpic et al. 2003; Prpic and Damen 2004), probably reflecting their morphological similarity. Pattern formation in the chelicera, however, is different from the legs and pedipalps, suggesting that the differences in pattern formation correlate with the specific morphology of the chelicera (Prpic and Damen 2004). However, these studies have only involved representatives of the higher spiders, the Araneomorphae (for an overview of spider phylogeny, see Fig. 1a). Members of the two other spider groups, the Mesothelae and the Mygalomorphae, have not yet been studied. Thus, it is currently unclear whether the patterns in the araneomorph members studied so far are representative of the spiders as a whole. The Mesothelae (less than 100 described species) are a basally branching spider group and display a number of primitive traits including a fully segmented opisthosoma (hence, their common name segmented spiders). Unfortunately, these spiders are rare, very difficult to breed and embryos are not yet available for molecular studies. We have, therefore, used a member of the spider group branching after the Mesothelae, the Mygalomorphae (bird spiders). The studied species, Acanthoscurria geniculata (Fig. 1d), is a large and colourful bird spider distributed in Brazil (South America). We have studied the expression of several leg patterning genes in Acanthoscurria embryos and we show that the patterns are largely conserved between mygalomorph spiders and the higher spiders of the Araneomorphae. However, A. geniculata also shows traits like embryonic endite rudiments on the legs and leg-like morphology and pattern formation in the posterior spinnerets, which we interpret as primitive characters lost in the Araneomorphae. Materials and methods Embryo collection and fixation Embryos of A. geniculata were obtained from a female in the private collection of the first author. Only a single cocoon was available for study. The embryos of Cupiennius salei and Achaearanea tepidariorum were obtained from our laboratory stocks in Gttingen. Embryos of all three species were fixed according to the published protocol for Cupiennius embryos (Prpic et al. 2008a). Total RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instructions and cDNA was synthesised Fig. 1 Overview of the phylogenetic relationships of the spider species used in this work. a Simplified phylogenetic tree showing the relationships of the three major spider groups: Mesothelae, Mygalomorphae, and the higher spiders Araneomorphae (boxed). The names of the species in this study are given in parentheses below the names of the larger taxa. Photos of adult females of the species in this study: b A. tepidariorum, c C. salei and d A. geniculata. Scale bar is 1 cm in all panels with the Smart polymerase chain reaction (PCR) cDNA Synthesis Kit (Clontech). Fragments of the genes Dll, dac, exd and hth were isolated by PCR using the previously published primers (Prpic et al. 2001, 2003; Prpic and Tautz 2003). The hth-2 fragment, however, resulted from priming of the nested reverse primer only. This is the reason why it is shorter than the hth-1 fragment which resulted, as expected, from the priming of the nested forward and reverse primer pair. The orthology of all cloned fragments was assessed by phylogenetic analysis as described previously (Prpic et al. 2005). The (...truncated)