Metamorphosis in Craniiformea Revisited Novocrania Anomala Shows Delayed Development of the Ventral Valve Altenburger, Andreas; Wanninger, Andreas; Holmer, Lars E

  

Metamorphosis in Craniiformea revisited Novocrania anomala shows delayed development of the ventral valve Altenburger, Andreas; Wanninger, Andreas; Holmer, Lars E.

Published in: Zoomorphology

DOI: 10.1007/s00435-013-0194-3

Publication date: 2013

Document version Early version, also known as pre-print

Citation for published version (APA): Altenburger, A., Wanninger, A., & Holmer, L. E. (2013). Metamorphosis in Craniiformea revisited: Novocrania anomala shows delayed development of the ventral valve. Zoomorphology, 132(4), 379-387. https://doi.org/10.1007/s00435-013-0194-3

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Metamorphosis in Craniiformea revisited – Novocrania anomala shows delayed 1 2 development of the ventral valve 3 4 5 6 7 Running title: Metamorphosis in Novocrania 8 9 10 11 12 Andreas Altenburger, Andreas Wanninger, Lars E. Holmer 13 14 15 16 Authors’ names and addresses 17 18 19 Andreas Altenburger, University of Copenhagen, Natural History Museum of 20 21 Denmark – Zoological Museum, Universitetsparken 15, DK-2100 Copenhagen, 22 23 24 Denmark, [email protected], phone: +45 35321114 25 26 Andreas Wanninger, University of Vienna, Dept. of Integrative Zoology, Althanstr. 27 28 29 14, AUT-1090 Vienna, Austria 30 31 Lars E. Holmer, Uppsala University, Department of Earth Sciences, Palaeobiology, 32 33 SE-752 36 Uppsala, Sweden 34 35 36 37 38 Abstract 39 40 41 We revisited the brachiopod fold hypothesis and investigated metamorphosis in the 42 43 craniiform brachiopod Novocrania anomala. Larval development is lecithotrophic 44 45 46 and the dorsal (brachial) valve is secreted by dorsal epithelia. We found that the 47 48 juvenile ventral valve, which consists only of a thin layer that was previously 49 50 described as periostracal, is not a valve, and is not secreted by the same epithelia as 51 52 53 the dorsal valve. It is secreted by the attachment area of the larva at the posterior-most 54 55 tip of the posterior larval lobe. The same attachment area is used by larvae of 56 57 58 rhynchonelliform brachiopods during metamorphosis to cement their pedicle to the 59 60 substrate. N. anomala is therefore not initially attached by a valve but by material 61 62 63 64 65 2

corresponding to pedicle cuticle. This is different to previous descriptions, which had 1 2 led to speculations about a folding event in the evolution of Brachiopoda. We show 3 4 5 that the “brachiopod fold hypothesis”, which argues that brachiopods are transversely 6 7 “folded” across the ontogenetic anterior-posterior axis, should be rejected at least with 8 9 10 respect to the craniiforms. The data now suggest that the Craniiformea may be a 11 12 derived group within the Rhynchonelliformea. This interpretation suggests that the 13 14 15 last common ancestor of the Craniiformea has lost the pedicle and the ventral valve in 16 17 early juvenile development. Characters that have previously been considered to be 18 19 shared between the Craniiformea and the Linguliformea (clade Inarticulata), such as a 20 21 22 through-gut and missing hinge articulation, may thus be secondarily derived 23 24 characters of the Craniiformea within the Rhynchonelliformea. 25 26 27 28 29 Keywords 30 31 32 ontogeny, development, Craniiformea, phylogeny, morphology, body plan, evolution 33 34 35 36 Introduction 37 38 39 Brachiopoda is a marine animal phylum with a rich fossil record and approximately 40 41 370 recent species divided into the three clades, Craniiformea, Rhynchonelliformea, 42 43 44 and Linguliformea (Williams et al. 1996; Zezina 2008). The evolution of the 45 46 brachiopod body plan with two valves that enclose the soft tissue and a pedicle that 47 48 49 attaches the animal to the substrate has puzzled researchers since the naming of the 50 51 phylum by Duméril (1806). Brachiopods have since been deemed related to various 52 53 phyla such as molluscs, bryozoans, annelids and recently, based on molecular data, to 54 55 56 nemerteans (Grey 1848; Huxley 1853; Morse 1870; Dunn et al. 2008). 57 58 59 60 61 62 63 64 65 3

Brachiopod internal relationships between the three clades, and the relation of 1 2 brachiopods to phoronids, have not been completely resolved to date by molecular 3 4 5 methods (Cohen et al. 1998; Cohen 2000; Cohen and Weydmann 2005; Sperling et al. 6 7 2011; Cohen 2013). Also, the comparative morphological analyses found an equal 8 9 10 amount of characters shared among the Craniiformea and the Linguliformea (formerly 11 12 the clade Inarticulata) and the Craniiformea and the Rhynchonelliformea (Carlson 13 14 15 1995). This has led to different outcomes in analyses concerning brachiopod internal 16 17 phylogeny, depending on the chosen character polarities (Carlson 1995; Holmer et al. 18 19 1995; Williams et al. 1996). Characters shared among the craniiforms and the 20 21 22 linguliforms include a through-gut, a circumferential mantle cavity, a muscular 23 24 system consisting of oblique and two pairs of adductor muscles, as well as a transient 25 26 27 median tentacle (Williams et al. 1996). Characters shared among the Craniiformea 28 29 and the Rhynchonelliformea include a proteinaceaous calcitic shell, a single row of 30 31 32 tentacles on trocholophous lophophores, gonads suspended in mantle sinuses, and 33 34 lecithotrophic larvae (Holmer et al. 1995; Williams et al. 1996). 35 36 There are various hypotheses about the evolution of the brachiopod body plan. During 37 38 39 the years, four hypotheses have been put forward. 1. The ancestor of brachiopods was 40 41 a tube-dwelling polychaete-like metameric worm with a hydraulic body construction 42 43 44 (Gutmann et al. 1978). 2. The ancestor of brachiopods was an oligomerous worm with 45 46 setae, protocoel, mesocoel, and three segments in the metacoel (Temereva and 47 48 49 Malakhov 2011). 3. The ancestor of brachiopods was a halkieriid-like organism, 50 51 comprising a worm-like body with sclerites and shells at the anterior and posterior 52 53 end (Conway Morris and Peel 1995; Cohen et al. 2003). 4. Brachiopods evolved from 54 55 56 sclerite-bearing, tube-dewelling, worm-like organisms such as the tommotiids 57 58 59 60 61 62 63 64 65 4

Micrina, Eccentrotheca, and Paterimitra (Williams and Holmer 2002; Holmer et al. 1 2 2008; Skovsted et al. 2009a; 2009b; Holmer et al. 2011; Murdock et al. 2012). 3 4 5 In living brachiopods, members of each clade have a distinct mode of development. 6 7 The larvae of Craniiformea are lecithotrophic, bilobed, with three pairs of dorsal setal 8 9 10 bundles on the posterior lobe. The larvae of Rhynchonelliformea are lecithotrophic, 11 12 have three lobes and four setal bundles at the mantle lobe, while the Linguliformea 13 14 15 have planktotrophic, swimming juveniles (Nielsen 2005). Larvae of Craniiformea and 16 17 Rhynchonelliformea metamorphose upon settlement. Swimming juveniles of 18 19 Linguliformea do not undergo metamorphosis upon settlement because 20 21 22 metamorphosis is mainly completed at the end of the embryonic stage in lingulids or 23 24 shortly after hatching in the discinids (Lüter 2001, 2007; Popov et al. 2012). 25 26 27 Hypotheses concerning the putative body plan of the ancestor of Brachiopoda often 28 29 focus on the development of Novocrania anomala (Müller 1776) (previously assigned 30 31 32 to several genera and therefore also referred to as Crania anomala or Neocrania 33 34 anomala) (Lee and Brunton 1986, 2001), a member of the Craniiformea (Nielsen 35 36 1991; Popov et al. 1993; Conway Morris 1998; Cohen et al. 2003). It has been 37 38 39 claimed that both valves in juveniles of N. anomala are secreted by mantle epithelia 40 41 that originate at the dorsal side of the posterior lobe (Nielsen 1991). This notion has 42 43 44 led to the conclusion that a “folding-event” happened during brachiopod evolution 45 46 (Cohen et al. 2003; Malakhov and Kuzmina 2006). The concept was subsequently 47 48 49 termed “brachiopod fold hypothesis”, according to which brachiopods are assumed to 50 51 transversely “fold” across the larval anterior-posterior axis. According to this theory, 52 53 the brachiopod body plan needs to be conceptually “unfolded” in order to make useful 54 55 56 comparisons with the body plans of other animal phyla (Cohen et al. 2003). The body 57 58 plan of Halkieria evangelista as a possible stem group brachiopod was used to 59 60 61 62 63 64 65 5

support this view (Conway Morris and Peel 1995; Cohen et al. 2003). However, the 1 2 hypothesis that H. evangelista represents a brachiopod stem species has since been 3 4 5 questioned (Vinther and Nielsen 2005). 6 7 The metamorphosis of N. anomala was described in detail with the claim that prior to 8 9 10 metamorphosis ‘most larvae become inactive and lie curled together on the bottom 11 12 with the pair of ventral muscles maximally contracted’ (Nielsen 1991). We tested 13 14 15 whether these curled, inactive larvae are able to metamorphose by live observation, 16 17 the use of F-actin labelling, confocal laser scanning microscopy, and scanning 18 19 electron microscopy. 20 21 22 23 24 Materials and Methods 25 26 27 Collection of developmental stages 28 29 Adults of Novocrania anomala where obtained by dredging in the vicinity of the Sven 30 31 32 Lovén Centre for Marine Sciences, Gullmarsfjord, Sweden (58°15’921”N, 33 34 11°25’103”E), in September 2008. The rocks with attached N. anomala were kept in 35 36 running seawater at 14°C. Adults were removed from the rocks and dissected. Eggs 37 38 39 and sperm were removed with pulled glass pipettes, washed, and left in filtered 40 41 seawater in separate beaker glasses. Fertilized eggs were regularly checked for 42 43 44 germinal vesicle breakdown. Sperm cells were monitored for motility under a 45 46 compound microscope. When eggs were ready for fertilization (approximately 12 47 48 49 hours after dissection), 2 ml of a highly diluted sperm suspension were added. 50 51 Embryos and larvae were fixed at various stages from 4 hours after fertilization (hpf) 52 53 to 17 days after settlement in 4% paraformaldehyde in 0.1M phosphate buffer (PB) 54 55 56 for 90 min. Thereafter, larvae were washed three times for 15 min each in 0.1M PB 57 58 and finally stored in 0.1M PB containing 0.1% NaN at 4ºC. 59 3 60 61 62 63 64 65 6

1 2 Scanning electron microscopy 3 4 5 For scanning electron microscopy, the specimens were dehydrated in a graded ethanol 6 7 and acetone series, critical point dried, sputter coated with gold, and investigated 8 9 10 using a Zeiss Supra 35VP scanning electron microscope. 11 12 13 14 15 Confocal laserscanning microscopy 16 17 The musculature was stained with phalloidin. Prior to staining, fixed larvae were 18 19 washed in phosphate buffer (PB) three times for 15 min each. Then, the larvae were 20 21 22 incubated for 1 h in PB containing 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, 23 24 USA) to permeabilize the tissue, left overnight at 4°C in 0.1M PB containing 0.2% 25 26 27 Triton X-100 and 1:40 diluted Alexa Fluor 488 phalloidin (Invitrogen, Molecular 28 29 Probes, Eugene, OR, USA), and subsequently washed in 0.1M PB that was changed 30 31 32 three times every 15 min. Finally, the specimens were embedded in Fluoromount G 33 34 (Southern Biotech, Birmingham, AL, USA) on glass slides. Negative controls 35 36 omitting the phalloidin dye were performed in order to test for autofluorescence and 37 38 39 rendered no signal. The stained larvae and juveniles were analyzed with a Leica DM 40 41 IRE2 fluorescence microscope equipped with a TCS SP2 AOBS laserscanning device 42 43 44 (Leica Microsystems, Wetzlar, Germany). Animals were scanned with 0.16 µm - 0.49 45 46 µm step size, and the resulting image stacks were merged into maximum projection 47 48 49 images. Light micrographs were recorded to allow overlay with the CLSM images for 50 51 exact orientation and localization of the muscle systems within the animals. 52 53 54 55 56 Schematic drawings and figure plates 57 58 Drawings and micrographs were assembled with Photoshop CS3 and Illustrator CS3 59 60 software (Adobe, San Jose, CA, USA). 61 62 63 64 65 7

1 2 Results 3 4 5 Overview of larval development at 14°C 6 7 Cleavage is radial and the first two divisions are holoblastic (Fig. 1A-B, 2A-B). The 8 9 10 blastula is spherical (Fig. 2C) and invagination starts at around 18 hours after 11 12 fertilization (hpf) (Fig. 2D). The blastocoel disappears completely at the gastrula stage 13 14 15 (Fig. 1C). Subsequently, the gastrula elongates and the blastopore comes to lie at the 16 17 postero-ventral side of the swimming larva (Fig. 1D, 2E). The elongated gastrula 18 19 differentiates into two larval lobes, i.e., an anterior lobe and a posterior lobe (Fig. 1E- 20 21 22 F, 2F-G). Larval development is complete with the establishment of three pairs of 23 24 dorsal setal bundles on the posterior lobe (Fig. 1G, 2H). Now the larvae are competent 25 26 27 for metamorphosis and swim driven by the cilia that are around the anterior lobe. At 28 29 this stage, the larvae have setae pouch muscles at the base of each bundle of setae. 30 31 32 These setae pouch muscles are interconnected by medioventral longitudinal and 33 34 transversal muscles (for a detailed description of myogenesis and larval myoanatomy 35 36 of N. anomala see Altenburger and Wanninger, 2010). 37 38 39 40 41 Metamorphosis 42 43 44 At a water temperature of 14 °C, metamorphosis takes place around six to ten days 45 46 after fertilization (dpf). During metamorphosis the larva lies flat on the surface with 47 48 49 the ventral side pointing towards the substrate. The anterior lobe and the posterior end 50 51 of the posterior lobe are bent ventrally, thus exposing the dorsal posterior lobe to the 52 53 environment (Fig. 4A-C). The larva attaches itself to the substrate with the posterior- 54 55 56 most tip of the posterior lobe. The dorsal mantle develops from an oval-shaped area 57 58 on the dorsal side of the posterior lobe and secretes the first-formed brachial valve 59 60 61 62 63 64 65 8

(Fig. 4D-F, 3B). The larval lobes are retained during the first steps of metamorphosis 1 2 and are subsequently remodeled to form the lophophore and other adult organs (Figs. 3 4 5 1H, 2I). 6 7 Prior to settlement, the larva swims along the bottom of the culture dish, probably in 8 9 10 order to test if the substrate is suitable for settlement. Larvae ready for metamorphosis 11 12 appear to be slightly curled, as they turn the anterior lobe and the posterior tip of the 13 14 15 posterior lobe ventrally. However, strongly curled larvae with maximally contracted 16 17 musculature are arrested in their development and unable to metamorphose (Fig. 3A, 18 19 3D). The larval musculature of healthy, settling, and metamorphosing individuals 20 21 22 remains elongated and relaxed (i.e., non-contracted) prior its resorption in the juvenile 23 24 (Fig. 3E). There is no ventral mantle at this stage of development (Fig. 2I, 3C). Some 25 26 27 days after settlement the larval lobes give rise to the adult tissues including the 28 29 lophophore (Fig. 3F). 30 31 32 33 34 Discussion 35 36 Development and regional specification during Novocrania anomala embryogenesis 37 38 39 were described previously, but interpreted differently (Nielsen 1991; Freeman 2000). 40 41 According to all observations (published previously and presented herein) the cilia on 42 43 44 the anterior lobe function as locomotory organ of the larva prior to metamorphosis. 45 46 Once the larva has found a suitable place for settlement it attaches itself to the 47 48 49 substrate with the posterior-most tip of the posterior lobe, which is bent ventrally. A 50 51 distinguishable domain of cells between the setal bundles on the dorsal side of the 52 53 posterior lobe participates in the formation of the mantle that will secrete the dorsal 54 55 56 valve during metamorphosis (Fig. 4 D-E) (Nielsen 1991; Freeman 2000). Nielsen 57 58 (1991, page 25) states moreover: “…the brachial (‘dorsal’) valve was secreted by the 59 60 61 62 63 64 65 9

anterior part of the dorsal side of the larva while the pedicle (‘ventral’) valve was 1 2 secreted by the posterior part of the dorsal side, i. e. both valves are actually dorsal!”. 3 4 5 We reject this interpretation for several reasons. First of all, the attachment area of the 6 7 larvae is the posterior-most tip of the posterior lobe, thus one cannot make a 8 9 10 distinction between dorsal or ventral at this point. It is further not clear which 11 12 epithelia form the ventral mantle that would be needed to secrete the ventral valve. It 13 14 15 was assumed that the ventral valve is secreted by and spreads from the attachment 16 17 area (Nielsen 1991). We agree with the assumption that cuticle spreads from the 18 19 attachment area. However, we disagree that it is a valve, as the juveniles in our 20 21 22 experiments did not develop a ventral mantle that could secrete the ventral valve 23 24 within the first 17 days after metamorphosis. This is seen in the specimen shown in 25 26 27 Fig. 4 D-F, which has a mantle on the dorsal side of the larva but not on the ventral 28 29 side. The juvenile specimens in Fig. 2I and 3C, which are shown from the ventral 30 31 32 side, likewise have a dorsal valve but neither a ventral valve nor a ventral mantle. 33 34 Only the attachment area is visible at this stage of development, which makes it clear 35 36 that the absence of a ventral mantle and a ventral valve is not due to experimental 37 38 39 procedures. 40 41 The development of the ventral valve in post-metamorphic stages of N. anomala is 42 43 44 delayed. A similar delay of ventral valve development is found in juvenile stages of 45 46 the Ordovician craniid Orthisocrania and in some palaeozoic rhynchonelliforms such 47 48 49 as Antigonambonites (Popov et al. 2007; Popov et al. 2010). Later stages of N. 50 51 anomala show a ventral valve consisting of a very thin periostracal layer with which 52 53 the animals cement themselves to the substrate (Rowell 1960). According to these 54 55 56 observations, the ventral mantle does not begin to secrete calcite until the animals are 57 58 in the “12 – 14 pairs of cirri stage” and about 1 mm wide (Rowell 1960). 59 60 61 62 63 64 65 10

Generally, the ventral valve of adult N. anomala show “a predominantly organic layer 1 2 … survives within the central area of the pedicle valve of living Crania” (Williams 3 4 5 and Wright 1970, page 48). In mature adults, over 10 mm in diameter, a calcitic 6 7 thickening exceeding 0.5 mm is limited to an anterior arc which forms a dorsally 8 9 10 convex submarginal rim so that a central area, between 3 and 5 mm across, remains 11 12 only partially covered by the merest mineral film (Williams and Wright 1970). 13 14 15 It is well established that the Craniida occur in the fossil record not before the Lower 16 17 Ordovician with the clade Pseudocrania (Bassett 2000). This is late compared to the 18 19 Linguliformea and the Rhynchonelliformea which are found much earlier in the fossil 20 21 22 record, namely already in the Lower Cambrian (Williams et al. 2007). Many 23 24 phylogenetic analyses hint towards the Craniiformea as being derived within 25 26 27 Brachiopoda, based on both morphological and molecular data (Carlson 1995; 28 29 Holmer et al. 1995; Cohen 2000; Cohen and Weydmann 2005). It thus makes no 30 31 32 sense to hypothesize a potential body plan ground pattern based on the development 33 34 of N. anomala, because of the risk to take an apomorphy of a subclade as a character 35 36 for the whole taxon (Wägele 2005). 37 38 39 The attachment area of N. anomala is at the posterior-most tip of the posterior lobe 40 41 (Nielsen 1991). Also the lecithotrophic larvae of rhynchonelliform brachiopods attach 42 43 44 themselves with the posterior-most tip of their pedicle lobe to the substrate at the 45 46 onset of metamorphosis (Altenburger and Wanninger 2009). This pedicle lobe 47 48 49 contains the primordium of the juvenile pedicle that forms after metamorphosis 50 51 (Stricker and Reed 1985a). In the rhynchonelliform larva the dorsal and ventral valves 52 53 are secreted by mantle epithelium that is formed on the underside of the skirt-like 54 55 56 mantle lobe, which is reverted over the apical lobe during metamorphosis (Stricker 57 58 and Reed 1985b, c). In N. anomala no inversion takes place. Here, the dorsal valve is 59 60 61 62 63 64 65 11

secreted from the dorsal mantle after settlement, and there is no ventral valve at this 1 2 stage. In our view the cementing cuticle of N. anomala may represent the same 3 4 5 material that is used to attach the pedicle to the substrate in rhynchonelliform 6 7 brachiopods. Rhynchonelliform brachiopods glue themselves to the substrate by a 8 9 10 sticky secretion for tentative attachment, followed by secretion of a layer of cuticle 11 12 which is secreted between the glued pedicle and substrate for permanent attachment 13 14 15 (Stricker and Reed 1985a; Chuang 1996). From this perspective, the lecithotrophic 16 17 larvae of Rhynchonelliformea and Craniiformea are not that different. Because, if one 18 19 takes the skirt-like mantle that constitutes the mantle lobe of three-lobed 20 21 22 rhynchonelliform larvae away, and consequently the ability to invert the skirt-like 23 24 lobe over the apical lobe during metamorphosis, the remaining larva is bilobed. The 25 26 27 body plan of N. anomala could resemble an early stage rhynchonelliform larva before 28 29 it separates the mantle from the pedicle lobe. In the rhynchonelliform Terebratalia 30 31 32 transversa (Sowerby, 1846), the mantle lobe forms during a second constriction as an 33 34 outgrowth from the pedicle lobe from the dorsal side of the larva and slowly grows 35 36 around both sides of the larva towards the ventral side (Stricker and Reed 1985b; 37 38 39 Freeman 2003). Larva of N. anomala might keep the prospective mantle cells, which 40 41 later also develop the setal sacs on their dorsal side, without developing a mantle lobe. 42 43 44 These considerations lead us to the conclusion that Craniiformea might be a derived 45 46 group within Rhynchonelliformea that has lost the pedicle and the ventral valve in 47 48 49 early postmetamorphic development. This view is supported by the following 50 51 characters uniting the craniiforms and the rhynchonelliforms: 1. Lecithotrophic larvae 52 53 with an attachment area at the posterior-most tip of the larva. 2. Calcitic shells with 54 55 56 punctae (Williams 1997). 3. The gonads are placed within the mantle canals (Popov et 57 58 al. 1993). 4. One pair of oblique muscles is present (Popov et al. 1993). 59 60 61 62 63 64 65 12

The following characters have previously been used to unite the Craniiformea with 1 2 the Linguliformea (to form the clade Inarticulata), but these may more likely represent 3 4 5 apomorphies of the Craniiformea within the Rhynchonelliformea: a secondary 6 7 through-gut at the posterior body wall, and loss of hinge articulation because of loss 8 9 10 of the ventral valve in early postmetamorphic development. The craniiform body plan 11 12 is thus derived from the rhynchonelliform body plan and the Craniiformea may 13 14 15 represent a subclade of the Rhynchonelliformea. A naming of this clade has already 16 17 been proposed as the Calciata (Popov et al. 1993). 18 19 Our observations contribute significantly to the understanding of brachiopod 20 21 22 interrelationships which are still hotly debated, especially with regard to the position 23 24 of Phoronida (Helmkampf et al. 2008; Sperling et al. 2011; Cohen 2013; Cohen and 25 26 27 Bitner 2013). In relation to our findings this is relevant because in a study based on 28 29 nuclear housekeeping genes combined with three ribosomal genes, the craniids 30 31 32 behave as a “rogue taxon” that is placed with little statistical resolution at the base of 33 34 Brachiopoda (Sperling et al. 2011). 35 36 Coming back to the metamorphosis of N. anomala we want to point out a further 37 38 39 important difference to the previously described mechanisms. This concerns the 40 41 ability of maximally contracted curled larvae to metamorphose. Although we 42 43 44 observed curled larvae, these are arrested in development and do not metamorphose. 45 46 While curled larvae have contracted muscles (Fig. 3D), all individuals that 47 48 49 successfully completed metamorphosis in our study (N = 15) showed relaxed, i.e., 50 51 expanded medioventral longitudinal muscles after settlement and prior to resorption 52 53 (Fig. 3E; see also Altenburger and Wanninger 2010 for a general description of the 54 55 56 musculature in N. anomala). Also the anterior-posterior alignment of the gut of the 57 58 adults is incompatible with a “folding” event of the body axis during metamorphosis 59 60 61 62 63 64 65 13

(Williams and Carlson 2007). Another argument is the presence of complete ventral 1 2 and dorsal mesenteries in N. anomala (Chuang 1960). Phoronids, which are ‘folded’ 3 4 5 animals, have developed lateral mesenteries instead (Bailey-Brock and Emig 2000). 6 7 Especially the hypothesis of a “brachiopod fold” put forward by Cohen et al. (2003) 8 9 10 focuses on the “curling” event prior to metamorphosis. However, if one wants to 11 12 make this comparison, the “fold” would be between the anterior and the posterior lobe 13 14 15 of the larva. To arrive at a brachiopod body plan from this folding event, one valve 16 17 had to be secreted from the dorsal anterior lobe and the other one from the dorsal 18 19 posterior lobe. But this is not the case and it was never reported that way. 20 21 22 Accordingly, the larvae do neither fold before nor during metamorphosis, they only 23 24 bend the anterior lobe and the posterior-most tip of the posterior lobe ventrally. Thus, 25 26 27 hypotheses arguing for a “brachiopod fold event” during brachiopod evolution are not 28 29 supported by larval development and metamorphosis of N. anomala and should 30 31 32 therefore be rejected. 33 34 35 36 Acknowledgements 37 38 39 We thank Andreas Schmidt-Rhaesa for helpful comments that significantly improved 40 41 the manuscript. We are grateful to the staff at the Sven Lovén Centre for Marine 42 43 44 Sciences in Kristineberg, Sweden, for providing laboratory space. Gary Wife 45 46 (University of Uppsala) is thanked for assistance with the scanning electron 47 48 49 microscope. Lars Holmer acknowledges support from the Swedish Research Council 50 51 (VR 2009-4395, 2012-1658). 52 53 54 55 56 57 58 59 60 61 62 63 64 65 14

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Figures 1 2 Fig. 1. Sketch drawings of developmental stages of Novocrania anomala. Numbers 3 4 5 indicate the age in hours after fertilization for all stages at 14°C, except for H where it 6 7 represents hours after the onset of metamorphosis. Size of all stages is around 130 µm 8 9 10 in diameter. Anterior is oriented upwards. Cilia were omitted for clarity. A, 11 12 Unfertilized oocyte (black) with fertilization membrane (grey). B, Apical view of a 13 14 15 four cell stage with the egg shell and two polar bodies (small circles). C, Frontal view 16 17 of a gastrula with blastopore (asterisk), ectoderm (ec), and endoderm (en). The 18 19 gastrula starts to swim at this stage. D, Lateral view of an elongated gastrula with 20 21 22 ectoderm (ec) and endoderm (en). The blastopore (asterisk) is situated at the posterior 23 24 pole of the gastrula. E, Dorsal view of an elongated gastrula with anterior lobe (AL). 25 26 27 F, Ventral view of an early two-lobed larva with anterior lobe (AL) and posterior lobe 28 29 (PL). The blastopore is closed at this stage. Larval setae pouch muscles (red circles) 30 31 32 start to develop, and larval setae (se) have started to grow on the posterior side. G, 33 34 Dorsal view of a fully developed larva with anterior lobe (AL), posterior lobe (PL), 35 36 and three pairs of dorsal setal bundles (se) that are moved by fully developed setae 37 38 39 pouch muscles (red circles). The setae pouch muscles are ventrally interconnected by 40 41 two strands of medioventral longitudinal muscles (red longitudinal bars). The 42 43 44 medioventral longitudinal muscles are interconnected by three bundles of transversal 45 46 muscles (red transversal bars). See also Fig. 1 in Altenburger and Wanninger 2010 for 47 48 49 the original data on myoanatomy. H, Ventral view of a juvenile after metamorphosis. 50 51 The larval anterior lobe (AL) and posterior lobe (PL) are still visible. The juvenile 52 53 valve (v) is formed on the dorsal side with larval setae (se) extending from it. The 54 55 56 larval setae are pushed outwards by the juvenile valve and the larval musculature is 57 58 resorpt. 59 60 61 62 63 64 65 24

1 2 Fig. 2. Scanning electron micrographs of Novocrania anomala developmental stages. 3 4 5 Anterior is oriented upwards, scale bars equal 25µm, numbers in the upper right 6 7 corner of each specimen indicate the developmental time in hours after fertilization 8 9 10 (A-H) or hours after settlement (I) at 14°C. A, Four cell stage. B, Eight cell stage with 11 12 polar bodies and radial cleavage pattern. C, Coeloblastula stage. D, Lateral view of a 13 14 15 spherical gastrula with blastopore (arrowhead). E, Ventral view of a wedge-shaped 16 17 gastrula with blastopore (arrowhead). F, Ventral view of a wedge-shaped gastrula 18 19 with first anlage of the anterior lobe (AL) and almost closed blastopore (arrowhead). 20 21 22 G, Dorsolateral view of an early larva with anterior lobe (AL) and posterior lobe (PL). 23 24 Three sets of larval setal bundles (se) start to grow on the dorsal side of the posterior 25 26 27 lobe. H, Ventral view of a fully developed larva with anterior lobe (AL), posterior 28 29 lobe (PL), and three pairs of setal bundles (se). I, Ventral view of a settled juvenile 30 31 32 with dorsal valve (v) seen from ventral, anterior lobe (AL), posterior lobe (PL), and 33 34 the attachment area on the substrate (arrow). 35 36 37 38 39 Fig. 3. The musculature in larvae of Novocrania anomala prior to and after 40 41 metamorphosis. Anterior is up in all micrographs and scale bars equal 25µm. A-C, 42 43 44 scanning electron micrographs. A, Dorsolateral view of a folded larva as described by 45 46 Nielsen (1991) with anterior lobe (AL), posterior lobe (PL), and three pairs of setal 47 48 49 bundles (se). This folded larva is not able to metamorphose. B, Dorsal view of a 50 51 settled juvenile with dorsal valve (v) and remaining larval setae (se). C, Ventral view 52 53 of a settled juvenile with dorsal valve (v), anterior lobe (AL), posterior lobe (PL), and 54 55 56 attachment area on the posterior lobe (arrow). D-E, Confocal micrographs of a larva 57 58 (D) and a juvenile (E), corresponding to the stages shown in A and B. Muscles appear 59 60 61 62 63 64 65 25

red in the micrographs. D, Ventrolateral view of a folded larva corresponding to the 1 2 stage shown in A with anterior lobe (AL), posterior lobe (PL), and strongly contracted 3 4 5 musculature (empty arrow). In healthy specimens, the larval musculature is elongated 6 7 between anterior and posterior lobe. E, Ventral view of a settled juvenile with dorsal 8 9 10 valve (v) and elongated larval longitudinal musculature (empty arrows). The larval 11 12 lobes, i.e., the anterior lobe (AL) and the posterior lobe (PL), are still recognizable 13 14 15 under the valve. F, Light micrograph of a settled juvenile with remaining larval setae 16 17 (se), dorsal valve (v), anterior lobe that is remodeled to form the lophophore (AL/Lo), 18 19 and remnants of the posterior lobe (PL). 20 21 22 23 24 Fig. 4. Larvae of Novocrania anomala during metamorphosis. A-C, early stage just 25 26 27 after settlement seen from dorsolateral (A), lateral (B), and ventrolateral (C) with 28 29 first-formed coat (c) on the dorsal posterior lobe, three pairs of dorsal setal bundles 30 31 32 (se), anterior lobe (AL), and posterior lobe (PL). The first-formed coat (c) is the first 33 34 material that is simultaneously secreted by the epithelial cells situated on the dorsal 35 36 side of the posterior lobe. D-F, Later stage seen from dorsolateral (D), lateral (E), and 37 38 39 ventral (F) with one pair of anterior setae bundles (se), first-formed valve (fv), dorsal 40 41 mantle (m), anterior lobe (AL), and posterior lobe (PL). Note that the posterior setae 42 43 44 bundles have been shed at this stage and the left part of the ventral side in F was 45 46 slightly damaged during preparation of the specimen. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image