Calcitic Sclerites at Base of Malacostracan Pleopods (Crustacea) – Part of a Coxa Verena Kutschera*, Andreas Maas†, Gerd Mayer† and Dieter Waloszek†

Kutschera et al. BMC Evolutionary Biology (2015) 15:117 DOI 10.1186/s12862-015-0357-6

RESEARCH ARTICLE Open Access Calcitic sclerites at base of malacostracan pleopods (Crustacea) – part of a coxa Verena Kutschera*, Andreas Maas†, Gerd Mayer† and Dieter Waloszek†

Abstract Background: Cuticular specialisations such as joints and membranes play an important role in the function of arthropod limbs. This includes sclerotisations and mineral incrustations of cuticular areas to achieve either more rigidity or flexibility. The anterior eight thoracopods of Malacostraca have limb stems comprising a coxa and a basipod, which carries the two rami. Their pleopods, the limbs of the posterior trunk part, have for long been regarded to lack a coxa. Several calcitic sclerites occur in the area between ventral body and limb stem. This raises the questions: do these elements represent specialisations of the membrane due to functional requirements, and do they originate from an originally larger limb portion, i.e., the coxa, or in fact represent it. Results: We investigated 16 species of selected malacostracan taxa from all major in-groups. Calcitic sclerites occur in constant numbers and position within a species (no individual variation, and independent of specific modification such as in genital appendages). These are even constant within a supra-specific taxon, which facilitates comparisons. In general the sclerites connect via two pivot joints to the sternite medially and the tergopleura laterally, and two more to the limb stem. Based on this, we reconstructed putative ground-pattern conditions for the sclerites of the examined taxa of Malacostraca. Conclusions: The pattern of sclerites is characteristic for each monophyletic malacostracan taxon. The highest number of sclerites most likely represents the plesiomorphic state. Reduction of sclerite numbers occurs in Caridoida and its in-groups. Sclerite arrangement in these taxa provides an important character complex for phylogenetic studies. The presence of pivot joints to the body proximally and basipod distally demonstrates the existence of a coxa, which is just slightly less sclerotised, particularly on its posterior side. This can be explained by enhanced flexibility of the pleopods evolved in the course to their major role as swimming devices. Both the pivot joints and the proximal and distal extension of the calcitic sclerites demarcate the minimum area of the coxa. With this, sclerites appear as very valuable also in shedding more light on the putative relationships between Malacostraca, Myriapoda, Insecta, and Remipedia. Keywords: Proximal endite, Coxa, Basipod, Pleon, Alizarin Red, Fluorescence microscopy

Background membrane. These reinforcements occur in the form of The membranous cuticle in the joint or articulation be- sclerites within the membrane [1]. Another option is that tween body proper and appendage of sclerotised arthropods more sclerotised cuticular elements approach each other by is an important structure that intermediates between rigid- the formation of simple to very complex joints [7]. ity and flexibility in order to facilitate the operability of an A well-developed basal membrane did not appear before appendage. Numerous variations occur among modern ar- the euarthropod level in connection with the development thropods ([1] and references therein for Chilopoda, Progo- of a rigid, antero-posteriorly flattened stem portion, the neata, and Hexapoda; [2] for Trilobita; [3] for Chelicerata, basipod, which carried the two rami endopod and exopod and crustaceans in particular [4-6]). One possibility to mod- [8,9]. This design permitted a more elaborate ex- and in- ify the design of arthropod joints is to stiffen parts of the trinsic musculature to extend from inside the body and inserting into the limb stem. Initially, the limb stem mainly * Correspondence: [email protected] moved in anterior and posterior direction. With the newly † Equal contributors formed basal membrane, interactions could now take place Biosystematic Documentation, University of Ulm, Helmholtzstraße 20, 89081 Ulm, Germany between the body and the stiffer limb stem, allowing

© 2015 Kutschera et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 2 of 27

inward-outward and rotating movements. Such a "eu- demanded for more data to be collected, compared and arthropodium" was a multi-functional unit, with the limb interpreted. Therefore, it seemed of high relevance for us to stem medially armed with setae and spines to serve for food study the details of the pleon morphology with particular manipulation, and endopod and exopod serving more for regard to the basal joint and its structures associated with food gathering (grabbing things) and locomotion (walking it. We started to particularly search for structures that as much as swimming) ([9], see [10] for an extensive dis- mighthintattheoriginalsituationatthebaseofthepleo- cussion of the evolution of crustacean appendages). pods of malacostracans. Our goal was to find hints for the With few changes, this limb type was continued into the original presence of either the "proximal endite", or a "coxa". euarthropodan in-group lineages including Crustacea. This We examined what appears to be the exclusively membran- allowed the evolution as one of the crustacean novelties, a ous area extending between the ventral body surface and setae-bearing endite, the proximal endite, which occurred the comprehensive pleopod limb stem ("the pleopod-body medially just below the limb stem [11-16]. articulation") in a wide array of malacostracan taxa with In the stem species of Labrophora, the taxon that in- Alizarin-Red staining (which highlights calcified areas) and cludes Phosphatocopina and Eucrustacea, the proximal fluorescence microscopy (Figures 1, 2, 3, 4 and 5; Table 1). endite of the anterior two postantennular appendages We detected several small, calcified areas (termed "sclerites" became modified into a distinct ring-like sclerotised herein) within the pleopod-body articulation (Figures 2, 3 proximal unit, the coxa [6,9,11,17-19]. and 4; Table 1). Considering up to eight individuals for each In entomostracan crustaceans, the occurrence of a coxa is species and similar patterns in closely related species indi- restricted to exactly those appendages, the antenna and cate that the patterns presented here are real and not arte- mandible. The maxillula, the still trunk-limb shaped maxilla, facts produced either by fixation methods, or by the time lag and the trunk limbs retain the proximal endite. Malacos- to the next moult. We also used normal light and scanning traca in this context are characterised by calcification of the electron microscopy to check the proximal limb area for its cuticle, which apparently was a major trigger for the devel- structural composition. Besides discussion of the possible opment of different life strategies within the diverse lineages. role of the sclerites in the functionality of the articulations, In all malacostracans, the eight appendages of the anterior we will also evaluate their potential for phylogenetic con- part of the two-part thorax (thoracopods 1–8ofthoraxI siderations (Figure 6; Table 2 and Table 3) and provide a [20]) have a divided limb stem, comprising a coxa and basi- suggestion about the morphology of the pleopods. pod [4,5]. However, it is still unclear if the coxa of these limbsisderivedfromaproximalenditeorisevolvedina Results different way. By contrast, the six appendages of the second Details revealed on the ventral side of a pleomere and its part of the limb-bearing trunk region of Malacostraca body–limb articulations include the shape of tergo- (thorax II [20]), the so-called pleopods or swimmerets are pleurae, sternites, and insertion of limbs, as well as the traditionally thought to have an undivided limb stem number, shape, and position of the sclerites within the [21-24]. In fact, details of the limb stem are even omitted in arthrodial membranes. In all species we examined, the the descriptive literature. With regard to the common oc- morphology is generally similar for the first to fifth currence of a coxa for all postantennular head limbs and pleomeres (4 in Nebalia bipes (Fabricius, 1780) and those of thorax I of Malacostraca, one might expect an ori- Nebaliopsis sp.) (Figure 1). Therefore, we describe the ginal coxa also on the pleopods, but which is absent, maybe situation in a general way for each species, applicable to it has been lost. Other scenarios are also possible. For ex- pleomeres 1–5(1–4) (see below, Table 1). Additionally, we ample,acoxaoriginallybepresentonthepleopodsmight indicate sexual dimorphism occurring in some of these have fused to the limb stem [15,25-27]; or perhaps it was species, including the sternites, and also record excep- originally absent [8], which would reflect the phylogenetic- tions we uncovered from what one can read in general- ally older situation of the euarthropod condition of a limb ised descriptions. comprising only three limb parts, i.e., even lacking the prox- imal endite developed in the stem lineage of Crustacea. An- Anaspidacea (Figures 2 and 3) other alternative is the possible degeneration of either the Anaspides tasmaniae Thomson, 1893 proximal endite, or a coxa related to the loss of feeding (Figures 1A, 2A–N; Table 4) functions. Calcified tergopleura (tgpl) borders pleomere (plm) laterally In fact, the pleon is a functionally highly important body (Figure 2A–D); calcified, rhombic plate medially interpreted tagma of malacostracan crustaceans, e.g., for swimming, as sternite (st) (Figure 2C, E, G, I, K, M); st subdivided at caridoid escape reaction, brood care, but the pleon has been plm1 (Figure 2E), undivided plm2–5 (Figure G, I, K, M); st strangely little considered so far for phylogenetic purposes, drawn out posteriorly into sternitic wings (stw) (Figure 2D). except [28] who demonstrated the potential of the pleon Limb stem (ls) truncated cone inserting ventrally at plm and its morphological details for phylogeny analyses and (Figure 2A–N); carries multi-annulated, distally-tapering Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 3 of 27

Figure 1 Examined species in lateral view. Scale bars 1 cm. A. Anaspides tasmaniae Thomson, 1893. B. Allanaspides hickmani Swain, Wilson & Ong, 1970. C. Dikerogammarus haemobaphes (Eichwald, 1841). D. Erugosquilla massavensis (Kossmann, 1880). E. Euphausia superba Dana, 1850. F. Gnathophausia gigas Willemoes-Suhm, 1873. G. Gonodactylus chiragra (Fabricius, 1781). H. Hyperia sp. I. Lophogaster typicus Sars, 1857. J. Mysis sp. K. Nebalia bipes (Fabricius, 1780). L. Nebaliopsis sp. M. Neosergestes semissis (Burkenroad, 1940). N. Paranaspides lacustris Smith, 1908. O. Penaeus monodon Fabricius, 1798. P. Thysanopoda tricuspidata Milne-Edwards, 1983. exopod (ex) (Figure 2A, B) and comparably small, cylin- carrying multi-annulated, distally tapering ex (Figure 3A–B); drical endopod (en) (Figure 2E, G, I, K, M); pleopod (plp) no en at plp 1–5. 5 without en (Figure 2M); postero-laterally, ls proximally A single sclerite is present laterally in the articulation excavated (Figure 2F, H, J, L, N). membrane of Al. helonomus (Figure 3A–B; Table 3). The A single semicircular sclerite lies laterally within the mem- sclerite is semicircular and anteriorly bifurcate brane between ls and tgpl (Figure 2C–N; Table 1, also for (Figure 3A–B). The median half of the bifurcated end more details). The sclerite is anteriorly bifurcate; at the split encompasses the ls anteriorly and forms a joint with it, of the fork, the ls articulates with this sclerite 1 laterally. while the lateral half articulates with the tgpl via a pivot Medially, the ls articulates directly with the st via a pivot joint (Table 1). Medially, the ls articulates directly with the joint(Figure2E,G,M;blackarrowheads;Table1).Theme- st via a pivot joint. Examination of internal apodemes was dian half of the bifurcated end of sclerite 1 encompasses not possible (Table 1 and Table 3). Differences between the the ls anteriorly while the lateral half articulates with sexes were not recognised. the tgpl via a pivot joint. No apodemes arise from the inside of this sclerite. Sexual dimorphism: plp 1–2 modified into petasma in Paranaspides lacustris Smith, 1908 males, yet both with single lateral, anteriorly bifurcate (Figures 1N, 3C–E, Table 4) sclerite forming joints with ls and tgpl. Calcified tgpl borders pleomere laterally (Figure 3D–E); divided, median plate interpreted as st with stw extending antero-laterally; plp insert postero-laterally (Figure 3C–E); ls Allanaspides helonomus Swain, Wilson & Ong, 1970 truncated cone inserting latero-ventrally (Figure 3C–E); ls (Figures 1B, 3A–B, Table 4) carries multi-annulated, distally tapering ex (Figure 3C) Tgpl similar to An. tasmaniae (Figure 3A); st barely calci- and comparably small, lentiform en (Figure 3C, E); ex- fied thus weakly fluorescent and barely visible; ls truncated ceptions are plp1–2 in males forming petasm with en cone inserting ventrally at the pleomere (Figure 3A); ls (Figure 3C–D); ls overlaps st medio-proximally. Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 4 of 27

Figure 2 Photographs of female Anaspides tasmaniae Thomson, 1893 (Anaspidacea) stained with Alizarin Red. Scale bars in A & B 1 cm; C–N 1 mm. a1 = antennula; ba = basipod; en = endopod; ex = exopod; plp = pleopod; sc = sclerite; st = sternite; stw = sternitic wing; tgpl = tergopleura; thp = thoracopod; urp = uropod. A. Whole animal from ventral. Rectangle corresponds to area documented in B. B.Pleon from ventral. Rectangle illustrates documented area in C & D. C. Detailed view on plp articulation, here of plps 3 pointing posteriorly. Rectangle illustrates documented area in E, G, I, K, and M. Rest of image black and white except sc 1. D. Detailed view on articulation of plp 3 pointing anteriorly. Rectangle illustrating documented area in F, H, J, L, and N. Rest of image in black and white except sc 1. E. Articulation of plp 1 pointing posteriorly. Sc 1 covered by tgpl. F. Articulation of plp 1 pointing anteriorly with sc 1. G. Articulation of plp 2 pointing posteriorly with sc 1. H. Articulation of plp 2 pointing anteriorly with sc 1. I. Articulation of plp 3 pointing posteriorly with sc 1. J. Articulation of plp 3 with sc 1. K. Articulation of plp 4 pointing posteriorly with sc 1. L. Articulation plp 4 pointing anteriorly with sc 1. M. Articulation of plp 5 pointing posteriorly with sc 1. N.Articulation of plp 5 pointing anteriorly with sc 1.

A single sclerite is present laterally in the articulation forms articulations with both the tgpl and the ls (Table 1). membrane of Pa. lacustris (Figure 3D–E; Table 1). The We could not determine the presence of internal apodemes sclerite is semicircular and anteriorly bifurcate and is (Table 1). Differences between the sexes were not located directly between ls and tgpl (Figure 3D–E). It recognised. Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 5 of 27

Figure 3 Fluorescence micrographs of Anaspidacea. A–B female Allanaspides hickmani Swain, Wilson & Ong, 1970 and C–EmaleParanaspides lacustris Smith, 1908. Scale bars A & B 0.5 mm; C–E 1 mm. Abbreviations as before. A. Detailed view on articulation of plp 3 pointing posteriorly. Note sc 1. B. Articulation membrane of plp 3 pointing anteriorly with sc 1. C. Ventral side displaying plp 1 & 2 with en modified to petasma. D. Detailed view on articulation area of plp 2 pointing posteriorly and with modified en. Note sc 1. E. View on articulation of plp 3 pointing posteriorly with sc 1.

Amphipoda (Figure 4A–H) calcified (Figure 4B–C); stw merge into tgpl laterally The pleon of Amphipoda is divided into a pleosome and (Figure 4B–C); ls sub-cylindrical, inserts on pleomere urosome (Figure 4A, E) [32]. Accordingly, the pleopod ventrally (Figure 4B–C);ramimulti-annulated,lsdrawn articulations show different morphologies and are de- out proximo-medially and proximo-laterally into knobs scribed separately. Although the sixth pleopod is part of forming pivot joints. the urosome, it is omitted here. There are five sclerites in the articulation membrane of the pleopods (Figure 4B–C; Table 1). Sclerite 1 lies Dikerogammarus haemobaphes (Eichwald, 1841) laterally in the articulation membrane, is semicircular and (Figures 1C, 4A–D, Table 4) forms a bifurcated fork anteriorly. The latero-proximal Pleosome. – Calcified tgpl borders pleomere laterally process of the fork forms a pivot joint with the knob of (Figure 4B–C); st, anterior and posterior stw strongly the fusion area of tgpl and stw (Figure 4B; white Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 6 of 27

Figure 4 (See legend on next page.) Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 7 of 27

(See figure on previous page.) Figure 4 Pleon details of malacostracan representatives. A–D Dikerogammarus haemobaphes (Eichwald, 1841) (Amphipoda). E–H Hyperia sp. (Amphipoda). I–J Fluorescence micrographs of Neosergestes semissis (Burkenroad, 1940) (Dendrobranchiata). K–L Penaeus monodon Fabricius, 1798 (Dendrobranchiata). M–N Euphausia superba Dana, 1850 (Euphausiacea). O–P Thysanopoda tricuspidata Milne-Edwards, 1983. A–D, K–P Photographs of Alizarin-Red stained specimens. E–J fluorescence micrographs. Scale bars A = 1 cm; B–P = 1 mm. Abbreviations as before. A. Pleon from lateral displaying tagmatisation. B. Articulation of plp 3 pointing posteriorly with sc 1– 3. White arrowhead = joint tergopleura–sc 1. C. Articulation of plp 3 pointing anteriorly with scl 1–2, 4–6. White arrowhead as in B. D. Articulation of plp 5 pointing posteriorly. White arrowhead = joint. Note darker area anterior to basipod. E.PleonofHyperia sp. from lateral displaying tagmatisation. F. Articulation of plp 3 pointing posteriorly with sc 1–3. Tgpl slightly deformed. White arrowheads = joints of tgpl–sc 1, st–sc 2. Black arrowhead = joints of ba–sc 1. G. Articulation on plp 3 pointing anteriorly with sc 1–2, 4–6. Arrowheads as in F. H. Articulation of plp 5 pointing posteriorly. I. Articulation of plp 3 pointing posteriorly with sc 1 anchored deeply in tergopleura (white arrowhead). J. Articulation of plp 3 pointing anteriorly with sc 1–3. Note uparching sternite on lateral side marked by white star. Black arrowhead = joint ba–sc 1. K. Articulation of plp 3 pointing posteriorly with sc 1, 2. White arrowhead = joint tgpl–sc 1. L. Articulation of plp 3 pointing anteriorly with sc 1–2. Tgpl removed. M. Articulation of plp 3 pointing posteriorly with sc 1–2. White arrowhead = joint st–sc 2. N. Articulation of plp 3 pointing anteriorly with sc 1–3. White arrowhead = joint tgpl–sc 1. Black arrowhead = joint ba–sc 2. O. Articulation of plp 3 pointing posteriorly with sc 1–2. White arrowhead = joint tgpl–sc 1. P. Articulation of plp 3 pointing anteriorly with sc 1–3. White arrowhead as in O.

arrowhead; Table 1). Distally, sclerite 1 forms a pivot There are five sclerites in the articulation membrane joint with the ls (Table 1). Sclerite 2 lies on the median of pleopods 1–3 of the pleosomal segments (Figure 4F– side (Figure 4B–C). It is semicircular and has a proximal G; Table 1). Sclerite 1 lies laterally in the articulation outgrowth that forms a joint with the st (Figure 4B; membrane, is semicircular on the lateral side, and ex- white arrowhead). Distally, sclerite 2 articulates with the tends rather straight, latero-medially orientated at the ls. A third sclerite is sited on the anterior side of anterior side of the articulation membrane (Figure 4F). the articulation membrane between sclerites 1 and 2 Its latero-proximal part forms an articulation with the (Figure 4B). It is round and lies directly proximal to the tgpl (Figure 4F–G; white arrowheads; Table 1). Sclerite ls. On the posterior side two additional sclerites are 1 forms a pivot joint with the ls latero-distally found (Figure 4C): sclerite 4 is oval and lies median to (Figure 4G; black arrowhead; Table 1). Sclerite 2 lies sclerite 1; sclerite 5 is located directly proximal to sclerite medially (Figure 4F–G). It is semi-circular and has a prox- 1 and 4. The antero-lateral half of the sub-rectangular, imal outgrowth that contacts the st and forms a pivot elongated sclerite 5 contacts the postero-median margin jointwithit(Figure4F–G; white arrowhead; Table 1). of sclerite 1. Sclerite 3 and 5 possess apodemes. Sclerite 2 forms a pivot joint with the ls postero- Urosome. – The morphology of the urosomal segments medially (Figure 4G, black arrowhead; Table 1). Sclerite differs from that of the pleosomal ones enormously. The 1 and 2 contact each other antero-medially (Figure 4F). calcified ventral areas are sub-rectangular with two oval A third elongate sclerite 3 lies distal to this connection holes posteriorly (Figure 4D). Thus the posterior stw is (Figure 4F). It is crowbar-shaped, latero-medially orien- very slender compared to the anterior wing; ls is cylin- tated, and contacts sclerite 2 with its median side dis- drical, carrying non-annulated rami, inserting latero- tally (Figure 4F). On the posterior side two other ventrally at urosomal segment (Figure 4D). sclerites are present (Figure 4G): sclerite 4 is elongated, No sclerites are present in the articulation membranes latero-medially orientated, and lies centrally, close to of pleopods 4 and 5. However, there is a strongly calcified the ls in the articulation membrane contacting sclerite 1 area antero-laterally at the pleopod insertion. This area laterally and sclerite 2 medially. The crescentic sclerite forms a pivot joint with the limb stem (Figure 4D; white 5 lies laterally, proximal to the connection of sclerite 1 arrowhead). Speculatively, the strong calcification and the and 4. Whether internal apodemes are present could joint might be hints that a sclerite is fused to the sternitic not be examined (Table 1). wing. No differences between the two sexes of D. haemo- Urosome. – The urosomal segments do not have as baphes could be recognised. distinctive tgpl as the pleosomal segments. The tgpl merge smoothly with the st. Nevertheless, the st seems to be sub-rectangular with two semicircular, Hyperia sp. (Figures 1H, 4E–H, Table 4) postero-lateral recesses. At these recesses the plp insert Pleosome. – Calcified tgpl borders pleomere laterally at the latero-posterior margin of a urosomal segment (Figure 4F); st and stw relatively slender (Figure 4F–G); (Figure 4E, H). The ls is cylindrical and carries two non- stw merge into tgpl laterally (Figure 4F–G); ls sub- annulated portions, endopod and exopod. No sclerites cylindrical (Figure 4F–G), carrying multi-annulated rami; ls are present in the articulation membranes of pleopods 4 drawn out proximo-medially and proximo-laterally into and 5. No differences have been recognised between the knobs forming pivot joints (Figure 4F–G; black arrowheads). two sexes. Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 8 of 27

Figure 5 Pleopod articulations of different Malacostraca. A–B Lophogaster typicus Sars, 1857 (Lophogastrida). C–D Neognathophausia gigas (Willemoes-Suhm, 1875) (Lophogastrida). E–F Mysis sp. (Mysida), male. G–H Nebalia bipes (Fabricius, 1780) (Phyllocarida). I–L Nebaliopsis sp. (Phyllocarida). M–N Erugosquilla massavensis (Kossmann, 1880) (Hoplocarida) O–P Gonodactylus chiragra (Fabricius, 1781) (Hoplocarida). A–H, M–P Photographs of Alizarin-Red stained specimens. I–L fluorescence micrographs. Scale bars 1 mm. Abbreviations as before. A. Plp 3 pointing posteriorly with sc 1– 3. White arrowhead = joint tgpl–sc 1. White star = setae at sc 2. B. Plp 3 pointing anteriorly with sc 1–2, 4. Black arrowhead = joint ba–sc 1. White arrow marks position sc 5. White stars = setae at sc 2. C. Plp 3 pointing posteriorly with sc 1–3. White arrowhead = joint of tgpl–sc 1. White star = setae at sc 2. Black arrowhead = joint ba–sc 2. D. Plp 3 pointing anteriorly with sc 1–2, 4. Black arrowheads = joints ba-sc 1, ba-sc 2. White arrow marks position sc 5. White star = setae at sc 2. E.Plp4 pointing posteriorly with sc 1–2. F. Plp 4 pointing anteriorly with sc 1–2. G. Plp 3 pointing posteriorly with sc 1– 3. Sc 1 overlapped by tgpl. H. Plp 3 pointing anteriorly with sc 1–2, 4–8. I. Plp 3 pointing posteriorly with sc 1–2. White arrowhead = joint tgpl–sc 1. J. Plp 3 pointing anteriorly with sc 1–6. White arrowheads = joints ba–sc 1, ba–sc 2. K. Plp 4 pointing posteriorly with sc 1–2. white arrowhead = joint tgpl–sc 2. L. Plp 4 pointing anteriorly with sc 3. M.Plp 3 pointing posteriorly with sc 1–4. Position of sclerite 5 marked by black arrow. N. Plp 3 pointing anteriorly with sc 1–2, 6–8. White arrowhead = joint tgpl-sc 1. O. Plp 3 pointing posteriorly with sc 1–5. P. Plp 3 pointing anteriorly with sc 1–2, 6–8. White arrowhead = joint tgpl–sc 1.

Decapoda (Figure 4I–L) merge into tgpl laterally (Figure 4F–G ls cylindrical and Neosergestes semissis (Burkenroad, 1940) thickened in proximo-distal mid-section; ls carrying (Figures 1M, 4I–J; Table 4) annulated rami; ls drawn out proximo-medially and The calcified tgpl contacts ventral surface of pleomere proximo-laterally into knobs forming pivot joints (Figure 4J; laterally; st and stw strongly calcified (Figure 4I–J); stw black arrowhead). Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 9 of 27

Table 1 Summarising table containing main results of sclerite examination Species plp Number Scheme of sclerites Pivot joints apo Laterally Medially Anaspides tasmaniae Thomson, 1893 1–51 tgpl-sc 1 st-ba - sc 1-ba

Allanaspides hickmani Swain, Wilson & Ong, 1970 1–51 tgpl-sc 1 st-ba ? sc 1-ba

Dikerogammarus haemobaphes (Eichwald, 1841) 1–35 tgpl-sc 1 st-sc 2 sc 3 sc 1-ba sc 2-ba sc 5

Erugosquilla massavensis (Kossmann, 1880) 1–58 tgpl-sc 1 st-sc 2 sc 1 sc 1-ba sc 2-ba sc 4 sc 6 sc 8 Euphausia superba Dana, 1850 1–53 tgpl-sc 1 st-sc 2 - sc 1-ba sc 2-ba

Gnathophausia gigas Willemoes-Suhm, 1873 1–55 tgpl-sc 1 st-sc 2 sc 3 sc 1-ba sc 2-ba sc 4 sc 5

Gonodactylus chiragra (Fabricius, 1781) 1–58 tgpl-sc 1 st-sc2 sc1 sc1-ba sc2-ba sc4 sc6

Hyperia sp. 1–35 tgpl-sc 1 st-sc 2 ? sc 1-ba sc 2-ba

Lophogaster typicus Sars, 1857 1–55 tgpl-sc 1 st-sc 2 sc 3 sc 1-ba sc 2-ba sc 4 sc 5

Mysis sp. 1–52 tgpl-sc 1 st-sc 2 - sc 1-ba sc 2-ba

Nebalia bipes (Fabricius, 1780) 1–38 tgpl-sc 1 st-sc 2 sc 3 sc 1-ba sc 2-ba sc 4

Nebaliopsis sp. 1–36 tgpl-sc 1 st-sc 2 ? sc 1-ba sc 2-ba Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 10 of 27

Table 1 Summarising table containing main results of sclerite examination (Continued) Neosergestes semissis (Burkenroad, 1940) 1–53 tgpl-sc 1 st-sc 2 sc 1 sc 1-ba sc 2-ba

Paranaspides lacustris Smith, 1908 1–51 tgpl-sc 1 st-ba ? sc 1-ba

Penaeus monodon Fabricius, 1798 1–52 tgpl-sc 1 st-sc 2 - sc 1-ba sc 2-ba

Thysanopoda tricuspidata Milne-Edwards, 1837 1–53 tgpl-sc 1 st-sc 2 - sc 1-ba sc 2-ba

Sclerites with apodemes with purple line. Abbreviations: apo apodemes, ba basipod, n number of scerites, plp given description valid for pleopods, sc sclerite, st sternite, tgpl tergopleura. *) scheme for large sclerites found on plp 3, 4 of males of Mysis sp.

Table 2 Ground-pattern situations reconstructed for the examined taxa Taxon scl gp n apo lat art disloc med art disloc Anaspidacea 1nono – ba – st no

Decapoda 31 ba– scl – tgpl no ba – scl – st post

Euphausiacea 3noba– scl – tgpl no ba – scl – st no

Mysida 2noba– scl – tgpl ant ba – scl + ext – st no

Amphipoda 52 ba– scl – tgpl no ba – scl + ext – st no

Lophogastrida 63 ba– scl – tgpl ant ba – scl – st post

Stomatopoda 83ba– scl – tgpl no ba – scl – st ant

Phyllocarida 72ba– scl – tgpl no ba – scl – st no

Schemes illustrate articulation membrane. Purple outline for a sclerites with apodemes. Abbreviations other than in Table 1: disloc, dislocation; gp, ground pattern; mart, median articulation; lat art, lateral articulation; med art, median articulation. Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 11 of 27

Figure 6 Mapping and ground-pattern reconstructions. A–B, D refer to phylogeny proposed by [35]. C refers to phylogeny proposed by [36]. Mapped are our findings (Table 2). Ground pattern states for higher taxa were reconstructed accordingly. Red = possible autapomorphic states. Purple = sclerites with apodemes. Note that Decapoda here is short term of Decapoda sensu lato. A. Relationships of basal Malacostraca. B. Phylogeny of Caridoida after [35]. C. Phylogeny of Caridoida after [36]. D. Relationships in Neocarida. See also [42], also for the putative position of Bathynellacea.

Threescleritesarepresent(Figure4I–J; Table 1). Penaeus monodon Fabricius, 1798 Sclerite 1 lies laterally and is semicircular with a tipped (Figures 1O, 4K–L, Table 4) anterior end (Figure 4I). This tipped end is anchored in Calcified tgpl borders pleomere laterally; st and stw the tgpl (Figure 4 I; white arrowhead). The lateral side strongly calcified (Figure 4K–L); stw merge into tgpl lat- arches upward resulting in a fold when the pleopod is erally (Figure 4K–L); ls cylindrical tapering proximally; ls bent anteriorly (Figure 4J; white star). The distal margin carries annulated rami; ls drawn out proximo-medially and of sclerite 1 forms a pivot joint with the ls posteriorly proximo-laterally into knobs forming pivot joints; ls not (Figure 4J; black arrowhead; Table 1). Sclerite 2 lies pos- fully calcified, only median and lateral sides calcified and teriorly and is crescentic (Figure 4J). Medio-proximally are, hence, deeply purple in the Alizarin-Red stained speci- it forms a pivot joint with the st, medio-distally with the mens (Figure 4K–L). ls (Table 1). Sclerite 2 contacts sclerite 1 on the poster- Two bulging sclerites are present in the articulation ior side of the articulation membrane (Figure 4J). Scler- membrane of pleopods 1–5 (Figure 4K–L; Table 1). ite 3 is C-shaped and lies proximal to the connection of Sclerite 1 lies latero-posteriorly in the articulation mem- sclerite 1 and sclerite 2 (Figure 4J). The tipped, anterior brane and is semicircular. Its anterior side forms the pivot end of sclerite 1 functions as an apodeme (Table 1). No joint with the tgpl (Figure 4K; white arrowhead; Table 1). other apodemes are present (Table 1). Only females The postero-distal part of sclerite 1 forms a pivot joint were available for study, thus we cannot offer any data with the ls (Figure 4I, white arrowhead; Table 1). Sclerite 2 for the males of Neo. semissis. lies medio-posteriorly and is semicircular (Figure 4K–L). Kutschera ta.BCEouinr Biology Evolutionary BMC al. et

Table 3 Symplesiomorphic conditions of sclerites

Phyllocarida Hoplocarida Euphausiacea Decapoda Anaspidacea Amphipoda Mysida Lophogastrida (2015) 15:117 Malacostraca Nebalia bipes Nebaliopsis Eumalacostraca Erugosquilla Caridoida Euphausia superba Neosergestes Penaeus Anaspides Peracarida Dikerogammarus Mysis Gnathophausia ingens (Fabricius, 1780) sp. massavensis (Dana, 1850) semissis monodon tasmaniae haemobaphes sp. Willemoes-Suhm, (Kossmann, 1880) Thysanopoda (Burkenroad, Fabricius, Thomson, 1893 (Eichwald, 1841) 1873 Lophogaster Gonodactylus chiragra tricuspidata Milne- 1940) 1798 Allanaspides Hyperia sp. typicus Sars, 1857 (Fabricius, 1781) Edwards, 1837 hickmani Swain, Ong & Wilson, 1970 Paranaspides lacustris Smith, 1908 la sc sc 1 sc 1 la sc sc 1* la sc sc 1 sc 1* sc 1 sc 1 la sc sc 1 sc 1 sc 1 me sc sc 2 sc 2 me sc sc 2 me sc sc 2 sc 2 sc 2 - me sc sc 2 sc 2 sc 2 an sc* sc 3* - an sc* sc 3* an sc* - - - - an sc* sc 3* - sc 3* po-di sc sc 5 sc 4 ------po-me sc* sc 4* sc 3 pome sc* sc 6* pome sc 3? sc 3? - - po-di sc? sc 4 - sc 4 sc? po-ce sc sc 6 sc 5 poce sc sc 7 poce? sc 3? sc 3? - - po-pr sc? sc 5* - sc 5* po-pr sc sc 7 sc 6 poce sc sc 8* (E.m.) -? ------Left column malacostracan ground pattern. Corresponding sclerites given in right columns for each taxon/ species. Abbreviations: an anterior, ce central, di distal, (E.m.) Apodeme only in Erugosquilla massavensis Kossmann 1880, la lateral, me median, po posterior, pr proximal, sc sclerite, ? = uncertain homologisation; * = sclerite with apodeme. ae1 f27 of 12 Page Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 13 of 27

Table 4 Species examined, taxon affiliation, and applied methods for examination of pleopod-body articulation Species Taxon Reg AR Mph FM Di Anaspides tasmaniae Thomson, 1893 Caridoida Hessler, 1982 Anaspidacea Calman, 1904 - ++ - + Allanaspides hickmani Swain, Wilson & Caridoida Hessler, 1982 Anaspidacea Calman, 1904 --- + - Ong, 1970 Dikerogammarus haemobaphes Caridoida Hessler, 1982 Peracarida Calman, 1904 Amphipoda, Gammaridea - ++ - + (Eichwald, 1841) Erugosquilla massavensis (Kossmann, Hoplocarida Calman, 1904 Stomatopoda Latreille, 1817 - ++ -- 1880) Euphausia superba Dana, 1850 Caridoida Hessler, 1982 Eucarida Calman, 1904 Euphausiacea Dana, 1852 - ++ - + Gnathophausia gigas Willemoes-Suhm, Caridoida Hessler, 1982 Peracarida Calman, 1904 Lophogastrida Sars, 1870 - ++ - + 1873 Gonodactylus chiragra (Fabricius, 1781) Hoplocarida Calman, 1904 Stomatopoda Latreille, 1817 + + + - + Hyperia sp. Caridoida Hessler, 1982 Peracarida Calman, 1904 Amphipoda, Hyperiidae ---+- Lophogaster typicus Sars, 1857 Caridoida Hessler, 1982 Peracarida Calman, 1904 Lophogastrida Sars, 1870 - ++ - + Mysis sp. Caridoida Hessler, 1982 Peracarida Calman, 1904 Mysida Boas, 1883 - ++ - + Nebalia bipes (Fabricius, 1780) Phyllocarida Packard, 1879 Leptostraca Claus, 1880 - ++ - Nebaliopsis sp. Phyllocarida Packard, 1879 Leptostraca Claus, 1880 --- +- Neosergestes semissis (Burkenroad, Caridoida Hessler, 1982 Eucarida Calman, 1904 Decapoda Latreille, 1802 --- ++ 1940) Dendrobranchiata Bate, 1888 Paranaspides lacustris Smith, 1908 Caridoida Hessler, 1982 Anaspidacea Calman, 1904 --- +- Penaeus monodon Fabricius, 1798 Caridoida Hessler, 1982 Eucarida Calman, 1904 Decapoda Latreille, 1802 - ++ - + Dendrobranchiata Bate, 1888 Thysanopoda tricuspidata Milne- Caridoida Hessler, 1982 Eucarida Calman, 1904 Euphausiacea Dana, 1852 - ++ - + Edwards, 1837 Abbreviations: AR Staining with Alizarin Red, Di Dissection, FM Fluorescence microscopy, Mph Macrophotography, Reg Regeneration in NaCl.

Postero-medially, it is drawn out proximally forming a a pivot joint (Figure 4M, black arrowhead; Table 1). Sclerite pivot joint with st (Table 1). Distally, sclerite 2 forms a 1 is drawn out postero-laterally and forms a pivot joint with pivot joint with the ls (Table 1). The two sclerites lie next the tgpl (Figure 4N; white arrowhead; Table 1). The distal to each other on the posterior side of the articulation margin on the posterior side is also somewhat zigzag, giving membrane. No apodemes were present (Table 1). space for the lateral curve of the limb stem (Figure 4N). Although the first pleopods form a petasma in males (as is Sclerite 2 lies medially (Figure 4M–N). Antero-medially, it autapomorphic for Dendrobranchiata) no differences articulates with the st via a pivot joint (Figure 4M; white concerning the studied structures were found in the two arrowhead; Table 1). The pivot joint of sclerite 2 and the ls sexes. is sited directly distal to this articulation (Figure 4M, black arrowhead). Posteriorly, sclerite 2 is gently excavated prox- Euphausiacea imally to give space for the median parts of sclerite 1 as well Euphausia superba Dana, 1850 as distally giving space for the tip of the ls and forming a (Figures 1E, 4M–N; Table 4) pivot joint there with it (Figure 4N; black arrowhead; Table Calcified tgpl borders pleomere laterally; st and stw less 1). Sclerite 3 lies on the posterior side between sclerite 1 calcified than tgpl (Figure 4M–N); stw merge into tgpl and sclerite 2 proximal to the ls (Figure 4N). It starts with a laterally (Figure 4F–G); ls truncated cone, with slender relatively broad lateral side, extends further medio-distally, proximal part carrying multi-annulated rami; proximal and ends in a tipped median side (Figure 4N). No apo- margin of ls relatively straight on anterior side (Figure 4M), demes were present (Table 1). Although pleopods 1 and 2 whereas uneven posteriorly (Figure 4N); ls drawn out form a petasma in males, no differences in sclerite morph- proximo-medially and proximo-laterally into knobs form- ologywerepresentinthetwosexes. ing pivot joints (Figure 4N; black arrowhead). Three sclerites lie in the articulation membrane of pleo- Thysanopoda tricuspidata Milne-Edwards, 1837 pods 1–5ofEu. superba (Figure 4M–N; Table 1). Sclerite 1 (Figures 1P, 4O–P; Table 4) lies laterally (Figure 4M–N). Its anterior part has a straight Calcified tgpl borders pleomere laterally; st and stw proximal margin, whereas the distal margin is zigzag, the compact and calcified (Figure 4F–G); stw merge into tgpl most distal one of the three cusps articulates with the ls via laterally (Figure 4F–G); ls truncated cone carrying multi- Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 14 of 27

annulated rami. The proximal margin is relatively straight Apodemes were found at sclerite 3, 4, and 5 (Table 1). No on the anterior side (Figure 4O), whereas it is uneven differences were found in the two sexes. posteriorly (Figure 4P). Proximo-medially and proximo- laterally the ls is drawn out into knobs forming pivot Lophogaster typicus Sars, 1857 joints. (Figures 1I, 5C–D; Table 4) Three sclerites are present in the articulation membrane Calcified tgpl borders pleomere laterally; st and stw strongly of pleopods 1–5ofT. tricuspidata (Figure 4O–P; Table 1). calcified (Figure 5C–D); stw merge into tgpl laterally Sclerite 1 lies laterally, is semicircular, and drawn out prox- (Figure 5C–D); ls truncated cone carrying multi-annulated imally where it forms a joint with the tgpl (Figure 4O–P; rami; ls laterally and medially grown out forming pivot white arrowhead; Table 1). Distally, it forms a pivot joint joints (Figure 5D; black arrowheads; Table 1), lateral out- with the ls (Table 1). Sclerite 2 lies medially (Figure 4M, N). growth being the much larger one. It articulates with the st antero-medially (Table 1). Poster- Five sclerites lie in the articulation membrane of the pleo- iorly, sclerite 2 is gently excavated distally giving space for pods (Figure 5C–D; Table 1). Sclerite 1 lies laterally and is the tip of the limb stem and forming a pivot joint with it tripartite: it has a tipped anterior part, which forms a pivot there (Table 1). Sclerite 3 lies on the posterior side between joint with the tgpl (Figure 5C; white arrowhead; Table 1). sclerite 1 and sclerite 2 proximal to the limb stem The bulgy middle part lies most laterally and contacts the (Figure 4N). From posterior it appears like a curved spindle tgpl at the lateral extension (Figure 5D) and the ls at its lat- (Figure 4P). No apodemes are present (Table 1). Only eral side. Both sclerite 1 and ls being articulated to each females were available, thus no statement for males of T. other there (Figure 5D; black arrowhead; Table 1). The pos- tricuspidata can be made. terior part is sub-oval and extends medially (Figure 5D). Sclerite 2 lies medially and consists of a strongly calcified Lophogastrida (Figure 5A–D) distal part exhibiting a tip that points medially and forms a Gnathophausia gigas Willemoes-Suhm, 1873 pivot joint with the limb stem (Figure 5C–D; black arrow- (Figures 1F, 5A, B; Table 4) heads;Table1).Theproximalpart is less calcified, identifi- Calcified tgpl borders pleomere laterally; st and stw able by its brighter purple colour, and runs towards the st strongly calcified (Figure 5A–B); stw merge into tgpl lat- forming a pivot joint with it (Figure 5D; Table 1). Sclerite 2 erally (Figure 5A–B); ls bulgy cylinder carrying multi- carries few setae pointing medially (Figure 5C–D; white annulated rami; proximal end of ls drawn out laterally stars). Sclerite 3 lies anteriorly in the articulation membrane ending in long outgrowth nested deeply in articulation and looks like a filled eight from anterior (Figure 5C). membrane (Figure 5A–B). Sclerite 4 lies posteriorly between sclerite 1 and sclerite 2 Five sclerites lie in the articulation membranes of the ple- (Figure 5D). It is L-shaped, its lower side being latero- opods (Figure 5A–B; Table 1). Sclerite 1 is sited laterally medially orientated and lying between sclerite 1 and the st (Figure 5A–B). Its anterior part is tipped, points anteriorly, (Figure 5D). The upper part is proximo-distally orientated and articulates with the tgpl (Figure 5A; white arrowhead; and contacts the ls (Figure 5D). The oval sclerite 5 lies Table 1). Laterally, sclerite 1 arches distally lying right be- latero-posteriorly (Table 1) and is overlapped by sclerite 1. tween tgpl and ls (Figure 5B). From there it extends further Sclerite 3 has a large apodeme (Table 1). Another apo- medially on the posterior side as a roundly W-shaped struc- deme is found at sclerite 4 and 5 (Table 1). No differ- ture forming a pivot joint with the ls (Figure 5B; black ences were present in the two sexes. arrowhead; Table 1). Sclerite 2 lies medially and encom- passes the ls there forming a median pivot joint with it Mysida (Figure 5E–F) (Table 1). From anterior and from posterior it is triangular, Mysis sp. (Figures 1J, 5E–F; Table 4) forming altogether a rounded rhombus (Figure 5A–B). Females of Mysida have rudimentary pleopods consisting Medio-posteriorly, sclerite 2 forms a pivot joint with the st only of one portion and being comparably small. The same (Table 1). In the antero-posterior middle a proximo-distally applies to pleopods 1, 2, and 5 of male mysids. Pleopods 3 oriented row of densely set setae is located (Figure 5A–B; and 4, however, are modified in males into a petasma for white star). Sclerite 3 lies anteriorly in the articulation sperm transfer and are comparably large. According to membrane and is sub-rectangular (Figure 5A). Sclerite 4 these two various states the morphology of the sternites lies posteriorly between sclerite 1 and sclerite 2 (Figure 5B). and sclerites differs. First, we describe the morphology oc- Its complex shape is caused by a distal loop, visible in curring in female Mysis sp.andpleomeres1,2and5in Figure 5B. Proximal to this loop lies a latero-medially orien- males. Then we describe the morphology appearing in the tated bar, which is tipped on both ends. From this bar arises petasma of male Mysis sp. another proximo-distally orientated bar, which is nested Female 1–5; male 1, 2, 5. – The tgpl emerges from the deeply in the articulation membrane. Sclerite 5 lies latero- ventro-lateral side of the segment. It extends further posteriorly and is hidden by sclerite 1. It is oval (Table 1). posteriorly, still running ventrally. In the posterior Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 15 of 27

quarter of its length, the tgpl bends concavely laterally. used for swimming. In segments 5 and 6 the pleopods Centrally, the less calcified sternite is sited, which is consist of only two portions and are much smaller than drawn out into posterior sternitic wings. The pleopod pleopods 1–4. Sclerites are only present at the bases of consists only of one portion and is club-shaped with a pleopods 1–4. Segment 7 is limbless. The last body part is rounded tip. The proximal margin is laterally excavated the conical telson carrying large furcal rami. on the anterior side, but rather straight posteriorly and drawn out medially. The ls is drawn out into knobs Nebalia bipes (Fabricius, 1780) forming pivot joints proximo-medially and proximo- (Figures 1K, 5G–H; Table 4) laterally. Calcified tgpl borders pleomere laterally; st and stw Two sclerites are present in the articulation membrane of strongly calcified (Figure 5G–H); posterior stw set-off the pleopods of females and males (only pleopods 1, 2, and from st (Figure 5H); ls cylindrical carrying spine-bearing 5) (Table 3). Sclerite 1 lies laterally and is drop-shaped in rami; proximal anterior margin of ls convex (Figure 5G); ventral view, the tip pointing laterally. It forms a pivot joint whereas posterior margin rather concave (Figure 5H); ls with the limb stem antero-laterally (Table 1). Sclerite 2 lies drawn out proximo-medially and proximo-laterally into medially and is sub-rectangular being latero-medially orien- knobs forming pivots. tated. It forms a pivot joint with the limb stem antero- Eight sclerites are present in the articulation membranes medially (Table 1). No apodemes were present (Table 1). of pleopods 1–4(Figure5G–H; Table 1). Sclerite 1 lies Male 3–4. – The tgpl emerges from the ventro-lateral antero-laterally (Figure 5G–H). It is elongated and its main side of the segment. It extends further posteriorly, still part extends anteriorly (Figure 5G). Sclerite 1 encompasses running ventrally. In the posterior quarter of its length, the ls laterally, by what a small part of sclerite 1 lies also the tgpl bends concavely laterally (Figure 5E). The st is posteriorly (Figure 5H). Sclerite 1 forms an articulation rhombic, one tip pointing anteriorly. The anterior margins with the tgpl and the ls (Table 1). Sclerite 2 lies medially, are concave and end in the two lateral tips (Figure 5E). is C-shaped and curves around the ls (Figure 5G–H). The From there, the more concavely bent posterior margins whole distal margin of sclerite 2 runs over the ridge of the run medio-posteriorly, forming a kind of bar-like posterior st (Figure 5H). Proximally, it forms an articulation with end (Figure 5E). From there, semicircular posterior wings the ls (Table 1). The sub-rectangular sclerite 3 lies laterally arise running laterally towards the tgpl (Figure 5F). Pleo- in the articulation membrane between sclerite 1 and the pod 3 is rather large compared to one-portioned other limb stem (Figure 5G). Sclerite 4 lies laterally to sclerite 2 pleopods and its limb stem is cylindrical and carries one on the posterior side of the articulation membrane ramus. Proximo-medially and proximo-laterally the ls is (Figure 5H). It is sub-oval with a latero-distal outgrowth drawn out into knobs forming pivot joints. Pleopod 4 is (Figure 5H); sclerite 4 is tripartite in pleopod 1. Sclerite 5 approximately 6 times as long as pleopod 3 and has also a is sited lateral to sclerite 4 and right proximal to the mar- cylindrical ls, which carries the endopod and a long exo- gin of the ls (Figure 5H). It is crescent-shaped. The sub- pod, eight times as long as the endopod and reaching till rectangular sclerite 6 is sited distal to sclerite 5 and is the uropods. Proximo-medially and proximo-laterally the flanked latero-posteriorly by sclerite 7 and medially by the ls is drawn out into knobs forming pivot joints. elongated sclerite 8 (Figure 5H). The single sclerites, how- Two sclerites are present at pleopods 3 and 4 in male ever, stick very closely together and fit like puzzle pieces Mysis sp. (Figure 5E–F; Table 1). Sclerite 1 lies laterally into each other. Apodemes are present at sclerite 3 and and is semicircular (Figure 5E–F). Antero-laterally, it is sclerite 4 (Table 1). No differences were found between drawn out and articulates to the tgpl via a pivot joint the two sexes. (Table 2). Laterally, it forms a pivot joint with the ls (Table 1). Sclerite 2 lies medially and is semicircular in Nebaliopsis sp. (Figure 1L, 5I–L; Table 1) ventral view (Figure 5E–F). Its anterior part is, however, Calcified tgpl borders pleomere laterally; st lies medially very deep proximo-distally (Figure 5E) whereas the pos- lies (Figure 5I–J); stw set off from st (Figure 5I–J); ls cylin- terior part is comparably slender (Figure 5F). Medio- drical carrying rami; ls drawn out proximo-medially and proximally, it is drawn out and forms a pivot joint with proximo-laterally into knobs forming pivots (Figure 5J; the posterior stw (Table 2). Medio-distally, it forms an white arrowheads). articulation with the ls (Table 1). No apodemes are Six sclerites are present in pleopods 1–3 (Figure 5I–J; present (Table 1). Table 1). Sclerite 1 lies laterally and extends further medi- ally into the anterior articulation membrane (Figure 5I). It Phyllocarida (Figure 5G–L) is C-shaped and encompasses the ls laterally (Figure 5J). It The pleon of Phyllocarida is tagmatised: the anterior four articulates to the tgpl (Figure 5J, white arrowhead; Table 1) segments form a unit characterised by comparably large and the ls (Figure 5J, black arrowheads; Table 1). Scler- pleopods with limb stem, endopod, and exopod and being ite 1 is centrally located, sub-rectangular and latero- Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 16 of 27

medially oriented. Laterally it contacts sclerite 2. Scler- insertion (Figure 5M). Sclerite 3 and sclerite 4 are compar- ite 2 lies medially (Figure 5I–J). It is C-shaped and en- ably small, oval, and lie distal to sclerite 1 at the anterior compasses the ls, and articulates to st and ls (Figure 5J; side (Figure 5M). A fifth, equally small sclerite 5 lies fur- white arrowhead; Table 1). Sclerite 2 is C-shaped, lies ther laterally being hidden in a membrane fold (Figure 5M; laterally and articulates with tgpl and ls. On the poster- black arrow). Posteriorly, an elongated sclerite 6 ior side a single large, in ventral view drop-shaped scler- contacts sclerite 1 with its lateral end and sclerite 2 ite 3 is present (Figure 5L). Sclerite 3 lies laterally to medially (Figure 5N). Proximal to sclerite 6 lies the sub- sclerite 2, but still in the median half of the posterior ar- rectangular sclerite 7, which is proximally surrounded ticulation membrane (Figure 5J). It is sub-triangular and by the L-shaped sclerite 8 (Figure 5N). Apodemes were medially overlapped by sclerite 2 (Figure 5J). Sclerite 4 present on the insides of sclerites 1, 4, 6, and 8 (Table 1). lies latero-distally and is sub-rectangular with rounded No differences were found between the two sexes. edges (Figure 5J). Sclerite 5 is comparably large and lies centrally in the posterior part of the articulation mem- Gonodactylus chiragra (Fabricius, 1781) brane (Figure 5J). It consists of an elongated, crescent- (Figure 1G, 5O–P; Table 4) shaped median part and a semicircular lateral part being Calcified tgpl borders pleomere laterally; tgpl starts lat- attached to it (Figure 5J). Proximal to sclerite 5 is sclerite 6, erally on anterior side of the pleomere adjoining former which is triangular and contacts sclerite 5 and the tgpl segment and extending latero-posteriorly where curving (Figure 5J). Only three sclerites are present in the articula- dorsally forming a latero-posterior tip; st and stw strongly tion zone of pleopod 4 (Figure 5K–L): Anteriorly, two scler- calcified (Figure 5O–P); stw merge into tgpl laterally; ls ites lie in the articulation membrane. Sclerite 1 is centrally short, slightly compressed cylinder carrying annulated ex, located, sub-rectangular and latero-medially oriented. Lat- a gill and equally multi-annulated en; anterior and poster- erally it contacts sclerite 2. Sclerite 2 is C-shaped, lies lat- ior margin of ls concave (Figure 5O–P); ; ls drawn out erally and articulates with tergopleura and limb stem. On proximo-medially and proximo-laterally into knobs form- the posterior side a single large, in ventral view drop- ing pivots. shaped sclerite 3 is present (Figure 5L). Eight sclerites are present in the articulation membranes The presence of apodemes was not examinable. No of the pleopods (Figure 5O–P; Table 1). Sclerite 1 lies differences between the two sexes were noted. antero-laterally (Figure 5O–P). It is elongated and its main part lies anteriorly (Figure 5O). Sclerite 1 encompasses the Stomatopoda/Hoplocarida (Figure 5M–P) limb stem laterally, by what a small part of sclerite 1 lies Erugosquilla massavensis (Kossmann, 1880) also posteriorly (Figure 5P). Sclerite 1 forms an articulation (Figure 1D, Figure 5M–N; Table 4) with the tgpl and ls (Figure 5P; white arrowhead; Table 1). Calcified tgpl borders pleomere laterally; st and stw Sclerite 2 lies medially and is C-shaped encompassing the ls strongly calcified (Figure 5M–N); stw merge into tgpl (Figure 5O–P). Its anterior part is very long and is located laterally; ls short, slightly compressed cylinder, ls carries very close and proximal to the limb stem. Sclerite 2 articu- multi-annulated ex also a gill and the equally multi- lates with the ls but it does not contact the median knob in annulated en; anterior and posterior margins of ls the insertion of the pleopod (Figure 5O–P; Table 1). Sclerite slightly concave (Figure 5M, N); ls drawn out proximo- 3 and sclerite 4 are comparably small, oval, and lie distal to medially and proximo-laterally into pivots. sclerite 1 at the anterior side (Figure 5O). A fifth sclerite 5 Eight sclerites are present in the articulation membranes is present antero-medially being elongated and running of the pleopods (Figure 5M–N; Table 1). Sclerite 1 lies parallel to the margin of the pleopod insertion (Figure 5O). antero-laterally (Figure 5M–N). It is elongated and the an- Posteriorly, the elongated sclerite 6 contacts sclerite 1 with terior part is somehow dolphin-shaped in ventral view its lateral end and sclerite 2 medially (Figure 5P). Proximal (Figure 5M). However, laterally it extends into a C-shaped to sclerite 6 lies the sub-rectangular sclerite 7 (Figure 5P). part, which encompasses the limb stem and extends Proximal to sclerite 7 lies the sub-oval sclerite 8. Apodemes onto the posterior side of the articulation membrane were found on the insides of sclerites 1, 4, and 6 (Table 1). (Figure 5M–N). This is also the part forming articulations No differences are present between the two sexes. with the tgpl (Figure 5N; white arrowhead; Table 1) and the ls. Sclerite 2 lies medially and is C-shaped. It is built of Reconstruction of the ground-pattern states for the a small anterior part (Figure 5M) and a rather bulgy part sclerite conditions of taxa within Malacostraca followed by a comparably slender part extending further Calcified sclerites occur in the pleopod-body arthrodial laterally until the extension of the st on the posterior side membrane in all examined species (Figures 2–5; Table 1). (Figure 5N). The sternitic extension and the postero- They occur in constant number and position within one lateral end of sclerite 2 form an articulation (Table 1). It is species on pleopods 1–5 (resp. on pleopods 1–4 in phyllo- not connected to the median knob of the pleopod carids). As an exception, in species with a tagmatised pleon Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 17 of 27

the sclerite pattern is at least similar in one tagma, e.g. Diker- two of its sclerites correspond to our anterior sclerite 3 and ogammarus haemobaphes and Hyperia sp. (Figure 4A–H posterior sclerite 5 both with apodemes (see his figure 4B for Amphipoda; or Nebalia bipes and Nebaliopsis sp. insertion of M6 = apodeme sclerite 3; figure 4A insertion of Figure 5G–L for Phyllocarida). The sclerite patterns are even M8 = apodeme sclerite 5). similar in representatives of one supra-specific taxon We conclude that the ground-pattern for Amphipoda (Table 1; Table 4). Therefore, the sclerites seem to be very includes five sclerites (Table 2 and Table 3): a large lateral conservatively preserved structures, as they appear even in one forming joints with the tergopleura and the basipod, a strongly modified pleopods, for instance gonopods forming large median one also forming joints and having a proximal petasms in, e.g., Paranaspides lacustris (pleopods 1–2; outgrowth, an anterior third sclerite between lateral and me- Figure 3C) or Mysis sp. (pleopods 3–4; Figure 5E–F). Almost dian sclerite, and two posterior sclerites lying compactly ag- no literature includes detailed information on this gregated rather laterally. Based on the data of [29] and our character complex for comparisons. Exceptions are rare observations on D. haemobaphes we assume there is an [29]. These species-specific data (Table 2 and Table 3) apodeme for the anterior scleriteandtheproximalsclerite formed the basis for a reconstruction of the ground- on the posterior side (Table 1, Table 2 and Table 3). We re- pattern states with regard to the sclerites of all major construct this ground-pattern state despite the various life malacostracan taxa. styles realised in different amphipod species (e.g. [23,29,33]). Sclerites in the ground pattern of Anaspidacea. – In the Sclerites in the ground pattern of Decapoda. – In Deca- three species of Anaspidacea – Anaspides tasmaniae, Alla- poda (sensu lato, including Amphionides reynaudii H. naspides hickmani, Paranaspides lacustris – there is one Milne Edwards, 1832 [34]), Neosergestes semissis has three, lateral sclerite, that is anteriorly bifurcate and forms pivot Penaeus monodon two sclerites (Figure 4J–L; Table 1), thus joints with both the tergopleura and the basipod (sc 1 in there are two states for the sclerite pattern complicating a Figures2,3;Table1).Anotherjointisformedbybasipod ground pattern reconstruction for this taxon. Yet, the lat- and sternite (Figures 2, 3; Table 1). Despite the different life eral and median sclerites are very similar in both species: styles of these species [4,30,31] we find great similarities in The lateral sclerites 1 are semicircular and form joints limb morphology. We conclude that a single, lateral, anteri- with tergopleurae and basipods and exhibit an apodeme orly bifurcate sclerite forming a pivot joint with the tergo- (Table 1). The median sclerites 2 are both semicircular pleura and extending comparably much into the posterior and form joints with sternites and basipods; the articulation membrane was present in a common stem spe- sternite-sclerite 1 joint is shifted posteriorly (Table 1). cies and is therefore part of the ground pattern of Anaspi- In both species, the lateral and median sclerites touch dacea (Table 2; Table 3). each other posteriorly (Figure 4J, L), which means they Sclerites in the ground pattern of Amphipoda. – The lat- extend comparably far into the posterior articulation eral sclerites 1 are semicircular and form pivot joints with membrane. Remarkable is also that the lateral sclerite in tergopleurae and basipods in both species (Figure 4B, F, decapods does not extend much into the anterior black and white arrowheads; Table 1). They do not extend articulation membrane compared to all other species much into the posterior articulation membrane. The median that we examined (except for lophogastrids). The pres- sclerites 2 are semicircular and form joints with sternites ence of a third posterior sclerite in the stem species of and basipods in both species (Figure 4B–C, F–G; Table 1). Decapoda as visible in Neo. semissis seems likely. Yet, Interestingly, the median sclerites of both species have a this can only be revealed by an out-group comparison. proximal outgrowth forming the joint with the sternite Depending on the phylogenetic position assumed for (Figure 4B, F, G, white arrowhead, right ones respectively; Decapoda, two groups are favoured for an out-group Table 1). The anterior sclerites 3 differ in shape and comparison: either Euphausiacea (suggested as sister position in the two species (Figure 4B, F). Also the distal, taxon in [35]), or Xenommacarida (proposed in [36]). posterior sclerites 4 vary enormously in shape (sc 4 in Both of these presumptive sister taxa have a posterior Figure 4C, G), but the more proximal sclerites 5 again sclerite (see below), which is why we assume it also is >are very similar in shape

perfectly into each other posteriorly (Figure 4N, P), Posteriorly a large distal sclerite connects the which means they extend comparably far into the pos- median and lateral ones and has an apodeme (sc 6 in terior articulation membrane. Distal to this contact area, Figure 5M, O). Two smaller sclerites (sc 7 and 8 in a third sclerite is present. The similarity in the sclerite Figure 5M, O) are sited proximal to sclerite 6. Interest- pattern is probably as well due to the similar life styles ingly, the Recent benthic Hoplocarida are the only of both examined species [23,24]. We assume a ground Malacostraca that have gills on their pleopods [23,37]. pattern state comprising three bulging sclerites without This could explain why they have the highest number of apodemes for Euphausiacea (Table 2 and Table 3). apodemes; these might guarantee extra versatility to the Sclerites in the ground pattern of Lophogastrida. – In movement of the pleopods and facilitate sufficient both Gnathophausia gigas and Lophogaster typicus, we oxygenation. found five sclerites: Large lateral ones forming anteriorly Ground pattern of Phyllocarida. – The pleons of Neba- shifted pivot joints with the tergopleura laterally (sc 1 in lia bipes and Nebaliopsis sp. are tagmatised. The poster- Figure 5A, C, white arrowheads for joints; Table 1) and, ior pleopods (5 and 6) are small and appear reduced, via a medially pointing outgrowth, the basipod medially and the sclerite pattern of pleopod 4 differs also signifi- (Figure 5B, D, black arrowheads). Median sclerites 2 form cantly from that of the anterior three. Our reconstruc- posteriorly shifted pivot joints with the sternites (Table 1), tion is restricted to pleopods 1–3. with the basipods, and exhibit setae (Figure 5A–D, white The number of sclerites differs in both examined stars). Both species have a small anterior sclerite 3 species (Figure 4G–L; Table 1 and Table 3). Neverthe- (Figure 5A, C) and a complexly shaped sclerite 4 posteriorly less, similarities can be found, which consequently can (sc 4 in Figure 5B, D), both having internal apodemes be assumed for the common stem species (Table 2; (Table 1). Sclerite 5 lies latero-posteriorly and has another Table3):alargelateralscleriteandamediansclerite apodeme (Table 1, Table 2 and Table 3). Due to the similar both forming pivot joints with sternite, tergopleura, and life habits of lophogastrid species [23], we assume the the basipod (Figure 4G–J, white arrowheads; Table 1; ground pattern state for Lophogastrida correlates to the Table 3). The lateral sclerite does not extend much into sclerite pattern description above (Table 2 and Table 3). We the posterior articulation membrane (Figure 5H, J). An propose that the lateral sclerite does not extend very far antero-lateral sclerite is only found in Neb. bipes (Figure 5G; into the anterior articulation membrane in comparison to Table 1; Table 3). Yet, we assume this condition is of some the situation seen in all the other species we examined. significance for the phyllocaridan ground pattern because Sclerites in the ground pattern of Mysida. – Two scler- similar, antero-lateral sclerites are also found in a member ites appear on all pleopods of the examined Mysis sp., of the next closest relatives Eumalacostraca, the Hoplocarida independent from the gender. However, the sclerites at (seebelow).Fortheposteriorscleriteswededuceastatefor the modified pleopods 3 and 4 for males (Figure 5E–F) the stem species in form of a large lateral sclerite (sc 6 in are much larger than the sclerites found on other, rather Neb. bipes Figure 4H; sc 5 in Nebaliopsis sp. Figure 5J; reduced pleopods. We consider this significant for our Table 3) surrounded by a smaller distal sclerite (sc 5 in Neb. reconstruction of the ground pattern (Table 2): i.e. two bipes Figure 4H; sc 4 in Nebaliopsis sp. Figure 5J; Table 3), a sclerites present in Mysida, the lateral one forming an smaller proximal sclerite (sc 7 in Neb. bipes Figure 4H; sc 6 anteriorly shifted articulation with the tergopleura; the in Nebaliopsis sp. Figure 5J), and a smaller median sclerite median one has an outgrowth which forms a postero- (sc 4 in Neb. bipes Figure 4H; sc 3 in Nebaliopsis sp. laterally shifted articulation with the sternite; both scler- Figure 5J). Considering the dissection results of Neb. bipes ites lack apodemes. In general, all species of Mysida (Table 1), we assume apodemes for the anterior sclerite seem to share a similar life style [24], for which reason and the medio-posterior one (Table 2). Neb. bipes has an no drastic modifications from the ground pattern state additional posterior sclerite (Figure 5H; Table 1). Neba- are expected for other species. liopsis sp. lacks the small anterior sclerite (Figure 5I) of Sclerites in the ground pattern of Stomatopoda/Hoplo- Neb. bipes. Both conditions might be autapomorphies of carida. – In Hoplocarida the sclerite patterns in both ex- the respective taxon (if they are monophyletic [38]) what amined species are very similar, for which reason the should be illuminated by further studies). Differences of ground pattern is assumed as follows (Table 2 and Table 3): the examined species from the state in the ground pattern a large lateral and a large median sclerite participating of the Phyllocarida might be explained by the different life in pivot joints (sc 1, sc 2 in Figure 5M, O). The lateral styles: Nebaliopsis is a bathypelagic swimmer [39] whereas sclerite has an apodeme and does not extend much into Neb. bipes is benthic [40]. the posterior articulation membrane (Figure 5N, P). Anteriorly, two small sclerites lie distally to the lateral Phylogenetic mapping sclerite, the more median one having an apodeme, and a We mapped the reconstructed ground-pattern states onto slightly larger sclerite lies medially (sc 3–5inFigure5M,O). two recently published phylogenetic hypotheses [35,36] Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 19 of 27

(Figure 6). Our expectation was that this should help us to these two taxa in Eucarida (Figure 6B). The sister taxon to reconstruct the ground-patterns of higher taxa and, with Eucarida (N. N. in Figure 6B) should comprise the Anaspi- this, unravel more aspects of the evolution of the sclerite dacea and Neocarida (Thermosbaenacea + Peracarida). complex within Malacostraca. Wirkner & Richter [36] on the other hand suggested Both phylogenetic hypotheses are in agreement concern- Decapoda as the adelphotaxon to a taxon, which they ing the relationship of the basal divergence of Phyllocarida called Xenommacarida, which comprises the Anaspidacea and Eumalacostraca, the latter including Hoplocarida and +N.N.(Euphausiacea+Neocarida)(Figure6C). Caridoida (Figure 6A). The position of Bathynellacea re- Sclerite condition in the ground pattern of Eucarida. – mains uncertain because in these animals the pleopods are The assumption of a common origin of Euphausiacea and strongly reduced, in some species even being absent (except Decapoda (Figure 6B) would imply that three sclerites for the uropods, see e.g. [28]). Accordingly bathynellids were probably present in the common stem species. This were left unconsidered in our study. suggests the presence of only one posterior sclerite in the Taking the basal relationships of Malacostraca for granted, stem species of Eucarida. As in both, Decapoda and the ground-pattern of sclerites in both Phyllocarida and Euphausiacea, the lateral and median sclerites extend far Hoplocarida is crucial for the reconstruction of the sclerite into the posterior articulation membrane and touch each pattern in the ground pattern of Malacostraca and Eumala- other posteriorly, this was most likely also a feature of the costraca. In fact, the situations in Phyllocarida and Hoplo- eucaridan stem species because this condition is found in carida (Table 2: and Table 3) may be taken as basis for the no other taxon and is most likely an autapomorphy of reconstruction of the sclerite condition in the stem spe- Eucarida. Yet it is unclear whether the posterior sclerite in cies of Malacostraca (Figure 6A; Table 3). Accordingly, the eucaridan stem species lies more distally as in Euphau- our homologisation of the sclerites in relation to the siacea or more proximally as in Decapoda. malacostracan ground pattern below is given in Table 3. It is reasonable to assume that the shape of the sclerites Sclerites status in the ground pattern of Malacostraca in Euphausiacea is an autapomorphy (indicated by the (Figure 6A; Table 3). – For malacostracan pleopods we can red-grey marbling of the euphausiacean sch. in Figure 6B), assume that one comparably large sclerite lies laterally and while the anterior shortening of the lateral sclerite and the articulates with the tergopleura and the basipod. It extends posterior shift of the median sclerite-sternite articulation more into the anterior aspect of the arthrodial or articula- are autapomorphies for Decapoda (emphasised by red tion membrane than into the posterior aspect. Another colour in Figure 6B). large sclerite lies medially and participates in joints with Sclerite condition within Neocarida. – Inside Neocarida sternite and basipod. At least one additional smaller sclerite relationships are heavily disputed (see e.g. [36,41,42]), so we lies anteriorly, located slightly lateral and distal to the lateral focused on the phylogeny of Tabacaru & Danielopol [35]. sclerite. In the posterior articulation membrane are four In the ground patterns of all examined neocaridan taxa, sclerites: one sclerite relatively larger than the other three, Amphipoda, Lophogastrida, and Mysida, a lateral and a by which it is surrounded. The phyllocarids largely reflect median sclerite is present. In all these ground patterns, the this condition. articulation between the medianscleriteandthesterniteis In Hoplocarida, the condition has slightly changed in a more posterior position than in other examined taxa. compared to that of the ground-pattern state of Mala- However, in the three neocaridan taxa this condition is costraca (Figure 6A; Table 3): Two additional smaller achieved either by a posteriorly positioned simple pivot in sclerites are located in the anterior joint membrane and Lophogastrida, or by a posteriorly extending outgrowth in only three sclerites (instead of four) are present poster- Amphipoda and Mysida. iorly. Whether the fourth posterior sclerite became lost Sclerites in the ground pattern of "Mysidacea" (Figure 6D). or fused with another cannot be said. Furthermore, the – The lateral sclerite articulates with the basipod and with most distal of the posterior sclerites is elongated and the the tergopleura; the latter articulation is shifted anteriorly. articulation of sternite and median sclerite has shifted The median sclerite articulates with the basipod and the anteriorly. All these features are probably autapomor- sternite, the latter articulation being shifted posteriorly and phies of Hoplocarida except for the presence of only formed by an outgrowth of the median sclerite, as present three posterior sclerites (Table 2; Table 3). The small in Amphipoda. The anterior sclerite lies slightly laterally number of posterior sclerites probably had evolved in and has an apodeme. Two posterior sclerites occur, the dis- the stem species of Eumalacostraca, as no other in- tal one with an apodeme. group taxon exhibits more than two posterior sclerites Lophogastrida would have changed some details of this (Figure 6A; Table 3). ground pattern situation (red elements in their sch. in More complex and challenging is the situation within Figure 6D): The lateral sclerite runs anteriorly and is par- Caridoida. Tabacaru & Danielopol [35] assume a sister allel to the tergopleura; it does not bend around the basi- group relationship of Decapoda and Euphausiacea, uniting pod (Figure 6D, dashed red lines). The median sclerite Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 20 of 27

Figure 7 Images of the basal part of pleopods. Abbreviations as before. A. Palinurus elephas Latreille, 1803. Note that the sclerites are separated from each other by a small membranous area and almost solely reflect the outline of the coxa. B, C. Gammarus roeselii Gervais, 1835. The limb stem comprises the short coxa (artificially highlighted by shrinking effects during the drying process for SEM) and the elongated tubular basipod (cf. Figure 4A–H). B. Posterior view. C. Anterior view. Note that the coxal outline is rather well determinable. D, E. Nebalia bipes (cf. Figure 5G–L). D. Posterior view. E. Anterior view. The coxal outline is not as clear as in the amphipod. F. Pleomeric segment of the crayfish Astacus leptodactylus exhibiting the coxal portion of the pleopod. Note that the coxal outline is almost only reflected by the presence of sclerites. bears setae and articulates directly with the sternite; not via the lateral sclerite is involved with the articulations with the an outgrowth. Mysida have retained only the lateral and the basipod and tergopleura; this sclerite does not extend as median sclerites. In general, Amphipoda and Lophogastrida much into the posterior articulation membrane as it does are more similar to each other than to Mysida, both exhi- into the anterior one. The median sclerite articulates with biting five sclerites against two in Mysida. Yet, a closer rela- the basipod and sternite, the latter articulation being shifted tionship of Lophogastrida and Mysida (= Mysidacea) has posteriorly and formed by an outgrowth of the median scler- been assumed [35,43] (Figure 6D) but also rejected [44-46]. ite. One sclerite with an apodeme lies in the anterior articu- A further reconstruction of sclerite patterns for Neocarida lation membrane; two other sclerites lie posteriorly, the is uncertain, hindered by the absence of data from and taxa proximal one having an apodeme. within Mancoida, in part caused by lack of pleopods (most The enormous extension of the lateral and median scler- likely reduction) in many in-groups, e.g. Cumacea, and un- ites into the anterior articulation membrane found in clear in-group relationships [36]. Adequate out-group com- Amphipoda is regarded as an autapomorphy of this taxon parison is not possible, because Thermosbaenacea have only (red elements in their sch. in Figure 6D), albeit depending reduced pleopods 1 and 2 and these appear to lack any on the state in Mancoida. sclerites (personal observations). The state most parsimoni- The reconstruction of a possible ground-pattern of the ously assumed for Peracarida is also valid for Neocarida as sclerites for N. N. (Anaspidacea + Neocarida, Figure 6B) is no further character polarisation can be performed with difficult because the state in Anaspidacea differs enor- Thermosbaenacea (Figure 6D, question mark). We conclude mously from the one found in our sampling of Neocarida. for the ground pattern of Neocarida and/or Peracarida that Compared to the sclerite state assumed for Eucarida, the Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 21 of 27

stem species of N. N. must have featured at least the follow- We have a similar situation when mapping our ground- ing postulated character states. pattern reconstructions on the phylogeny of Wirkner & Ground-pattern for N. N. (Anaspidacea + Neocarida). – Richter [36] (Figure 6C): We cannot unambiguously po- The lateral sclerite forms a central articulation with the larise the scleritic situation in Anaspidacea vis-à-vis a tergopleura and the basipod. It extends comparably far common stem pattern with Euphausiacea and Neocarida into the posterior articulation membrane. The median (Figure 6C). We would have to assume three sclerites as sclerite articulates via pivot joints with sternite and basi- the most parsimonious condition, the lateral sclerite ex- pod, both being located centrally. One other sclerite lies tending far into the anterior and posterior articulation posteriorly. Whether this latter sclerite had an apodeme membranes. Consequently, the inflated condition of the remains uncertain, due to character-state polarisation with euphausiacean sclerites would be an autapomorphy, as Hoplocarida. would be the posterior shortening of the lateral sclerite, This implies that the single, anteriorly bifurcate sclerite in the presence of an anterior sclerite with apodemes, and an Anaspidacea, which forms an anteriorly shifted joint with additional posterior sclerite in Neocarida. It is most parsi- the tergopleura and the median articulation is formed by monious to assume that the stem species of Xenommacar- the direct contact of the sternite and basipod, are autapo- ida possessed the same condition as seen in N. N. morphies of Anaspidacea (Figure 6B). Consequently, auta- (Euphausiacea + Neocarida) (Figure 6C), as well Caridoida. pomorphies of Neocarida would then have to be: the This would mean that in Anaspidacea the situation auta- posterior shortening of the lateral sclerite, the addition of pomorphically reduced to the single and anteriorly bifur- an anterior sclerite with apodeme, the addition of a laterally cate sclerite, and the median articulation directly with the shifted posterior sclerite, the lateral shift of the remaining basipod and sternite evolved autapomorphically. In Deca- posterior sclerite, and the posterior shift of the median ar- poda the anterior extension of the lateral sclerite and the ticulation of the median sclerite, and the sternite developed posterior position of the median articulation would be as an outgrowth. autapomorphic. The autapomorphies of Caridoida would The ground-pattern states concerning sclerites of Eucar- be the loss of the anterior sclerite, as well as of two poster- ida and N. N. (Anaspidacea + Neocarida) are almost iden- ior ones, the median shift of one posterior sclerite, and the tical. This is why we can assume the following sclerite posterior extension of the lateral sclerite. state for Caridoida. Ground pattern of Caridoida. – The lateral sclerite forms a central articulation with the tergopleura and the Discussion basipod. It extends relatively far into the articulation Value of sclerite patterns membranes, anteriorly and posteriorly. The median Calcified sclerites appear to occur at the base of the pleo- sclerite forms a central joint with sternite and basipod. pods, at least for the malacostracan taxa we investigated. If lateral and median sclerite are touching each other Some of the sclerites form pivot joints between the ster- posteriorly is unclear. Posteriorly another sclerite is nites medially and tergopleurae laterally. The limb stem present, but whether it exhibits apodemes remains also forms corresponding depressions or knobs proximally. In uncertain. addition, some internal apodemes act as attachment sites Based on what we reconstruct for the eumalacostracan for muscles. The pattern of the sclerites is not only rather state (Figure 6A), this would imply the following autapo- invariable between individuals of a species and within the morphies for Caridoida: 1) the extension of the lateral scler- series of pleopods of a species, but also conservative ite into the posterior articulation membrane, 2) the loss of within members of putatively monophyletic groups. Ac- the anterior sclerite, 3) the loss of two posterior sclerites, cordingly we were able to reconstruct ground-pattern and 4) the median shift of one of the posterior sclerites. states of the according taxa. However, this reconstruction does not seem parsi- Mapping our data onto existing phylogeny hypotheses for monious because the conditions found in the eumala- Malacostraca mirrors the basal sister-group relationship of costracan and neocaridan stem species would appear Phyllocarida and Eumalacostraca, long suggested before rather similar because: 1) the lateral sclerite extends [31,35,36,41-43,47,48], but see, e.g. [49]. Phyllocarida pos- more into the anterior articulation membrane than sess the highest number of sclerites, four well-developed into the posterior one, 2) the presence of an anterior lateral and median joints and no dislocation of these (Table 2), sclerite with apodeme, and 3) the appearance of more which we assume represents the ancestral condition of than one posterior sclerite in both. These features are this arrangement. This pattern of sclerites overlaps with absent in our reconstruction of the caridoid ground that of Hoplocarida (Table 2), in most hypotheses turning pattern (Figure 6B). Thus, Neocarida would have to out as the next branch in the system of Malacostraca and have made a step back and re-evolved these features, sister taxon to Caridoida. Accordingly we regard the scler- which is not parsimonious. ite pattern of Phyllocarida as the least derived one and Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 22 of 27

reflecting much of the character condition in the ground In addition to the petasma [52,53] and uropod morphology pattern of Malacostraca. [28], the sclerite pattern represents a third morphological Compared to this the number of sclerites and joint complex associated with the pleon available for more de- locations is slightly smaller but still large in the caridoid tailed investigations and analyses in the future. This is espe- taxa Lophogastrida and Amphipoda among the Peracar- cially important since [42] demanded for new character ida, both having five sclerites (Table 2). Within Peracarida, sources for resolving (eu)malacostracan relationships. the presence of five sclerites in Amphipoda points to a ra- ther basal position of this taxon. This differs from the Origin of the calcitic sclerites phylogeny proposed by Wirkner & Richter [36], Tabacaru Topologically, the sclerites on the malacostracan pleopods & Danielopol [37], and [50], in which Amphipoda are appear to be located within the basal arthrodial mem- deeply nested within Peracarida. However, the sclerite pat- brane, i.e., between the sclerotised ventral body proper tern is congruent with the peracaridan system promoted and sclerotised limb stem. Clearly all malacostracans pos- by Kobusch [51], in which Lophogastrida represent the sess a coxal limb portion on all their anterior eight thora- sister taxon to all other Peracarida, comprising Amphi- copods. Phyllocarida have antero-posteriorly flattened poda and the remaining taxa Mysida and Mancoida. Fol- limbs with likewise flat and large coxal and basipodal por- lowing this, Mysida exhibit the most derived condition with tions. The well-sclerotised and calcified coxae of all other two sclerites of the three peracaridan taxa investigated, but taxa, which are generally better calcified than those in interestingly linked to amphipods by a shared extension of phyllocarids (speaking only of the living taxa here) are one of the sclerites to the sternite, pointing to their separ- fairly short and possesses a well developed pivot joint ation from Lophogastrida (Table 2). Also in this respect, medially and laterally. Another such pair of pivot joints our data fit with the phylogeny of Kobusch [51]. The scleri- connects coxa and basipod, more the endopodal articles. ticextensiontothesternite(Table2)couldrepresentaspe- This articulation system includes membranes between all cific marker of evolutionary changes within the Peracarida, sclerotised portions, including the basal area. Even rather but this has to be investigated further in the remaining modified thoracopods share this morphology rather con- mancoidan in-group taxa as much as the low number of servatively and in a rather uniform design. This suggests sclerites, which might rather be taxon-specific due to the that a change from having neither a proximal endite nor a high reduction of the pleopods in Mysida. coxa should have occurred before or with the stem species Within the remaining Caridoida, Euphausiacea, and of all modern taxa, therefore the evolutionary transition Decapoda share a very specific sclerite pattern (Table 2). remains obscure. This hints at their sister-group relationship [35,48]. Here it Assuming that the calcitic sclerites on the malacostra- is even more strongly supported by no less than three de- can pleopods correspond to the phylogenetically old me- tails in the arrangement: lateral and median sclerites touch dian soft and setae-bearing proximal endite (feature of each other; absence of the anterior sclerite; no posterior Crustacea s. l., see [10]), this endite should have become sclerite (Table 2). Dislocation of the pivot joints, either to sclerotised, calcified and split into several sclerites before the tergopleura, or to the sternite, could be taxon-specific the evolution of the malacostracan stem species – and this and independently derived due to functional adaptations in line with the de novo formation of pivot joints. The start (Table 2). from a euarthropod limb situation lacking a proximal end- The pivot joint between lateral sclerite and tergopleura ite or coxa is likewise improbable since it also requires the occurs in the same fashion throughout all investigated taxa, evolution of the sclerites and pivots de novo. hence appears to be conservatively retained from the ances- At least it remains clear that four pivots have therefore tral state = ground-pattern of Malacostraca. This is true to be expected also for pleopods if they have or had a even for the Anaspidacea, in which, otherwise and autapo- coxal portion, and moreover two membrane areas. This morphically, an antero-lateral extension of the bilobed lat- scenario assumes a long series of thoracopods in the mala- eral sclerite articulates with the tergopleura instead of a costracan stem species, all equipped with a coxa-basipod simple pivot joint (Table 2). Also in other details, the situ- limb stem. With the tagmotic distinction into two sets ation in Anaspidacea, with onlyasinglebutbifurcatescler- and functional deviation of the two sets of limbs, also their ite, is autapomorphic, a condition most likely associated morphology became more distinct from that of the anter- with the specific orientation and movements of the pleo- ior set. One is the appearance of calcified sclerites, as in- pods, i.e., the limbs are oriented laterally during swimming vestigated here, instead of a well-visible coxa body. Yet of Anaspides tasmaniae. pleopods also retained conservative traits. The most ap- Summing up, our initial investigations of the sclerite pat- parent is the retention of the pivots. The proximal and tern in representatives of major malacostracan taxa demon- distal outlines of the sclerites form the minimum range of strate the value of this character complex, and potential as a the supposed coxal area, because these are located within phylogenetic marker for malacostracan in-group phylogeny. the sclerotised coxal cuticle. Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 23 of 27

All membrane between these lines is unsclerotised coxal more changes occurred on the pleopods [56-59], although cuticle, not belonging to the true joint membranes. These the specific functions of the basal joint have not yet been softer cuticular areas lie either anteriorly or posteriorly of touched (but see [29]). Further investigations on this the limb, while the basipod may have a deep excavation to would therefore be very helpful, also including life studies enlarge the joint area only posteriorly (Figure 7A). Ac- of the pleopods of the according taxa. cording to the degree of softness, the coxa may be more or less effaced in this outline, which may be the main rea- Conclusions and Outlook son to have mostly been overlooked in the past. The pres- Our study of scleritic elements in the proximal-most limb ence of a coxal outline is therefore dependent on the joint area of malacostracan pleopods does not only yield degree of sclerotisation, in our studied species maximally new data about this structural complex, but it also demon- visible in e.g. the pleopods of amphipods (Figure 7B, C), in strates its potential to enlighten us about phylogeny. Calci- a medium state in phyllocarids (Figure 7D, E), and in its fied sclerites are not simply present in the body–limb minimum visible in just the sclerites, as in stomatopods or articulation of malacostracan pleopods, but also participate decapods (Figure 7A, E). in the formation of joints with limb basipod and sternites The entire structural complex around the sclerites, now medially and tergopleurae laterally by providing the corre- identified as a modified coxa, should have evolved before sponding depressions or knobs to those of the associated the stem species of all modern Malacostraca, but neither elements. Some of the sclerites have, in addition, internal ontogenetic nor palaeontological data are available to elu- apodemes for internal muscles to attach there. Number and cidate this problem. Hence, to reflect the question of the position of the sclerites turned out to be taxon-specific and evolutionary origin of the coxal body of malacostracan constant, even conserved in strongly modified pleopods. A thoracopods, no matter if the anterior eight or the pleo- higher number of sclerites (7 or 8) seem to be part of the pods, cannot be discussed here at present. ground pattern for Malacostraca. Another, though more indirect, evidence are the ob- Caridoida are characterised by a reduced number of served apodemes. These belong to the morphology of the sclerites, but retain all major components. Within Cari- coxae of the anterior thoracopods too and serve as attach- doida, the sister-group relationship of Euphausiacea and ment devices for musculature running into the limb [4,5]. Decapoda is supported by the occurrence of a similarly Such apodemes are also present on some of the sclerites, reduced and specific pattern of sclerites; we interpret and scleritic elements might even comprise several pieces this as autapomorphic for Eucarida. forming a ring (e.g. in Meganyctiphanes norveciga (Sars, Within Peracarida the original sclerite pattern seems to 1857); see [5], their figure 3A). Thus there are further have become simplified, with the appearance of a specific similarities between the basal area of pleopod limbs and extension connecting the median sclerite and sternite in thoracopodal coxae. Amphipoda and Mysida points to a further specialisation. However, we refrain from confirming earlier authors, Independent evolution of Lophogastrida and Mysida is who had applied the term "coxa" to proximal (= scleritic) supported. structures on malacostracan pleopods [54,55]. Sclerites Furthermore, we consider these sclerites as calcitic com- themselves do not represent the coxa (or are its re- ponents in the cuticle proximal to the basipod. In most mains), but are solely part of it, calcifications within the cases, however, this coxa area is not a completely sclerotised cuticle of the coxa. Only in some cases they are equiva- ring element; sclerotised areas are mainly limited to the me- lent to the sclerotised region of the coxa, i.e. when the dian and lateral edges – also more or less the sites of the cal- rest has become little sclerotised. These are apomorphic citic sclerites – the posterior area is membranous. states of a particular taxon, but not in the sense of de- Further studies on this character complex are demanded generation. In this respect it is also unclear if the "prox- and promising: Particularly the functional morphology of imal segment" of the pleopods Ungerer & Wolff [27] the calcitic sclerites in their role to assist the joints and in- identified in embryos of Orchestia cavimana represent fluence to movability of the pleopods in line with the role the same structure recognised here as the coxa. of the decalcified areas of the coxa should be examined in The limb stems of pleopods are generally elongated and more detail. Further investigations of representatives of tubular. Modification of their connection to the body by a other in-group Peracarida, i.e. of the Mancoida would also de-sclerotisation of parts of the coxa and by parallel en- be helpful in order to improve the resolution of the system hancement of rigidity by the calcitic sclerites led to the within Peracarida. specific movements of the pleopods (independent if the In a wider array, the identification of a coxa on now all presence of sclerites originates from development or re- thoracopods of Malacostraca might also be interesting for tention). Initially leaving all four pivot joints, movement of the ongoing discussion about tracheate or insect relation- the pleopods was mainly in antero-posterior direction. In ships with crustaceans or even malacostracans and the connection to other life styles and modes of locomotion problematic role of Remipedia. Relationships have been Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 24 of 27

supposed in a whole suite of papers, also based on sclerot- dissected but examined with fluorescence microscopy ised elements present at the limb base in these taxa (Table 4). [1,60-65], which makes it even more challenging to com- Macrophotography. – Most overview images of whole pare more scleritic data between all these groups. Indeed specimens were obtained with a Canon EOS 450D camera closer examination of the situation in Remipedia and the with an EFS 60 mm objective under crossed polarised filters Tracheata seems promising to us, not least due to the [68-70]. The depth of field was improved by recording presence of scleritic elements in their limb joints. image stacks and combining the single images into one sharp photograph with the software Combine ZP [69]. The Methods overview images of the other specimens (Al. helonomus, Material Hyperia sp., Pa. lacustris,andNeo. semissis;seeTable4) We examined 3–8 individuals of 16 species representing were documented with fluorescence microscopy and com- different malacostracan taxa (Figure 1, Table 4). Alla- posite imaging [35,69]. naspides hickmani (museum number: 26633) and Para- Images of the pleopod-body articulations of Alizarin- naspides lacustris (museum number: 26632) were from Red stained specimens were either made with a Canon the Naturkundemuseum Berlin, Germany. Nebaliopsis EOS 450D camera with a MP-E 65 mm objective on a sp. specimens were from the Senckenberg Naturmu- light table to enhance the contrast, or by attaching the seum Frankfurt, Germany (museum number: SMF- camera to a Zeiss Axioskop microscope photographing 43552) and the Zoological Museum of Copenhagen, with transmitted light (Table 4, column AR). The depth of Denmark. All Nebaliopsis specimens were damaged due field was improved accordingly. All images were further to capturing them in the deep sea; only their cepha- processed in Adobe Photoshop CS3 resp. CS4. lothoracic shields and complete pleons were present, Fluorescence microscopy. – Specimens borrowed from which was sufficient for our study. museums were not stained with Alizarin Red. To guarantee We studied the body–limb articulation of the pleopods 1– an equally distinct documentation, the pleopod-body articu- 5 in each species, except for the phyllocarid representatives, lations of these specimens (Hyperia sp., Nebaliopsis sp., Neo. as pleopods 5 and 6 are small and reduced. The pleopods 6 semissis, Pa. lacustris,andAl. hickmani; Table 4) were docu- are often modified into uropods and were therefore ex- mented via fluorescence microscopy ([34,70], Table 4). Calci- cluded here, as they were subject of another study [28]. Taxa fied sclerites are fluorescent whereas the membranous parts without pleopods or with strongly reduced pleopods, i.e. are not. Thus, the sclerites are displayed bright and whitish Bathynellacea, Thermosbaenacea, and in-group Peracarida, in a fluorescence micrograph, whereas the membranous as well as in-group Decapoda were omitted. parts are comparably dark. The specimens were docu- mented with a Zeiss Axioskop 2 with an Axiocam under Methods UV light (356 nm). Regeneration. – Specimens of Gonodactylus chiragra were Scanning electron microscopy. – Preparation and docu- available as dried samples. They were regenerated with a mentation followed the protocol given by [33]. 5% NaCl solution (Table 4), in which the specimens were Dissection. – Those specimens, which were stained deposited [66] till the segments and appendages were with Alizarin Red and were not loaned from a museum, movable again what took three days. The specimens of were further dissected with pointed forceps and fine Go. chiragra were further processed for staining with Ali- scissors (Table 4). By that it was possible to isolate the zarin Red (Table 4). sclerites and detect presence of apodemes, the internal Staining with Alizarin Red. – Alizarin Red tinges calci- attachment points of muscle fibres. fied parts of the cuticle purple while membranous areas Terminology. – The terminology applied was taken remain white. This allowed detection of small, calcified mainly from Walossek [6,8] and collaborators [10,12]. areas ("sclerites") in the pleopod-body articulation. We The sclerites were numbered consecutively in each spe- followed the protocol given by Brösing et al. [67]: speci- cies. The various numbers do not intend inter-specific mens (Table 4) were cooked at 100°C in a 10% KOH- homologisation. For the ground-pattern reconstructions solution for 1 h, which dissolves the inner organs while numbering was omitted, but the positions of the sclerites only the cuticle remains. A spatula point Alizarin Red was were used to specify them (detailed explanations for our added and the specimens were cooked for further 15 mi- schs. in Table 1, Table 2 and Table 3). In most cases only nutes, during which the actual staining took place. After- the left half of a pleomere was illustrated, and the sterni- wards the specimens were washed with distilled water and tic wings omitted. stored in 70% ethanol for documentation. This was per- Remarks concerning the term "sternite": A sternite is formed with representatives of several species (Table 4), here understood as the median, sclerotised area between but not all. The specimens loaned from different mu- the insertions of a pair of post-oral appendages [71]. This seums were neither stained with Alizarin Red nor sclerotised area is part of the entire ventral region of a Kutschera et al. BMC Evolutionary Biology (2015) 15:117 Page 25 of 27

segment, the rest of the area is membranous. The sternite Acknowledgements provides the median attachment point of the appendages, We are very grateful for the material received for this study: Anaspides tasmaniae was made available by Roy Swain, Sandy Bay, Australia; better their basipods, the outer attachment is between Erugosquilla massavensis waskindlyprovidedbyWafaaSalamIsmail,Egypt. basipod and tergopleura, both enforcing an antero- Euphausia superba was made available by the best sister taxon in the world posterior swing of the limb. In the well-sclerotised and Charlotte Havermans Brussels, Belgium; Gnathophausia gigas and Lophogaster typicus were genially received from Kenneth Meland, Bergen, calcified malacostracans the attachments are likewise im- Norway; Gonodactylus chiragra was made available by Timmy Grohrock, proved and more rigid, in the simple form consisting of Büchenbach, Germany (www.fangschreckenkrebse.de). Kindly pivot joints, ball-and-socket joints that connect the coxa acknowledged is also the loan of museum specimens, organised by C. – Oliver Coleman, Museum für Naturkunde, Berlin, Germany, Jørgen Olesen, of thoracopods 1 8 with the sternite and tergopleura. Natural History Museum Denmark, Copenhagen, Denmark, and Michael Sternites may fuse with one another along the body, often Türkay, Senckenberg Research Institute and Natural History Museum within entire tagmata, but never in the pleon of Malacos- Frankfurt, Germany. This research received support from the SYNTHESYS Project http:// traca. There modifications may occur in the form of www.synthesys.info/, financed by the European Community Research extensions around the limbs anteriorly and posteriorly Infrastructure Action under the FP 6 "Capacities" Programme (DK-TAF-1024). into so-called wings, sometimes even encompassing the Additionally, we are grateful to Susanne Maas and Ina Schmidt for inspiring us to continue their early studies on this topic. Special thanks go to Stefan complete insertion areas of the limbs. The two wings might Liebau, meanwhile Tübingen, Germany, for providing access to the even fuse laterally with the tergopleura, forming a rigid skel- fluorescence microscope. Thanks are also due to the programmers of freely etal system around the pleopodal insertion areas, such as in available software Combine ZP and to the two reviewers of the manuscript, which spent much effort to improve language and structure. a lobster. However the indications of the pivots are still present. 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