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. 2021 Dec 20;240(6):1034–1047. doi: 10.1111/joa.13617

The evolutionary relationship between arm vertebrae shape and ecological lifestyle in brittle stars (Echinodermata: Ophiuroidea)

Mona Goharimanesh 1,2,✉, Fereshteh Ghassemzadeh 1, Barbara De Kegel 2, Luc Van Hoorebeke 3, Sabine Stöhr 4, Omid Mirshamsi 1, Dominique Adriaens 2
PMCID: PMC9119616  PMID: 34929059

Abstract

Ophiuroidea are one of the most diverse classes among extant echinoderms, characterized by their flexible arms composed of a series of ossicles called vertebrae, articulating with each other proximally and distally. Their arms show a wide range of motion, important for feeding and locomotion, associated with their epizoic and non‐epizoic lifestyles. It remains to be explored to what degree the phenotypic variation in these ossicles also reflects adaptations to these lifestyles, rather than only their phylogenetic affinity. In this study, we analyzed the 3D shape variation of six arm vertebrae from the middle and distal parts of an arm in 12 species, belonging to the intertidal, subtidal and bathyal zones and showing epizoic and non‐epizoic behaviors. A PERMANOVA indicated a significant difference in ossicle morphology between species and between lifestyles. A principal component analysis showed that the morphology of epizoic ophiuroids is distinct from non‐epizoic ones; which may reflect variation in arm function related to these different lifestyles. The Phylogenetic MANOVA and phylogenetic signal analysis showed that shape variation in the vertebral articulation seems to reflect ecological and functional adaptations, whereas phylogeny controls more the lateral morphology of the vertebrae. This suggests a convergent evolution through ecological adaptation to some degree, indicating that some of these characters may have limited taxonomic value.

Keywords: convergent evolution, CT scan, geometric morphometric, ossicles, prehensility

It is the first study to link morphological characteristics to lifestyle using 3D shape quantifications (micro CT) and analyses (geometric morphometrics) on ophiuroid vertebrae. It shows that characteristics related to the aboral process and lateral vertebrae are essential for phylogenetic interpretations, while dorsal and central articular structures may have limited taxonomic value, as they seem to reflect adaptations to distinct functional/ecological lifestyles.

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1. INTRODUCTION

Ophiuroidea represent the most species rich class of echinoderms, occupying various intertidal to subtidal marine habitats (O'Hara et al., 2017; Smith et al., 1995). They hide in/under rocks or live on the seafloor, as well as on corals, sponges and artificial structures (Boissin et al., 2016; de Castro Manso et al., 2018; Fujita & Ohta, 1990). Their arms play a crucial role in locomotion and feeding (Magnus, 1967; Pentreath, 1970), as they are used to grasp food, attach to other organisms or to crawl over various substrates (Clark et al., 2019; Jangoux, 1982). The arms are composed of a series of ossicles (called vertebrae), articulating with each other proximally and distally (LeClair, 1996). Ophiuroid arms grow from the distal tip proximalwards, with new vertebrae added proximal to the terminal plate, which means that distal segments are younger and less developed than proximal ones (Stöhr, 2005; Sumida et al., 1998). Distal vertebrae are also smaller, with less defined articulating structures (Stöhr & Martynov, 2016).

Two main types of vertebral articulations were recognized by Mortensen (1927), i.e. streptospondylous vs. zygospondylous articulation (referring to the articular structures on the distal face of a vertebra), based on which the class Ophiuroidea was organized into two orders: the Euryalida and Ophiurida. Recently, the classification of Ophiuroidea was revised and arm vertebrae are no longer used as the main character for the delimitation of higher level taxa. The Euryalida and Ophiurida now belong to the superorder Euryophiurida (O'Hara et al., 2018). The Euryalida currently includes three families, where all species possess streptospondylous vertebrae, whereas the Ophiurida consists of five families (Goharimanesh et al., 2021; O'Hara et al., 2017) with zygospondylous vertebrae. Within the superorder Ophintegrida, a variety of streptospondylous, zygospondylous and intermediate types can be found (O'Hara et al., 2017, 2018). Both the zygospondylous and streptospondylous vertebrae have distinct zygocondyles, but with the zygosphene—a ventral articulatory peg on the distal side—being absent in the streptospondylous type (Figure 1) (Hotchkiss & Glass, 2012; Hotchkiss et al., 2007). In general, non‐euryalid ophiuroids (clades Ophiurida and Ophintegrida), encompassing over 90% of all ophiuroid species, are taxonomically and ecologically more diverse than the euryalid ones (LeClair, 1996; LeClair & LaBarbera, 1997; O'Hara et al., 2017; Stöhr et al. 2012; Warner, 1982). This could be related to the variability of the zygospondylous articular structure on the vertebrae (Byrne et al, 1994; Hendler & Miller, 1991), with the hourglass shape being simpler than the interlocking zygospondylous ones (LeClair, 1994; LeClair & LaBarbera, 1997). About 60 years later, Litvinova (1989a) identified five secondary divisions of vertebral articulations (referring to the articular structures on the proximal face of a vertebra). They represent an evolutionary gradient from an ancestral knob‐shaped type (undeveloped articulating knobs and depressions—see figure 1 in Litvinova [1994]) to a more universal zygospondylous, hourglass‐shaped streptospondylous, comb‐shaped zygospondylous and an aberrant type. Each shape has been assumed to be associated with arm motion in different directions, with either horizontal or vertical restrictions (Litvinova, 1994). Within‐family variation in the morphology of the proximal and distal articulatory faces exists as well, and has been linked to ecological and functional adaptations (Stöhr, 2012). For instance, in some families (e.g., Ophiotrichidae) epizoic taxa, habitually attaching to other surfaces, have vertebrae that closely resemble the euryalid streptospondylous shape (Alitto et al., 2020; Litvinova, 1989a), erroneously termed transspondylous by Alitto et al. (2020), which however is a morphologically different type limited to the genus Ophiosmilax Matsumoto, 1915 (Štorc, 2004). We propose the term ‘semi‐streptospondylous’ instead.

FIGURE 1.

FIGURE 1

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(a) Anatomy of the vertebra in Ophiocoma scolopendrina (terminology partially adopted from Hotchkiss et al. [2007]). (b) Schematic images of vertebral articular structures; zygospondylous based on Bray (1985), semi‐streptospondylous based on the Ophiothela image in Alitto et al. (2020) (here renamed semi‐streptospondylous due to its different morphology from transspondylous), streptospondylus (euryalid) based on Mortensen (1933) and transspondylous based on Štorc (2004)

Considerable attention has been paid to the vertebral shape, but on a limited number of taxa and usually in a taxonomic context (Alitto et al., 2018, 2020; Fujita, 2003; Hotchkiss & Glass, 2012; Hotchkiss et al., 2007; Matsumoto, 1917; O'Hara et al., 2018; Smith et al., 1995). However, qualitative descriptions alone face limitations that hinder a detailed study of vertebral morphology across taxa. Morphometric analyses on the two‐dimensional vertebral shape showed inter‐ and intra‐specific differences in several zygospondylous (non‐euryalid) ophiuroids, indicating that the ossicles become narrower and longer along the proximal‐distal axis of the arm (LeClair, 1996). Moreover, species could be grouped mainly based on the absence/presence of the keel structure (i.e. “extended keel” as defined in Goharimanesh, et al. [2021]) on the distal dorsal face of the vertebrae, a structure of which the functional role is not known (LeClair, 1996). Besides, despite the important role of prehensility (grasping an object partly or wholly with combined ventral and lateral flexion) for balance, feeding, and locomotion (Organ, 2010), the morphological differences between prehensile and non‐prehensile ophiuroid arms are not yet fully understood.

Studies on ophiuroid vertebrae have so far mainly relied on two‐dimensional shape, leaving ossicle shape variation partially unexplored. Scanning electron microscope (SEM) imaging provides more detailed 2D images for shape analysis than light microscopy but requires destructive physical dissection and chemical preparation. Micro CT (µCT) has gained much attention as a non‐destructive method to generate three‐dimensional images of brittle stars and their ossicles (Clark et al., 2018; Okanishi et al., 2017; Stöhr et al., 2019; Tomholt et al., 2020). However, to date, no µCT data have been used for a 3D geometric morphometric (GM) analysis on ophiuroids. As Stöhr et al. (2019) recently highlighted, an in‐depth quantitative analysis, such as geometric morphometrics on vertebrae, is required to investigate their variation in relation to their lifestyle and phylogenetic context.

This is the first study to provide a 3D GM investigation of vertebral variation in relation to different functional and ecological aspects of ophiuroid lifestyles. In addition, compared with the earlier 3D studies on ophiuroids (Clark et al., 2018; Okanishi et al., 2017; Stöhr et al., 2019; Tomholt et al., 2020) a larger sample size (n = 12 species) was included in this study. Two hypotheses were tested, focusing on static and evolutionary allometry, which follow LeClair (1996): 1. Intra‐individually, the vertebrae show a shape variation along the proximal‐distal axis of the arm (static allometry) in a way that the distal parts show structural adaptations that allow an increased flexibility. 2. Vertebrae differ among species, where the main axis of variation combines traits associated with their phylogenetic and lifestyle affinities, such as prehensility (evolutionary allometry).

2. MATERIALS AND METHODS

2.1. Specimens

In this study, the species were selected to cover a wide phylogenetic range, as well as different functional lifestyles related to different use of their arms (prehensile and none‐prehensile). In addition, some species represented a close sister‐group relationship, which allowed us to test how similarities in morphology reflect lifestyle versus phylogenetic affinity. Species were categorized according to their ecological lifestyle (epizoic, endozoic, epiphytic, or free‐living) and according to their vertebral articular type (streptospondylous, semi‐streptospondylous or zygospondylous).

At least one specimen per species was used, belonging to seven families distributed across the superorders Ophintegrida and Euryophiurida (Figure 2). Specimens were collected from the Persian Gulf/Oman Sea (by M.G.) during December 2017–March 2018 and were obtained from the collection of the Swedish Museum of Natural History (SMNH). The following species are included: (1) Ophiotrichidae: Macrophiothrix hirsuta (Müller & Troschel, 1842), Ophiothrix (Ophiothrix) savignyi (Müller & Troschel, 1842), Ophiothela venusta (de Loriol, 1900), Ophiothela sp. [danae?] Verrill, 1867; (2) Ophiactidae: Ophiactis savignyi (Müller & Troschel, 1842), Ophiactis modesta Brock, 1888; (3) Ophionereididae: Ophionereis dubia (Müller & Troschel, 1842); (4) Ophiocamacidae: Ophiocamax vitrea Lyman, 1878; (5) Ophiocomidae: Ophiocoma scolopendrina (Lamarck, 1816), Ophiocomella sexradia (Duncan, 1887); (6) Ophiuridae: Ophiura sarsii Lütken, 1856; and (7) Asteronychidae: Asteronyx loveni Müller & Troschel, 1842.

FIGURE 2.

FIGURE 2

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Phylogenetic position of the 12 species used in this study, their vertebral morphology (left is distal view, right is proximal view), and corresponding articular type mentioned in the gray and yellow boxes with respect to distal and proximal views, respectively. Species highlighted in pink, yellow and green are epizoic, endozoic and epiphytic species, respectively

In addition to personal field observations, information on habitats and lifestyle was gathered from the literature (Table 1). In this study, in accordance with previous studies (presented in Table 1), prehensile species were also reported as epizoic. For Ophiothrix savignyi and Ophiactis savignyi, which are either epizoic/free‐living or epizoic/endozoic, respectively (Figure 2), no information on whether they are prehensile or not could be found. Not much is known about their epizoic behavior either. In this regard, we first classified them as belonging to the “unknown” group and imported the data to perform a linear discriminant analysis. The robustness of the analysis was checked by a classifier analysis with Jackknife randomization, followed by a confusion matrix analysis. As a result, it showed a close relationship of these unknown species to the non‐prehensile rather than the prehensile group. Therefore, due to the lack of information regarding prehensility and vague information of being epizoic, we treated these two species as non‐prehensile in our analysis. We do clarify that non‐prehensile species are not necessarily assumed as being non‐epizoic and also not all epizoic species are necessarily referred to the prehensile group.

TABLE 1.

Information on habitat and lifestyle of the studied species. The articular structures on each vertebra are S = Streptospondylous, Semi‐S = Semi‐streptospondylous and Z = Zygospondylous. Fissiparous = asexual reproduction by fission (dividing) followed by regeneration. The species are classified as prehensile (or not) based on an analysis of literature descriptions, images and personal observation

Species Vertebra Ecological lifestyle Host and habitat Functional lifestyle References
Asteronyx loveni S Epizoic On gorgonians (Radicipes spp.) and pennatulids (Funiculina quadrangularis and Anthoptilum sp.), bathyal zone Prehensile Fujita and Ohta (1988)
Ophiothela venusta Semi‐S Epizoic Coils around gorgonians (Euplexaura sp., Echinomuricea sp. and solenocaulon sp.), bathyal zone Prehensile Price (1981), Goh and Chou (1994), Peyghan et al. (2018)
Ophiothela danae fissiparous, hexamerous Semi‐S Epizoic On macroalgae, soft and hard corals (Solenocaulon sp. and Millepora sp.), sponges and holothurians (Thelenota ananas), bathyal zone Prehensile Goh and Chou (1994), Price and Rowe (1996), Sastry (2001)
Ophiothrix savignyi Z Free‐living/epizoic Among sessile macroinvertebrate colonies, especially sponges such as Aplysinella rhax. Bottoms of subtidal rock, subtidal sand, mixed subtidal rock, and sand Non‐Prehensile Price (1981) George (2012), Sampey and Marsh (2015)
Ophiactis savignyi fissiparous, hexamerous Z Endozoic/epizoic Inside sponge canals (Spirastrella inconsfans and Spheciospongia), on sponges, corals (Stylocoeniella guentheri) or artificial structures. Rigid substrates in subtidal areas Non‐Prehensile Price (1981), James (1982), Sampey and Marsh (2015), Boissin et al. (2016)
Ophiactis modesta fissiparous, hexamerous Z Endozoic Inside sponge canals (Spirastrella inconsfans and Spheciospongia). Rigid substrates in subtidal areas Non‐Prehensile Price (1981), James (1982), Sampey and Marsh (2015)
Ophiocomella sexradia fissiparous, hexamerous Z Epiphytic

Associated with algae (Caulerpa sp.)

Subtidal areas, on hard substrates

 Non‐Prehensile James (1982), Sampey and Marsh (2015), Boissin et al. (2016)
Ophiura sarsii Z Free‐living Bathyal zone, muddy sediment and covering large areas of the seafloor Non‐Prehensile Fujita and Ohta (1990)
Ophiocamax vitrea S Free‐living Recorded in deep water to 130 m (Davis, 1972), but details of its habitat are still scarce. According to Lyman (1878), they live in muddy habitats, seemingly similar to that of O. sarsii Non‐Prehensile Lyman (1878) and Davis (1972)
Ophionereis dubia Z Free‐living Hard inter‐ and subtidal rock and coral reefs, and soft subtidal grass beds Non‐Prehensile Price (1981), Sampey and Marsh (2015)
Ophiocoma scolopendrina Z Free‐living Under rocks and coral rubble in shallow water of the upper intertidal zone Non‐Prehensile Personal observation
Macrophiothrix hirsuta Z Free‐living Intertidal sand/mudflats, space/crevices between rocks/pieces of coral rubble and underlying substrate, among sponges Non‐Prehensile Perosnal observation and Hoggett (1990)
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2.2. Visualization and segmentation

To allow for a higher magnification and thus obtain a better voxel resolution, segments from the middle and distal part of one arm per individual brittle star were cut out for CT scanning. In order to compare static allometry along the arm, we segmented the three proximal‐most vertebrae close to the middle part of the arm (referred to as proximal 1–3) to show the morphology of the keeled vertebrae, where present (following LeClair, 1996) and three others located proximal to the tenth most distal vertebrae (referred to as distal 1–3), thereby avoiding using newly formed and undeveloped vertebrae, since arms grow from their distalmost tip proximalwards (n = 6 ossicles per individual). Proximal 1 and distal 1 refer to the most proximal and the most distal vertebrae of each set of three vertebrae, respectively, and thus represent the most distantly positioned vertebrae. All specimens were μCT‐scanned at the center for X‐ray Tomography at Ghent University (UGCT—www.ugent.be/we/ugct/) using the HECTOR scanner (Masschaele et al., 2013). In this procedure, a voltage of 80 or 100 kV was used and 1400–1500 projections over 360o were recorded per scan. The pixel pitch of the detector was 400 μm and the resulting voxel sizes ranged between 7.26 and 21.35 μm (Table 2). The vertebrae were digitally segmented from the µCT slices and 3D meshes were generated using the software package Amira (Thermo Fisher Scientific v. 6). The obtained 3D‐surface of each individual vertebra was then converted into ply‐files, which were used for digitizing 3D anatomical point landmarks using Stratovan Checkpoint (v. 2020.02.05).

TABLE 2.

The parameters of μ‐CT scanning of the ophiuroid species studied

Species Number of projections Source voltage (kv) Voxel size (μm)
Ophiura sarsii 1501 100 7.26
Asteronyx loveni 1501 100 13.96
Ophiocamax vitrea 1501 100 9.01
Ophiothela venusta 1501 100 8.2
Macrophiothrix hirsuta 1501 100 12.97
Ophiocoma scolopendrina 1401 80 13
Ophiothela sp.[danae] 1501 100 21.35
Ophiactis savignyi 1501 100 15.04
Ophiactis modesta 1501 100 9.82
Ophiothrix savignyi 1501 100 13
Ophiocomella sexradia 1501 100 13
Ophionereis dubia 1501 100 13
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2.3. Morphometrics

Landmarks on the proximal face of the vertebrae were placed following LeClair (1994, 1996). For the distal, dorsal, and ventral aspects of the vertebrae, additional landmarks were added on homologous structures that could be identified in each individual brittle star. In total, 37 landmarks were put on each vertebra (Figure 3). On the dorsal face of the vertebrae, landmarks were positioned at the median dorsal groove (1–2 & 19) and at a v‐shaped depression beneath them (3–4), the median groove on the dorso‐proximal projection (17–18 & 20) and the projection on the dorso‐distal muscular fossae (29–30). On the distal side, landmarks were set on the distal projection above the zygosphene (5) and the zygocondyles (6–7). On the proximal side, landmarks were put on the distal tip of the epanapophyzes and zygapophyzes (34–37). On the ventral side, landmarks were placed at the ambulacral groove (8–10 distally, 13–14 & 33 proximally), and the projection and depression at the ventro‐distal muscular fossae (11–12 & 23–24). Additional landmarks were put on the depressions and projections on the proximal muscular fossae (15–16, 25–28), the dorso‐distal muscular fossae (31–32), and the lateral projection (21–22).

FIGURE 3.

FIGURE 3

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3D Landmark positions (yellow dots) on the vertebra of Ophiocoma scolopendrina. Left and right show oblique proximal and distal views, respectively

The acquired landmark coordinates were exported as Morphologica‐format and imported in MorphoJ 107a (Klingenberg, 2011). The data were treated as symmetrical 3D objects. A Generalized Procrustes Analysis (GPA) was performed to extract relevant shape data by removing the information of size, location, and orientation and to calculate the mean shape configuration. For the allometry tests, the centroid size was used, as it is the most comprehensive measure of the size commonly used in geometric morphometrics. Wireframes to visualize shape variation explained by each principal component were extracted using MorphoJ.

2.4. Multivariate analysis

A principal component analysis (PCA) was done on the variance‐covariance matrix of the obtained Procrustes coordinates using R (v. 1.1.463) to check the distribution of the species within morphospace. The phylogenetic tree (extracted from Christodoulou et al. [2019]) was plotted on the PCA morphospace using the “phylomorphospace” function in R. The PCA on 111 Procrustes coordinates reduced the variables to at least 71 dimensions as the most relevant ones for further analysis. Based on a scree plot with a broken stick analysis, we retained PC1–6 that cumulatively explained 80.3% of the variation. A multivariate normality test using the “mshapiro.test” in R showed that these six PCs were not normally distributed and a “LeveneTest” showed an unequal variance within the PC scores across species (p < 0.01) and lifestyle groups ([prehensile or none‐prehensile, p = 0.03) and (epizoic, endozoic, epiphytic or free‐living, p < 0.01]). We thus performed a nonparametric PERMANOVA on the PC scores for all vertebrae to test for differences between all species, between functional lifestyles (prehensile and non‐prehensile species), and between ecological lifestyles on how they cling onto or in other organisms (epizoic, endozoic, epiphytic, or free‐living). Then, a Chi‐Square test was used to check whether the differences in vertebral morphology are significantly correlated to the differences in lifestyles (ecological and functional). To test whether size and shape are correlated, the PC scores (PC 1–6) were regressed on centroid size, and a Pearson correlation analysis was done on all PC scores and centroid size, to identify which PC correlated most with centroid size (done in R). To correct the observed shape variation for phylogenetic affinity (Polly et al., 2013), a Phylogenetic MANOVA was performed on PC 1–6 combined, as well as for each PC separately to test whether functionality groups significantly differ after controlling for phylogeny. The phylogenetic tree of Christodoulou et al. (2019) was pruned toward the selected species of our study using the library “ape” in R. The Phylogenetic MANOVA was conducted on the average vertebrae shape per species using the “Geiger” and “Phytool” packages, with specimens classified as ‘prehensile (epizoic)’ and ‘non‐prehensile’. To explore the phylogenetic signal from a set of quantitative traits (PCA scores), we used the function “Phylosig” in R to calculate K after Blomberg et al. (2003) and λ after Pagel (1999). Bloomberg's K shows if there is a resemblance among closely related species under Brownian motion models of evolution (random walk), values of K range from 0 = no phylogenetic signal in the trait to infinity (K ≥ 1, strong phylogenetic signal). Lambda is a tree transformation that ensures the best fit of traits to a BM model. When the value = 0, the internal branches are removed, making the tree like a complete polytomy and thus there is no more resemblance among closely related species on average than between distant relatives (i.e. the trait does not contribute to the phylogenetic affinities). It ranges from zero (=no phylogenetic signal) in the trait to one (=strong phylogenetic signal) (Kamilar & Natalie, 2013; Paradis, 2014). In both tests, the null hypothesis assumes no phylogenetic signal among traits within all species (further information regarding the analysis can be found in Münkemüller et al. [2012] and Kamilar and Natalie [2013]).

3. RESULTS

The PCA on the Procrustes coordinates (n = 72 vertebrae) shows that PC1‐3 (explaining 65.5% of the total shape variation) could explain the major differences between the species (Figure 4). Thus, shape variation explained by PC4‐6 will not be described here. The majority of the shape variation explained by PC1 is localized in the dorsal and ventral part of the vertebrae (e.g., dorso‐proximal process and ambulacral groove), as well as the oral and aboral muscular fossae and the proximal articular structures. PC2 mainly represents variation in the dorsal part of the vertebrae and oral muscular fossae. PC3 represents variation in the aboral muscular fossae and the ventral and dorsal part of the vertebrae, as well as the distal articular structure (Figures 4 and 5).

FIGURE 4.

FIGURE 4

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(a) Plot of PC1–PC; and (b) PC2–PC3 for distal and proximal vertebrae of the 12 species; (c) their phylogenetic relationships superimposed on PC1–2. Colors reflect the best documented ecological lifestyle as indicated in Figure 1, and each point on plots a and b represents a single vertebra (the three distal and three proximal vertebrae have the lower and higher PC1 scores per species, respectively). Each line color on the phylogenetic PCA plot c represents the following phylogenetic clades (order): Green, Amphilepidida; Blue, Ophiacanthida; Purple, Ophiurida; and Black, Euryalida

FIGURE 5.

FIGURE 5

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Wireframes illustrating shape variation explained by PC1, PC2, and PC3, for the dorsal (top), lateral (middle) and frontal view (below). In both dorsal and lateral view the proximal face is on the left and in frontal view the dorsal face is up. Light blue wireframes show the consensus shape of all vertebrae; dark blue lines indicate the shape that corresponds to a PC‐score of 0.2

PC1 illustrates proximo‐distal variation both at an intra‐ and interspecific level (Figure 4a). Based on Figure 5, higher PC1 scores correspond to proximal vertebrae that are shorter in length but higher in overall shape, and longer at the ventro‐proximal, distal (landmarks 11, 12, 13 and 14) and lateral faces (landmarks 21, 22, 25, and 26), as well as the dorsal process (17, 18, 1, and 2).

PC1‐scores of proximal vertebrae are similar in A. loveni, O. venusta, M. hirsuta, Ophiactis savignyi and O. vitrea (Figure 4a), in which aboral and oral muscular projection is a prominent character, being wider compared with other species. The distal vertebrae are represented by lower PC1 scores in general, though distal vertebrae of O. vitrea have similar scores as the proximal vertebrae of O. sarsii (Figure 4a). In addition, the distal vertebrae in A. loveni, widely reported as a prehensile species (Fujita & Ohta, 1988; Manso, 2010), are also similar to the proximal vertebrae of Ophiothrix savignyi (Figure 4a). This similarity in PC1 scores shows the morphological similarities at the level of length versus width and height of distal vertebrae in species that show spiral twisting with the arms (such as A. loveni and O. vitrea), with the proximal vertebrae of species with and without spiral flexion (Figures 4 and 5).

PC2, rather than other PCs, shows a stronger correlation with centroid size (cor = 0.61, p < 0.01). Higher PC2 scores correspond to larger vertebrae that have a longer process at the dorsal projection (landmarks 17 & 18) bordering a narrower dorsal groove. Moreover, the muscular fossae on both proximal and distal faces get deeper, suggesting the insertion of larger muscles. The two epizoic taxa (Ophiothela venusta and Ophiothela sp.) cluster together along PC2 and also lie close to the other two species from the same family. Interestingly, the intertidal non‐epizoic M. hirsuta and the bathyal epizoic A. loveni, despite having a distinct phylogeny and lifestyle, share long arms and cluster in the morphospace. Also the non‐epizoic/endozoic/epiphytic (free‐living) subtidal/bathyal species (O. sarsii and Ophiocamax vitrea) cluster in this morphospace, as do the species belonging to the Ophiactidae and Ophiocomidae (Figure 4).

For PC3, positive scores represent less depressed dorsal and ventral muscular fossae on the proximal and distal faces of the vertebrae, as well as a shorter dorso‐distal projection with a narrower middle groove (landmarks 17, 18, and 20). PC3 also reflects a proximo‐distal pattern, in which distal vertebrae have higher scores than the proximal counterparts (Figure 4b). The articular structures on both faces are more projected. Asteronyx loveni shows a distinct position, having the highest PC3 scores.

In PCs 1–3, the three species with six arms (Ophiactis savignyi, O. modesta, and O. sexradia) are also similar in their vertebral shape (Figure 4). They differ from O. scolopendrina, belonging to the same family as O. sexradia (Ophiocomidae), as they have wider vertebrae with a substantially extended ventro‐distal process.

The studied species did not cluster according to their subtidal/intertidal/bathyal lifestyle and hard/soft habitat on the morphospace. The group along PC1 based on being epizoic or not, whereas differentiation between endozoic and epiphytic ones is explained by PC2. For PCs 1–3, prehensile species cluster together.

The PERMANOVA on the scores of the first six PCs (all six vertebrae included) shows a significant difference between species and also among both functional and ecological groups (p < 0.01). The result of the Chi‐Square test on vertebrae articular morphology (zygospondylous, semi‐streptospondylous and streptospondylous) and lifestyles (ecological and functional) was significant (p < 0.05), suggesting to reject the null hypotheses that vertebral type and ecological/functional lifestyle are independent. The allometry test indicated a weak positive relationship between centroid size and shape (R 2 = 0.32, p < 0.01). Also the Phylogenetic MANOVA reports a significant difference in shape variation of the most distal vertebra and all proximal vertebrae for all six PCs combined within the functionality lifestyle, irrespective of phylogeny. Especially PC2 and PC3 explained a significant difference between functional groups in the proximal vertebrae (Table 3) when their phylogeny was included, indicating that shape differences cannot be entirely explained by phylogeny alone.

TABLE 3.

Summary of the Phylogenetic MANOVA, showing p‐values (bold numbers indicate significant differences). Proximal 1 and distal 1 refer to the most proximal and the most distal vertebrae of each set of three vertebrae, respectively

Vertebrae PC (1–6) PC1 PC2 PC3 PC4 PC5 PC6
Proximal 1 0.02 0.9 0.05 0.01 0.6 0.8 0.7
Proximal 2 0.01 0.8 0.04 0.02 0.1 0.7 0.5
Proximal 3 0.007 0.8 0.03 0.01 0.1 0.6 0.6
Distal 1 0.04 0.1 0.06 0.09 0.8 0.8 0.4
Distal 2 0.1 0.05 0.07 0.1 0.9 0.9 0.2
Distal 3 0.1 0.07 0.31 0.2 0.9 0.7 0.5
Total (average) 0.008 0.2 0.04 0.04 0.6 0.7 0.5
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The phylogenetic signal in Table 4 shows that PC2 and PC3 mainly reflect phylogenetic relatedness in the shape variation, with significant and high Pagel's lambda values (0.99, p < 0.01). Blomberg's K, which allows comparisons of the amount of phylogenetic signal across traits and trees, similar to Pagel's lambda, shows a higher number (K > 1) for PC2 (p < 0.05, 1000 randomizations). So, our analysis shows that the traits controlling PCs 2–3 have both ecological signals and a phylogenetic one.

TABLE 4.

The summary of Phylogenetic signal (λ and K values) along PC1–6 using average PC values of total vertebrae (bold numbers indicate significant p‐values and high values of λ and K)

PC1 PC2 PC3 PC4 PC5 PC6
Pagel's (1999) λ 6.61e‐05 0.99 0.99 6.61e‐05 0.88 7.25e‐05
p‐value 1 0.001 0.008 1 0.4 1
Blomberg et al.'s (2003) K 0.18 1.4 0.7 0.3 0.4 0.2
p‐value 0.6 0.003 0.01 0.2 0.1 0.3
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4. DISCUSSION

Variation in 3D shape variability in the arm vertebrae supports our hypotheses with respect to proximo‐distal patterns and differences between species. In addition, the results show how the interspecific differences reflect either phylogenetic relationships or ecological and functional adaptations, such as being able to grasp around objects and other organisms. It should be noted that the inferences on functionality are based on (1) assumptions made in literature (Table 1), (2) observed pattern of shape variation, and (3) variation in relation to lifestyle but contrasted against phylogeny. The observed morphological variation along the proximo‐distal axis is consistent with those reported in LeClair (1996) and Clark et al. (2018), and the observed interspecific differences agreed with earlier morphometric analysis (LeClair, 1996).

The disparity between the proximo‐distal vertebrae confirmed the regional shape variation along the arm in the species studied (Figure 4a). LeClair (1996) reported that the vertebrae become smaller in size along the proximal to distal axis, which is also in line with our finding (shape changes represented by PC1—Figure 5). Yet, proximal vertebrae are characterized by a general broadening of the ventral muscular fossae, irrespective of their size (Figures 4 and 5). Proximal vertebrae are also wider and higher but shorter in length (Figures 4 and 5), which seems an evolutionary convergent trait as it is even observed in the tail of prehensile‐tailed vertebrates, such as chameleons and New World monkeys (Luger et al., 2020; Organ & Lemelin, 2011). Based on Organ and Lemelin (2009) and Organ (2010), shorter vertebrae with a higher aspect ratio (the ratio of width to length at the proximal end of the vertebra) and larger articular process can increase the robustness and stiffness at the proximal region of the tail in New World monkeys.

Convergently, as in those vertebrates, this tentatively supports our hypothesis that it is the distal part showing structural adaptations, which may increase flexibility by having smaller articular structures. The distal vertebrae have their articulation point at a larger distance from the insertion point of the muscles, which allows a relatively larger output force in this lever system (Glase et al., 1981). Consequently, despite a smaller insertion area for the muscles, the lever configuration contributes to a more efficient generation of output force. In this regard, our results show that the non‐prehensile O. sarsii has longer distal vertebrae than non‐prehensile O. vitrea and all prehensile species of the current study. According to Glase et al. (1981), longer vertebrae create a larger output force through the lever system, which can be essential for a hunting behavior, already reported for O. sarsii (Stancyk, 1998). However, further analysis of the musculoskeletal anatomy and functional performance is required to investigate the relation between the observed traits and actual prehension performance.

Based on the result there is a correlation between vertebrae articular morphology and ecological/functional lifestyles. The articular morphology is assumed to play a role in the arm's mechanical limits and, consequently, the organism's behavioral use of its arms and its associated lifestyle (Byrne et al., 1994; Hendler & Miller, 1991; Litvinova, 1989a, 1989b,1989a, 1989b). Based on Litvinova (1989a), O. sarsii has comb‐shaped zygospondylous vertebrae, which is similar to the zygospondylous articulations of M. hirsuta. However, contrary to M. hirsuta being a filter feeder, O. sarsii is an active predator (Harris et al., 2009). We propose that the articulation of O. sarsii is universal zygospondylous (it has been hypothesized to flex in different directions), instead of being comb‐shaped zygospondylous (only horizontal flexion), as reported by Litvinova (1989a). This could be supported not only by the shape of the vertebrae being similar to that of the universal type (Figure 2) but also because of the considerable flexion in different directions suited for their feeding behavior as reported in Stancyk (1998). In fact, it is still debatable whether O. sarsii has a comb‐shape zygospondylous articular structure with limited flexion, due to its various feeding modes, such as capturing live fish, squid, and krill by trapping them in an arm loop or by several ophiuroids together jumping (vertical flexions) onto larger prey to hunt (Stancyk, 1998). Thereby, it is suggested that not only the length but also the articulation shape of the vertebrae in O. sarsii has adapted for being a predator compared with filter feeder and infaunal ophiuroids such as M. hirsuta.

The shape of the articular structure seems to be an ecologically relevant trait, since A. loveni has an articular morphology similar to that of O. vitrea and Ophiothela sp. (streptospondylous and semi‐streptospondylous, respectively) rather than to closely related zygospondylous species (Figures 4b and 5). The vertebrae of A. loveni show the articular type that is hypothesized to be associated with highest flexibility (saddle‐shaped streptospondylous according to Litvinova [1994]), similar as in O. vitrea (Figure 2). Based on Alitto et al. (2020), epizoic fissiparous Ophiothela species have vertebrae with a semi‐streptospondylous articulation, with the zygosphene shortened, not extending beyond the long zygocondyles on the distal side. In contrast, according to Hotchkiss and Glass (2012), a zygosphene is present, but reduced in transspondylous vertebrae and has been separated from the zygocondyle, which cannot be confirmed for Ophiothela by this study. The misconception of transspondylous vertebrae by Alitto et al. (2020) may have been caused by vertebrae not being fully developed in incompletely regenerated animals after fission. Also, this type of vertebra is still only known from the fossil tentatively assigned to Ophiosmilax alternatus Kutscher & Jagt in Jagt, 2000 by Štorc (2004). The genus Ophiosmilax is a member of the family Ophiobyrsidae (order Ophiacanthida) and its type genus Ophiobyrsa Lyman, 1878 has streptospondylous vertebrae (B. Thuy, unpublished data), similar to Ophiocamax vitrea, a type common in Ophiacanthida.

According to Litvinova (1989a, 1989b, 1994,1989a, 1989b, 1994) and the phylogenetic hypothesis provided in Figure 2, it could be seen that the shape of vertebrae (zygopondylous universal type) underwent evolutionary changes to saddle‐type, semi‐streptospondylous, and comb‐shaped vertebrae or remained the same universal type in most clades. In other words, the evolutionary transformation sequence of vertebrae is from a zygopondylous type to a semi‐streptospondylous type, and then to a streptospondylous type, or from zygospondylous to comb‐shaped (Litvinova, 1989a, 1989b, 1994,1989a, 1989b, 1994; Thuy & Stöhr, 2018). Ophiuroid clades with streptospondylous vertebrae include Hemieuryalidae, Euryalidae, Gorgonocephalidae, Asteronychidae, Ophiobyrsidae, Ophioleucidae, and Ophiocamacidae (Goharimanesh et al., 2021). Streptospondylous and semi‐streptospondylous shapes have been hypothesized to allow the arm to bend in every direction and to twist spirally (Alitto et al., 2020; Litvinova, 1994). Contrary to a streptospondylous joint, the zygospondylous one has been hypothesized to be more limited in its mechanical movements (Byrne et al., 1994; Hyman, 1955). We observed O. scolopendrina (universal zygospondylous) also flexing vertically, apart from horizontal movements, during the fixation process. This feature might happen because of having the universal type of vertebrae articulation rather than being comb‐shaped zygospondylous, which has been assumed to let vertebrae bend in every direction but has been hypothesized to not allow for twisting spirally as seen in streptospondylous and semi‐streptospondylous vertebrae (Litvinova, 1989a). This thus confirms the limited degree of freedom, compared to streptospondylous/semi‐streptospondylous articulations.

Also, the dorso‐distal extension in the vertebrae (extended keel according to Goharimanesh et al. [2021]) may play a role in the function of arm movements, as proposed by LeClair and LaBarbera (1997) and Clark et al. (2018). The keeled species in the current research (belonging to Ophionereidae and Ophiotrichidae) are separated from non‐keeled species on PC2 (Figure 4), a distinction also observed by LeClair (1996). In fact, the central projections on the distal face are reduced in keeled vertebrae, but instead, a large depression on the dorso‐proximal surface and an extension on the dorso‐distal one, as well as the accessory aboral muscles between the proximal depression and distal extension are present (Goharimanesh et al., 2021; LeClair, 1996). Surprisingly, in A. loveni the dorso‐proximal projection is much extended into the distal depression of the adjacent vertebra, while it is quite the opposite for other species in this study (i.e. extended keel on the dorso‐distal face). This may explain the distinct position of this species along PC3, which explained variation in the dorsal region of the vertebrae (Figure 4b). A reduction in articular surfaces and additional muscles area on the dorsal face of the vertebrae could suggest a greater lateral bending in keeled species (Hendler & Miller, 1991). Although this structure might help improve the movement in the horizontal plane, it was not related to feeding type and did not show any significant difference in the position of maximal intersegmental rotation between keeled and non‐keeled species (LeClair & LaBarbera, 1997). According to LeClair and LaBarbera (1997) and Clark et al. (2018), the extensive, mean lateral flexibility observed in the species they studied (all universal zygospondylous, either with or without keel) was not linked to the vertebrae morphology or ecology, and thus the functional significance of this keel remains unknown. Clark et al. (2018) reported that the non‐keeled state is the ancestral state and that the keeled state might have evolved convergently in two clades. According to Figure 2 and as LeClair (1996) reported, Ophiotrichidae (keeled) is not closely related to Ophionereidae (keeled) but to Ophiactidae (non‐keeled). As expected, we did not see such a structure in our Ophiactis species, which was consistent with LeClair (1996). However, a keel is also present in Ophiopholidae (closely related to Ophiotrichidae), type genus Ophiopholis Müller & Troschel, 1842, which was not included in this study. Similar to M. hirsuta in our study, Ophiopholis species live in crevices among rocks and mussels, and are generally not epizoic (Drolet et al., 2004). Based on our observation on shape variation and reported ecological and functional lifestyles, we can conclude that the keel structure, apart from the central articular structure, is linked to ecological/functional adaptation, not to phylogeny. Such an extended keel, with smaller central articular structure and shorter ventral projections on the vertebrae are all seen in prehensile (epizoic) species. As the extended keel is present in prehensile species, but also in species that are known to show horizontal arm movements, it can be hypothesized that its presence influences the performance during horizontal flexing, while preventing vertebral dislocations.

Although in general, distantly related species show different vertebral shapes (LeClair, 1996), we observed some species that are not closely related (Ophiothela and A. loveni), but converged to a similar pattern in their vertebral articulation shape, as well as in having prehensile arms (Litvinova, 1989a). This suggests a convergent evolution through ecological adaptation of being epizoic or through conservation of an ancestral pattern through evolutionary time. Our analyses showed that trait variation is somehow correlated with phylogenetic relatedness, but not all (e.g. PC2 and PC3 representing both ecological and phylogenetic signals in shape variation). Especially for the shape pattern of the proximal vertebrae, ecological signaling seems prominent, whereas traits associated with the aboral process and lateral face of the vertebrae show a stronger phylogenetic signal. As shown by Fujita (2003) and Alitto et al. (2018), the aboral median groove and the relevant process are of taxonomic importance. Also Thuy and Stöhr (2011) have shown that the morphology of the lateral arm plate, which is associated with the lateral face of the vertebrae, reflects phylogenetic affinities. We found that dorsal (keel and corresponding depression) and central articular structures (zygosphene and zygocondyles) are more associated with ecological conditions, thereby refuting part of the observations by Smith et al. (1995) who assumed a phylogenetic signal in these structures. Our findings are in line with other studies that proposed that vertebral variation among both living and fossil ophiuroid taxa represent mechanical adaptations and thus depend on the various lifestyles or behaviors (Kokorin, 2015; Litvinova, 1989a, 1989b,1989a, 1989b). The current study provides more details and sheds light on the complex nature of vertebral morphological variation with respect to phylogeny and adaptations in ophiuroids. Our inferences made on functionality and adaptiveness only rely on correlated patterns between shape traits and lifestyle. In addition to a proper methodological tackling of functional performance hypotheses in relation to shape variation, future studies should also include larger sample sizes per species to better understand the importance of intra‐specific variation.

Various studies focused on the ophiuroid feeding mechanism (Boos, 2008; Fontaine, 1965; Gielazyn et al., 1999; Harris et al., 2009; Hollertz et al., 1998; Pentreath, 1970), but further research is still required to understand how the vertebral morphology controls the mechanics related to feeding in association with the different lifestyles. In addition, the ecological and functional status of Ophiactis savignyi and Ophiothrix savignyi is not clearly known in the literature. Although, in our study, we categorized them as non‐prehensile species supported by a statistical test, further investigation on ecological behavior of these two species is required. Vertebral morphology plays a major role in arm flexibility, where the range of motion may also depend on other factors such as the arrangement of intervertebral muscles, the morphology of the integument and external arm plates and the neurologically mediated stiffness of the connective tissue (Byrne & Hendler, 1988; Emson & Wilkie, 1982; Litvinova, 1994; Wilkie, 1992). Hence, it would be interesting to extend this work to musculoskeletal analyses and investigate the role of ophiuroid vertebrae on intersegmental flexibility, to understand how this feature affects an ophiuroid's ecological lifestyle.

5. CONCLUSION

Intra‐individually, the vertebrae show shape variation along the proximal‐distal axis of the arm (static allometry), with distal vertebrae having their articulation at a larger distance from the point of muscle insertion, which is beneficial for force transmission. This suggests structural adaptations that allow an improved arm functionality for grasping and locomotion. The increased vertebral height and large articular structures in the proximal vertebrae can be expected to limit the range of motion, making the proximal part of the arm stiffer. This pattern of variation in vertebral morphology in the studied ophiuroid species is phylogenetically and ecologically interesting, because it potentially indicates which skeletal characters are reliable for taxonomy and illuminates which skeletal features influence mechanical variations. The most striking results to emerge from the data are the patterns of how ophiuroid vertebral morphology is related to phylogenetic relationships and evolutionary adaptation (e.g., prehensility). It is the first study to link morphological characteristics to lifestyle using 3D shape quantifications (micro CT) and analyses (geometric morphometrics) on ophiuroid vertebrae. It shows that characteristics related to the aboral process and lateral vertebrae are essential for phylogenetic interpretations, while dorsal and central articular structures may have limited taxonomic value, as they seem to reflect adaptations to distinct functional/ecological lifestyles. It is concluded that the streptospondylous articulation, along with shorter ventral projections and an extended keel on the vertebrae are at least seen within prehensile arms. However, further study is required to investigate the musculoskeletal details and infer how those morphological characteristics are linked to arm function.

AUTHOR CONTRIBUTIONS

Mona Goharimanesh, Dominique Adriaens, and Fereshteh Ghassemzadeh provided the concept/design of the manuscript. Mona Goharimanesh sampled M. hirsuta and O. scolopendrina from Persian Gulf/Oman Sea and Sabine Stöhr provided the species used from Swedish Museum of Natural History. Sabine Stöhr contributed taxonomic and morphological information on the studied species. The scanning of the specimens was performed and supervised by Barbara De Kegel and Luc Van Hoorebeke, respectively. Mona Goharimanesh reconstructed 3D data, analyzed and interpreted the results. Mona Goharimanesh drafted the manuscript. Dominique Adriaens, Sabine Stöhr, Fereshteh Ghassemzadeh, Luc Van Hoorebeke and Omid Mirshamsi participated in writing the manuscript.

ACKNOWLEDGMENTS

We sincerely acknowledge Iván Josipovic for assisting with the scanning of the brittle stars at UGCT. We appreciate the Swedish Museum of Natural History for providing several species of the study. This project was funded by the FWO (grant 3G006716), INSF (no. 97012132) and the Ferdowsi University of Mashhad (no. 3/46018). The special research fund of the Ghent University (BOF‐UGent) is acknowledged for the financial support of the UGCT Centre of Expertise (BOF.EXP.2017.0007). The authors declare no conflicts of interest.

Goharimanesh, M. , Ghassemzadeh, F. , De Kegel, B. , Van Hoorebeke, L. , Stöhr, S. , Mirshamsi, O. & Adriaens, D. (2022) The evolutionary relationship between arm vertebrae shape and ecological lifestyle in brittle stars (Echinodermata: Ophiuroidea). Journal of Anatomy, 240, 1034–1047. Available from: 10.1111/joa.13617

DATA AVAILABILITY STATEMENT

The Micro CT data from this project (ID: 000396049) are available through MorphoSource (http://www.MorphoSource.org/).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The Micro CT data from this project (ID: 000396049) are available through MorphoSource (http://www.MorphoSource.org/).

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