Invertebrate Biology 133(3): 274–280. © 2014, The American Microscopical Society, Inc. DOI: 10.1111/ivb.12063 Evolutionary changes in the integument of the onychophoran Plicatoperipatus jamaicensis (Peripatidae) Ivo de Sena Oliveira,1,a Carsten L€ uter,2 Klaus W. Wolf,3 and Georg Mayer1 1 2 Animal Evolution and Development, Institute of Biology, University of Leipzig, D-04103 Leipzig, Germany Museum f€ ur Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, D-10115 Berlin, Germany 3 Electron Microscopy Unit, University of the West Indies (Mona), Kingston 7, Jamaica Abstract. The dorsal integument of nearly all species of Peripatidae—one of the two major subgroups of Onychophora (velvet worms)—typically exhibits 12 plicae (=annuli) per segment. The only exception might occur in Plicatoperipatus jamaicensis, from which 24 putative plicae per segment have been reported. Hence, the number of plicae might have been duplicated in this species. If so, one would expect that the structure of the duplicated plicae would resemble that of all other onychophoran species, and that the structures commonly associated with the plicae, including crater-shaped papillae and hyaline organs, would also have been duplicated. To clarify whether there was indeed such duplication, we compared the structure of the integument in embryos and adults of Pl. jamaicensis (with the putative number of 24 plicae per segment) and Principapillatus hitoyensis (with the common number of 12 plicae per segment). Our scanning electron microscopic data revealed that embryos of both species have 12 plicae per segment. While this number persists in adults of Pr. hitoyensis, 12 additional rows of papillae (=pseudoplicae) occur in the dorsal integument in adults of Pl. jamaicensis. These pseudoplicae differ from the typical plicae of other onychophorans in that they are not equipped with primary papillae and are situated in furrows between the true plicae, as evidenced by the position of hyaline organs and crater-shaped papillae. Our data further show that the number of the ventrolateral plicae, crater-shaped papillae, and hyaline organs is similar in the two species studied. This suggests that the plicae have not been duplicated in the integument of Pl. jamaicensis, but rather that additional pseudoplicae have been inserted between the 12 original plicae. These pseudoplicae might have led to a denser package of dermal papillae in the dorsal integument of Pl. jamaicensis, but the functional significance of this evolutionary change is unknown. Additional key words: annulation, annuli, cuticle, plicae, segment, velvet worms The body wall of many soft-bodied ecdysozoans (=molting animals), including priapulids, nematodes, and onychophorans, is micro-annulated (Fig. 1A,B). The presence of a similar cuticular organization into numerous rings or annuli in fossil ecdysozoans suggests that it might be an ancestral feature of the entire group (e.g., Whittington 1978; Budd 2001; Nielsen 2012; Ou et al. 2012). Among extant panarthropods, the annuli have persisted only in onychophorans, but have been lost in tardigrades and arthropods (Budd 2001; Nielsen 2012). The annuli of onychophorans, also called plicae (see Oliveira et al. 2010 for synonyms), are typically covered with a Author for correspondence. E-mail: ivo.de_sena_oliveira@uni-leipzig.de numerous dermal papillae, but they are still recognizable as separate folds or dermal ridges, which are clearly delineated from each other by transverse furrows (Fig. 1B; Read 1988a,b; Reid 1996; Oliveira et al. 2011, 2012, 2013a). Although the onychophoran cuticle and integument have been interpreted as being “essentially unsegmented” (Budd 2001) due to the lack of distinct segmental boundaries (Mayer & Whitington 2009), the plicae are clearly arranged in segmentally repeated sets along the antero-posterior body axis (Oliveira et al. 2011, 2012). For example, incomplete and/or anastomosing (=branching) plicae that exist in various onychophoran species are located in specific positions within each segment along the dorsum (Bouvier 1905; Zilch 1954; Ruhberg 1985; Oliveira The integument of Plicatoperipatus 275 Fig. 1. Examples of body annulation in two soft-bodied ecdysozoans. Scanning electron micrographs. Anterior is left. A. Body surface of an unidentified nematode from Chile in ventral view illustrating regular annulation. The nematode was collected from the body surface of specimens of the onychophoran Metaperipatus inae (see Mayer 2007 for details on the collection site). Scale bar=5 lm. B. Body surface of the onychophoran Principapillatus hitoyensis in dorso-lateral view. Note the numerous annuli (=plicae) covered with dermal papillae. Scale bar=250 lm. An, annulus; dp, dermal papilla; lg, leg/lobopod; pf, plical furrow; pl, plicae. et al. 2010, 2011, 2012). Evidence for a segmental organization of the onychophoran integument is also provided by other structures, such as ventral and preventral organs, interpedal structures, and sets of crater-shaped papillae, which are associated with specific plicae in each segment (Bouvier 1905; Oliveira et al. 2012, 2013a,b). While the number of plicae per segment varies from 12 to 18 among peripatopsid species, most species of the other major onychophoran subgroup, the Peripatidae, show a conserved number of twelve plicae per segment (Bouvier 1905, 1907; Clark 1913; Fuhrmann 1914; Froehlich 1968; Peck 1975; Ruhberg 1985; Reid 1996; Oliveira et al. 2010, 2011, 2012, 2013a). The only exception has been reported from the peripatid Plicatoperipatus jamaicensis (GRABHAM & COCKERELL 1892), which is believed to have 24 plicae per segment (Bouvier 1905; Clark 1913; Peck 1975; Oliveira et al. 2012). Thus, the number of plicae might have been duplicated in this species. In this case, one would expect that the associated structures, including the crater-shaped papillae and the hyaline organs (Bouvier 1905; Oliveira et al. 2012, 2013a), would also be duplicated. Moreover, close morphological resemblance of the duplicated and the original plicae would be anticipated. To clarify the evolutionary changes in the integument of Pl. jamaicensis, we applied scanning electron microscopy to embryos and adult specimens of this species. In addition, we carried out pepsin digestion of the body wall to reveal the internal structure of the cuticle and integument in adult specimens. For comparison, we analyzed embryos, adult specimens, and molted skins of another peripatid species, Principapillatus hitoyensis OLIVEIRA ET AL. 2012, which shows the common number of twelve plicae per segment. Methods Specimens of Plicatoperipatus jamaicensis (n=16) were obtained from leaf litter and in heaps of limestone in a banana plantation near Ecclesdown, Parish Portland, Jamaica, West Indies (18°020 09.2″N, 76°190 09.8″W). Specimens of Principapillatus hitoyensis were collected as described previously (Oliveira et al. 2012). For scanning electron microscopy, adult specimens of both sexes (n=4), as well as flexed-stage embryos (n=2) dissected from the female uteri, were fixed and preserved as described previously (Oliveira et al. 2012). After dehydration in an ethanol series, specimens were dried in a critical point dryer (CPD 030 Bal-Tec AG, Balzers, Liechtenstein, or K850, Emitech Ltd., Kent, England), coated with gold in a SCD 050 Sputter Coater (Balzers Union, Balzers, Liechtenstein) or SC 7640 Sputter Coater (Quorum Technologies, Polaron, Newhaven, UK), and examined with Quanta 200 (FEI, Hillsboro, Oregon, USA) or EVO LS 10 (Zeiss, Oberkochen, Germany) scanning electron microscopes. Trunk pieces (4–5 segments) from adult specimens of Pl. jamaicensis were rinsed twice in distilled water for 30 min and digested overnight in a solution containing 10% pepsin (0.3 g pepsin in 3 mL distilled water with Invertebrate Biology vol. 133, no. 3, September 2014 276 Oliveira, L€ uter, Wolf, & Mayer two drops of 2 mol L1 HCl). The detached skin was removed carefully from the rest of the body using fine forceps. The skins and the remaining body parts were postfixed in 4% formalin for 2 h, rinsed several times in distilled water, dehydrated in an ethanol series, and processed further for scanning electron microscopy as described above. Freshly molted skins obtained from living specimens of Pr. hitoyensis were spread on water surface according to Holliday (1944), placed on glass slides, and analyzed with a stereomicroscope (Wild M10, Leica Microsystems, Wetzlar, Germany) equipped with a digital camera (PCO AG SensiCam, Kelheim, Germany). For the terminology of morphological features used, see Oliveira et al. (2010, 2012). Embryos were staged according to Walker & Campiglia (1990). Light and scanning electron micrographs were processed and artificial colors added with Adobe (San Jose, CA, USA) Photoshop CS4. Final panels were designed with Adobe Illustrator CS4 and exported in the Tagged Image File Format. sponded to the segmental set of twelve plicae in Pr. hitoyensis (Fig. 2D), which were also equipped with relatively large primary papillae (Fig. 3D). Only seven of these twelve plicae passed to the ventral body surface in both species (Fig. 2E,F), whereas none of the pseudoplicae of Pl. jamaicensis did so (Fig. 2E). The pseudoplicae instead terminated laterally in each interpedal region. An analysis of the internal structure of the cuticle and integument of Pl. jamaicensis after digestion with pepsin revealed that the bases of the accessory papillae, which belonged to the pseudoplicae, were elongated and narrower than those of the plical papillae (Fig. 3A,B). Moreover, the accessory papillae did not form coherent, independent folds or ridges comparable to the typical plicae, but rather seemed to be positioned in furrows between the plicae (Fig. 3B). This inserted position was reflected by the corresponding arrangement of the cratershaped papillae and hyaline organs, which in Pl. jamaicensis were associated with the pseudoplicae (Figs. 2E, 3C), whereas they were clearly located in the plical furrows in Pr. hitoyensis (Figs. 2F, 3D). Results Scanning electron microscopy of the flexed-stage embryos of Plicatoperipatus jamaicensis and Principapillatus hitoyensis revealed a thin, smooth cuticle that covered the entire body, including the developing plicae and dermal papillae (Fig. 2A,B). At this developmental stage, the adjacent plicae were clearly separated from each other by prominent plical furrows (Fig. 2A,B). The embryos of both species each had twelve plicae per segment along the dorsum (Fig. 2A,B; herein we use the midline of each leg as a segmental landmark). In contrast to the regular arrangement of plicae in Pl. jamaicensis, a few incomplete plicae and anastomoses were evident in the dorsal integument of Pr. hitoyensis (Fig. 2B). While the initial arrangement of the integument (with twelve plicae per segment and no additional rows of papillae) is retained in adults of Pr. hitoyensis, it changes substantially in Pl. jamaicensis (Fig. 2C–F). The dorsal integument in adults of this species had a large number of small dermal papillae that were pine cone shaped and arranged in 24 transverse rows per segment (Fig. 2C). However, only twelve of these rows (Fig. 2C) were equipped with primary papillae, characterized by an apical piece and a sensory bristle (cf. Oliveira et al. 2010), whereas the remaining twelve rows (=pseudoplicae; Fig. 2C) consisted exclusively of accessory papillae (Fig. 3A–C). Thus, only the twelve rows bearing the primary papillae in Pl. jamaicensis (Fig. 2C) corre- Invertebrate Biology vol. 133, no. 3, September 2014 Discussion The putatively duplicated number of plicae in Plicatoperipatus jamaicensis has been used as a name-giving feature for this monotypic genus (plicato=plicated, folded, crumpled; Clark 1913; Grabham & Cockerell 1928; Brown 1956). Accordingly, one would expect that the additional plicae have a structure similar to the typical plicae of other onychophoran species and that the associated dermal structures, including the crater-shaped papillae and the hyaline organs, are doubled as well (Fig. 4A–D). However, our observations revealed that this is not the case. First, the structures associated with plicae are not doubled in Pl. jamaicensis, as the number of the crater-shaped papillae and hyaline organs in each body segment corresponds to that in other representatives of Peripatidae, including Principapillatus hitoyensis (Fig. 4A,B,E,F; Bouvier 1905; Oliveira et al. 2012, 2013a). Second, the number of plicae that pass to the ventral body surface between each two successive leg pairs is also the same as in other peripatid species (Fig. 4A,B,E,F; Bouvier 1905; Oliveira et al. 2012, 2013a). Third, only 12 of 24 transverse rows of papillae in the dorsal integument of Pl. jamaicensis (cyan in Fig. 4E,F) correspond in structure and position to the plicae of other peripatids (e.g., Bouvier 1905; Marcus & Marcus 1955; Froehlich 1968; Peck 1975; Read 1988b). In contrast, the remaining twelve rows in Pl. jamaicensis (orange in Fig. 4E,F), The integument of Plicatoperipatus 277 Fig. 2. Comparison of the integument in two onychophoran species. Scanning electron micrographs of flexed-stage embryos (A, B) and adults (C–F). Anterior is up in A–D; posterior is right in E and F. Note the segmentally repeated set of plicae (numbered 1–12), pseudoplicae (numbered 10 –120 ), and crater-shaped papillae. Note that only seven plicae extend to the ventral body surface in both species studied (numbered 1–7 in E and F). A, C, E. Plicatoperipatus jamaicensis. B, D, F. Principapillatus hitoyensis. Arrowheads point to the incomplete plicae; arrow indicates anastomosing plicae (see Oliveira et al. 2011, 2012 for more details). Scale bars=200 lm (A–D) and 100 lm (E, F). Cp, crater-shaped papilla; dp, dermal papilla; lg, leg/lobopod; pf, plical furrow. which we have dubbed pseudoplicae, cannot be regarded as true plicae because they lack primary papillae and are positioned in furrows between the actual plicae, as evidenced by the position of the crater-shaped papillae and hyaline organs in the two species studied (Fig. 4A,B,E,F). We therefore conclude that the structure of the dorsal integument in Pl. jamaicensis has not resulted from a duplication of annuli, but rather from an insertion of the pseudoplicae in furrows between the true plicae. This modification has led to a denser arrangement of dermal papillae in the dorsal Invertebrate Biology vol. 133, no. 3, September 2014 278 Oliveira, L€ uter, Wolf, & Mayer Fig. 3. Organization of the dorsal integument in two onychophoran species. Scanning electron micrographs from Plicatoperipatus jamaicensis (A–C) and stereomicrograph from Principapillatus hitoyensis (D). Anterior is up in all images. Arrows indicate primary papillae in A and B. A. Internal perspective of the skin after digestion with pepsin. B. Surface of the integument, from which the overlying cuticle has been removed after digestion with pepsin. Note that the pseudoplicae are narrow and positioned in furrows between the plicae. Note also that only the plicae bear primary papillae, recognizable by remnants of their apical pieces. C. External view of dorsal body surface. Note the position of hyaline organs on each pseudoplica. D. External view of dorsal body surface in molted skin. Note the position of hyaline organs within each plical furrow (arrowheads). Scale bars=50 lm (A–C) and 250 lm (D). Ap, accessory papilla; dm, dorsal midline; ho, hyaline organ; pp, primary papilla. integument of Pl. jamaicensis. Although the functional significance of this modification is unknown, the pseudoplicae are a derived feature of P. jamaicensis, as they have not been reported from any other onychophoran species. Intriguingly, we found that embryos of both species studied initially bear only Invertebrate Biology vol. 133, no. 3, September 2014 the twelve plicae per segment. While this arrangement persists in adult specimens of Pr. hitoyensis, additional pseudoplicae appear in the integument of Pl. jamaicensis late in development. Thus, the initial structure of the integument in embryos of Pl. jamaicensis, with twelve plicae per segment and no The integument of Plicatoperipatus 279 Fig. 4. Diagrams comparing the arrangement of plicae, hyaline organs, and crater-shaped papillae in Plicatoperipatus jamaicensis and the remaining species of Peripatidae. A, B. Pattern found in all species of Peripatidae, except for Pl. jamaicensis. C, D. Expected pattern in Pl. jamaicensis, if the number of plicae was duplicated. D, E. Arrangement of plicae and associated structures in Pl. jamaicensis revealed in this study. Arrowhead (in A, C, E) points to a cratershaped papilla. Dm, dorsal midline; ho, hyaline organ; lg, leg/lobopod; pl, plica; ps, pseudoplica. pseudoplicae, can be regarded as an ontogenetic recapitulation of the ancestral state, which was present in the last common ancestor of Peripatidae. Despite the evolutionary changes that have taken place in the integument of Pl. jamaicensis, our data show a strictly segmental organization of plicae and associated structures in the two onychophoran species studied. Both species exhibit 12 segmental plicae per trunk segment, only seven of which extend to the ventral body surface (Fig. 4A,E). Likewise, the rows of five to six crater-shaped papillae are also repeated in a segmental fashion in each interpedal region. Therefore, contrary to a previous view (Budd 2001; Mayer & Whitington 2009), our findings suggest that, at least in representatives of Peripatidae, the cuticle and the integument do have a Invertebrate Biology vol. 133, no. 3, September 2014 280 Oliveira, L€ uter, Wolf, & Mayer segmental organization, although the underlying segment patterning mechanisms, in particular in the dorsal body region, are unknown. Acknowledgments. The authors are thankful to Ms. Anke S€ anger (MfN, Berlin) for assisting with scanning electron microscopy, Vladimir Gross for proofreading the manuscript, and the staff of the Instituto Nacional de Biodiversidad (INBio, Heredia, Costa Rica) for providing the permits. This study was supported by a PhD fellowship of the Conselho Nacional de Desenvolvimento Cientıfico e Tecnol ogico (CNPq: 290029/2010-4) to ISO. 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