INTRODUCTION

Members of the family Diadumenidae Stephenson, 1920, are characterized by monotypic mesenteries, two types of nematocysts in the acontia, cinclides on the scapus, absence of marginal sphincter musculature, and ephemeral fighting tentacles. This monogeneric family is comprised of 10 valid species belonging to the genus Diadumene Stephenson, 1920, which is distributed in all but the Southern Ocean, albeit most species are restricted to the Atlantic Ocean (Fautin, 2013). Diadumene lineata (Verrill, 1869a), however, is a cosmopolitan species and one of the most widely distributed marine organisms (Minasian, 1982). The species is believed to be native to Southeast Asia (Uchida, 1932; Stephenson, 1935) and exotic in many regions of the world (see Fautin, 2013), possibly transported between localities by rafting on floating material or on the hulls of ships (Shick and Lamb, 1977; Gollasch and Riemann-Zürneck, 1996; Zabin et al., 2004; Farrapeira et al., 2007, 2010).

Diadumenidae has been recovered as monophyletic and closely related to members of Metridiidae Carlgren, 1893, in all molecular phylogenetic analyses including multiple species of the genus (Daly et al., 2008; Rodríguez and Daly, 2010; Gusmão and Daly, 2010; Rodríguez et al., 2012, 2014; Grajales and Rodríguez, 2016). Rodríguez et al. (2012) erected the clade Metridina for members of Diadumenidae and Metridiidae, emphasizing the morphological similarities among these families, which had been proposed by others (e.g., Stephenson, 1925). Similarly, a monophyletic Diadumene has been recovered with high support in recent studies including multiple species of the genus (Rodríguez et al., 2012, 2014; Lauretta et al., 2014; Grajales and Rodríguez, 2016). Although generic placement is straightforward, species-level identifications within Diadumene are difficult because species are defined by a mosaic of characters that vary in degree rather than kind.

Two species of Diadumene have been recorded in the southwestern Atlantic: Diadumene paranaensis Beneti et al., 2015, has been collected only on the southern coast of Brazil and D. lineata collected in the southeast and northeast of Brazil (Belém and Monteiro, 1977; Zamponi et al., 1998; Farrapeira et al., 2007; Da Silveira and Morandini, 2011; Rocha et al., 2013) as well as in Argentina (Acuña et al., 2004; Excoffon et al., 2004, Molina et al., 2009). Here we record Diadumene leucolena (Verrill, 1866) for the first time in the southern hemisphere and describe D. manezinha, sp. nov., raising to four the number of Diadumene species recorded in Brazil and to 11 worldwide. In addition, we incorporate microcomputed tomography (micro-CT) to the description of both species examined in the first application of the method for soft-bodied members of order Actiniaria Hertwig, 1882. Micro-CT reconstructions have efficiently delineated morphological features in other cnidarians (scleractinians: e.g., Kruszynski et al., 2006, Roche et al., 2010, Sentoku et al., 2015; jellyfish and staurozoans: Holst et al., 2016; octocorals: Morales Pinzón et al., 2014; hydrozoans: Puce et al., 2011) as well as other small soft-bodied invertebrates with no chitinous or calcified tissues (e.g., leeches: Tessler et al., 2016; earthworms: Fernández et al., 2014). Here we evaluate the utility of micro-CT imaging and its potential to generate fast, low-cost, and high-resolution datasets, despite the need for chemical staining due to the sea anemone's low-density soft tissue (Faulwetter et al., 2013a, 2013b). We present a protocol for sample handling, chemical staining, and scanning parameters that resulted in satisfactory imaging of both specimens examined. We compare advantages and limitations of using micro-CT over traditional techniques used in the taxonomy of sea anemones.

MATERIAL AND METHODS

Morphological study: Specimens of Diadumene leucolena were collected by hand in the intertidal zone of Praia do Farol Velho (0°35′33″S; 47°19′29.00″W) and Praia do Atalaia (0°35′36″S; 47°18′44.00″W) both in Salinópolis (Pará) on the north coast of Brazil (fig. 1). Specimens of D. manezinha, sp. nov., were collected by hand in Praia da Barra da Lagoa (27°35′4.49″ S, 48°26′10.45″ W) in Florianópolis (Santa Catarina) on the south coast of Brazil (fig. 1). Animals were relaxed using menthol crystals, photographed alive, and subsequently fixed in 10% seawater-formalin. Before fixation, small pieces of column or pedal disc from selected specimens were fixed in absolute ethanol for DNA analysis. The material was deposited in the Museu de Zoologia da Universidade de São Paulo (MZUSP) as follows: 10 formalin-fixed specimens of D. leucolena (MZUSP 002482) and 10 corresponding ethanol-fixed vouchers (MZUSP 2472–2481), and 20 formalin-fixed specimens of D. manezinha (MZUSP 002504) and six corresponding ethanol-fixed vouchers (MZUSP 2498–2503).

Formalin-fixed specimens were examined whole, dissected, and as serial sections. Fragments of three specimens of each species were dehydrated and embedded in paraffin. Histological sections 8–10 µm thick were stained with Heidenhain's Azan Triple Stain (Presnell and Schreibman, 1997). Cnidae capsules were identified and measured in squash preparations of tissue from tentacles, column, actinopharynx, filaments, and acontia of preserved specimens. These preparations were examined using differential interference microscopy (DIC) at 1000. magnification and, except for the rarer types, at least 20 undischarged capsules were measured to generate range, mean, and standard deviation for each type of cnida. Although the mean and standard deviation calculated are not statistically significant, they are shown to give an idea of size distribution in the length and width of undischarged capsules. The presence of each type of cnida in each tissue was confirmed on histological slides. We follow a nematocyst terminology similar to the one used by Sanamyan et al. (2012), which combines the classification of Weill (1934), modified by Carlgren (1940) and thus differentiating “basitrichs” from “b-mastigophores,” with that of Schmidt (1969, 1972, 1974), which captures the underlying variation seen in “rhabdoids.” Unlike Sanamyan et al. (2012), we do not modify Schmidt's (1969, 1972, 1974) nomenclature with den Hartog's (1995) notes on p-mastigophores B1 (see Discussion), but agree with the use of “mastigophore” instead of “rhabdoid” to preserve stability. We use a combination of classifications to better capture the underlying variation in cnida morphology given its imperative nature to actiniarian taxonomy, but do not imply any evolutionary relationship among these types. We include photographs of each type of nematocyst for reliable comparison across terminologies and taxa (see Fautin, 1988). Higher-level classification for Actiniaria follows Rodríguez et al. (2014).

Molecular methodology: Genomic DNA was isolated from approximately 25 mg of tissue using the Qiagen DNeasy kit. Whole genomic DNA was amplified using published primers and protocol detailed in Lauretta et al. (2014) for the mitochondrial markers 12S and16S. Sequences for nuclear 18S were amplified using ribosomal genes (see Grajales and Rodríguez, 2016, for details on amplification protocol and sequencing with newly designed primers specific to Actiniaria to avoid co-amplification of Symbiodinium Freudenthal, 1962). PCR products were cleaned using ExosapIT (Exonuclease I and FastAP thermosensitive alkaline phosphatase per manufacturer's specifications, except that shrimp alkaline phosphatase was replaced with FastAP). Cycle-sequencing reactions used a total of 5 µL of cleaned PCR product, at a concentration of 25 ng of product for every 200 base pairs (bp) of marker length. Cycle-sequencing products were cleaned using Centri-Sept columns (Princeton Separations; following the manufacturer's protocol) containing DNA-grade Sephadex (G-50 fine; GE Healthcare) and later sequenced using PCR amplification primers on an ABI 3770x at the in-house facilities of the American Museum of Natural History (AMNH). Forward and reverse sequences were assembled and edited in Geneious v.11.1.4 (Kearse et al., 2012) and blasted against the nucleotide database of GenBank to confirm that the obtained sequences corresponded to the target sequence/organism and not to their endosymbiotic algae. Sequences have been deposited in GenBank (see Supplementary Data:  https://doi.org/10.5531/sd.sp.33). DNA sequences for each of the three markers were combined and analyzed with sequences corresponding to superfamily Metridioidea Carlgren, 1893, from Rodríguez et al. (2014), using sequences from selected taxa within the superfamily Actinioidea Rafinesque, 1815, as outgroup. Sequences for each marker were separately aligned using MAFFT v.7.017 (Katoh et al., 2002) using the following settings: Strategy, E-INS-I; Scoring matrix for nucleotide sequences, 200PAM/k = 2; Gap open penalty, 1.53; Offset value, 0.05. Alignments for each marker were analyzed separately and as a concatenated dataset.

Phylogenetic inference: For each gene region, the best model of nucleotide substitution was chosen using the software PartitionFinder v.1.1.1 (Lanfear et al., 2012). Maximumlikelihood (ML) analyses were performed using RAxML v.7.6.3 (Stamatakis, 2006) as implemented on the CIPRES portal (Miller et al., 2010), using a different partition per gene. The majority rule criterion implemented in RAxML (-autoMRE) was used to assess clade support. Analyses were run with gaps treated as missing data.

CT-scan methodology: The staining protocols for micro-CT scan and image processing were modified from those of Tessler et al. (2016). Specimens fixed and preserved in 10% formalin since 2012, were transferred to seawater for 30 min and subsequently completely submerged in 0.2m cacodylate buffer. Samples were postfixed in 1% osmium tetroxide for 1.5 hr at room temperature. Due to the autolimiting nature of the osmium tetroxide, which prevents penetration, the mix of osmium-cacodylate buffer was injected into the gastrovascular cavity of the anemones using a microsyringe to ensure a homogeneous exposure to the stain. Samples were prepared for scanning by a series of washes; the first step consisted of 30 min in phosphate buffer, followed by a second step of 15 min in fresh phosphate buffer, and two washes of 100% ethanol for 30 and 15 min, respectively. Once stained and washed, samples were transferred to a 50 ml polyethylene tube filled with fresh 100% ethanol in which they were kept until scanning. Both specimens were scanned in the same tube with 100% ethanol on a phoenix v|tome|x s240 GE (General Electric, Fairfield, CT, USA) at 60 kV and 70 µA with a molybdenum target, for a resolution of 4.72 microns/voxel for Diadumene leucolena and 14.98 microns/voxel for D. manezinha. The exposure time for the detector varied from 500.155 ms for D. leucolena to 1,500.263 ms for D. manezinha. Reconstruction of the raw data was done using the software Phoenix datos|x (General Electric, Wunstorf, Germany) and the resulting files and images were processed and edited using the software VGStudio Max 3.0 (Volume Graphics, Heidelberg, Germany). Full micro-CT scan data and volumetric renderings are deposited in the Morphobase ( www.morphosource.org) under project MZUSP-IZ002504.

Source of Material: Specimens examined are retained in the following institutions: MNRJ, Museum Nacional/ Universidade do Rio de Janeiro; MZUSP, Museu de Zoologia da Universidade de São Paulo; YPM, Yale Peabody Museum; ZMUC, University of Copenhagen Zoological Museum.

RESULTS

FIG. 1.

Detail of collection sites for Diadumene leucolena (Verrill, 1866) (star) and type locality for Diadumene manezinha, sp. nov. (hexagon).

FIG. 2.

External anatomy of Diadumene leucolena (Verrill, 1866). A, lateral view of living specimen showing cinclides with protruding acontia (arrows); B, oral view of living specimen; C, lateral view of preserved specimen showing scapus with cinclides on top of raised projections arranged in longitudinal rows (arrows). Scale bars: A–B, 3.0 mm; C, 2.5 mm.

FIG. 3.

Internal anatomy and microanatomy of Diadumene leucolena (Verrill, 1866). A, longitudinal section through distal column of a partially retracted specimen showing differentiation between scapus (dotted line) and capitulum (solid line) with limit between the two indicated by horizontal bar; B, cross section through actinopharynx showing glandular epidermis; C, cross section through actinopharynx showing well-differentiated siphonoglyph; D, cross section through feeding tentacle showing ectodermal longitudinal musculature (arrow); E, cross section through distal scapus at actinopharynx level showing the three cycles of mesenteries (indicated by numbers); note a raised projection on endocoel corresponding to a pair of directive mesenteries (arrow); F, cross section through scapus at distal actinopharynx level showing the three cycles of mesenteries (indicated by numbers); note larger, more developed retractors; G, cross section through distal scapus showing unequal development between mesenteries of a second cycle pair; H, cross section through proximal column showing detail of weak parietobasilar musculature in first cycle mesentery (arrow); I, detail of a fertile mesentery of second cycle showing oocytes. Abbreviations: ac, actinopharynx; ca, capitulum; di, directives; ep, epidermis; ga, gastrodermis; oo, oocytes; sc, scapus; si, siphonoglyph; te, tentacles. Scale bars: A, D, G, 0.1 mm; B, C, I, 0.07 mm; E, F, 0.4 mm; H, 0.012 mm.

FIG. 4.

Micro-CT volumetric rendering and sections of Diadumene leucolena (Verrill, 1866). A, lateral view of the 3D rendering of a specimen showing cinclides in raised projections arranged in longitudinal rows (arrows); B, longitudinal section through whole specimen showing external and internal anatomy; notice raised projections on scapus (arrows); C, cross section through midscapus showing raised projection in endocoel corresponding to a second cycle pair of mesenteries (arrow); D, cross section through midscapus showing the cinclide (arrow) of the same second cycle pair of directive mesenteries shown in E ; E, cross section through midscapus showing cinclide in endocoel of a pair of directives (arrow); F, cross section through a feeding tentacle; G, cross section through distal scapus showing pairs of mesenteries of three cycles (indicated by numbers) including directive mesenteries; H, cross section through midscapus showing cycles of mesenteries (indicated by numbers); note filaments on mesenteries of first and some of second cycles (arrows); I, cross section through proximal scapus showing absence of mesenteries of third cycle (arrow); J, detail of a pair of mesenteries of first cycle with diffuse retractor and weak parietobasilar musculature (arrow); K, cross section through proximal scapus showing unequal development of a second cycle pair of mesenteries (indicated by number); L, cross section through pedal disc showing basilar musculature (arrow). Abbreviations: ac, actinopharynx; di, directive mesenteries; fi, filaments; ga, gastrovascular cavity; re, retractor. Scale bars: A, B, 2.0 mm; C–E, 0.25 mm; F, J, 0.2 mm; G–I, K, L, 0.5 mm.

Table 1.

Size ranges of the cnidae of Diadumene leucolena (Verrill, 1866).

, mean; SD, standard deviation; S, proportion of specimens in which each cnidae was found; N, Total number of capsules measured; F, frequency; +++, very common; ++, common; +, rather common; -, rare

FIG. 5.

Cnidom of Diadumene leucolena (Verrill, 1866). A, B, F, J, R, basitrich. C, G, K, L, M, P, Q, S, T, p—mastigophore B2a. D, H, I, holotrich. E, spirocyst. N, O, p—mastigophore B1.

FIG. 6.

External anatomy of Diadumene manezinha, sp. nov. A, lateral view of an extended living specimen showing row of cinclides (arrows); B, lateral view of preserved specimen showing capitulum retracted into scapus (arrow); C, lateral view of preserved specimen showing cinclide on scapus with protruding acontium (arrow) D, oral view of living specimen; E, oral view of preserved specimen showing fighting tentacles; notice fighting tentacle with autotomized tip (arrow). Scale bars: A, 3.0 mm; B, 2.5 mm; C, 3.5 mm; D, 1.5 mm; E, 2.0 mm.

FIG. 7.

Internal anatomy and microanatomy of Diadumene manezinha, sp. nov. A, longitudinal section through column showing differentiation between scapus (dotted line) and capitulum (solid line) with limit between the two indicated by horizontal bar; notice cinclide on scapus (arrow); B, longitudinal section through distal column showing a cinclide in detail (arrow); C, longitudinal section through mid-scapus showing endodermal musculature (arrow); D, cross section through a feeding tentacle showing ectodermal longitudinal musculature; E, cross section through distal scapus showing retractors of directive mesenteries and spermatic cysts; F, cross section through proximal scapus showing cycles of mesenteries (indicated by numbers) and a pair of directives; note mesenteries of first and second cycles with oocytes; G, detail of mesenteries of third cycle (indicated by number); H, cross section through pedal disc showing basilar musculature (arrows). Abbreviations: ca, capitulum; di, directive mesenteries; ep, epidermis; ga, gastrodermis; oo, oocytes; sc, scapus; si, siphonoglyph; sp, spermatic cysts. Scale bars: A, 0.25 mm; B, C, 0.05 mm; D, 0.025 mm; E, G, H, 0.2 mm; F, 0.4 mm.

FIG. 8.

Micro-CT volumetric rendering and sections of Diadumene manezinha, sp. nov. A, lateral view of 3D rendering of whole specimen showing external anatomy showing; note the long fighting tentacle (arrow); B, longitudinal section through whole specimen showing external and internal anatomy; C, cross section through distal column showing detail of capitulum; D, cross section through fighting tentacle and two feeding tentacles; E, cross section through distal scapus showing pairs of mesenteries of three cycles (indicated by numbers) including directive mesenteries; F, cross section through midscapus showing cycles of mesenteries (indicated by numbers) and gonads on first and second cycles; notice unpaired mesentery of third cycle (arrow); G, cross section through column at different levels showing unpaired mesentery of third cycle only distally (arrow); H, cross section through proximal scapus showing a pair of mesenteries of first cycle with diffuse retractor and no visible parietobasilar musculature; notice oocytes on pairs of mesenteries of second cycle; I, cross section through proximal scapus showing detail of a pair of mesenteries of second and third cycles; parietobasilar musculature is not visible (arrow); J, cross section through pedal disc showing basilar musculature (arrows). Abbreviations: ac, actinopharynx; ca, capitulum; di, directive mesenteries; fe, feeding tentacle; fi, filament; fit, fighting tentacle; ga, gastrovascular cavity; oo, oocytes; re, retractor musculature; sc, scapus. Scale bars: A, 1.5 mm; B, 2.0 mm; C, D, 0.4 mm; E, G–J, 1.0 mm; F, 2.0 mm; I, 0.8 mm.

Table 2.

Size ranges of the cnidae of Diadumene manezinha, sp. nov.

, mean; SD, standard deviation; S, proportion of specimens in which each cnidae was found; N, Total number of capsules measured; F, frequency; +++, very common; ++, common; +, rather common; -, rare.

FIG. 9.

Cnidom of Diadumene manezinha, sp. nov. A, B, G, K, P, T, basitrich; C, D, H, M, N, O, S, U, V, W, p-mastigophore B2a; E, I, J, holotrich; F, spirocyst; L, p-mastigophore A; Q, R, p-mastigophore B1. Scale bar: 15 µm.

DISCUSSION

Familial and generic placement: The combination of the presence of p-mastigophores B2a and basitrichs in the acontia and no marginal sphincter musculature places Diadumene leucolena and D. manezinha in the family Diadumenidae. The two species can be easily differentiated from members of families with similar morphology by the mesogleal sphincter and b-mastigophores in addition to p-mastigophores B2a in acontia of Metridiidae and by presence of only basitrichs in the acontia of Acricoactinidae.

All examined specimens of Diadumene leucolena and D. manezinha exhibited features that in combination characterize Diadumene (see genus diagnosis). In addition, fighting tentacles were morphologically differentiated in three out of the 20 specimens of D. manezinha examined. Unlike feeding tentacles, fighting tentacles were opaque, thicker, and with blunt tips as described by Williams (1975). A single fighting tentacle lacked part of its tip (fig. 6E), which might suggest autotomization, a known result of agonistic behavior (Williams, 1975; Purcell, 1977). Absence of visibly differentiated fighting tentacles in most specimens of D. manezinha and all specimens of D. leucolena is not exceptional within Diadumenidae, as these structures have never been observed in some species (e.g., D. fransciscana and D. lighti) and when present may appear in frequencies as low as 1.7% in some populations (e.g., populations of D. lineata in England: Williams, 1975). Likewise, fighting tentacles are ephemeral during an individual's lifespan, being induced by territorial threats from non-clonemates (Williams, 1975; Purcell, 1977; Watson and Mariscal, 1983a; Östman et al., 2010a) and restricted to individuals along the border of an aggregation facing other aggregates (Purcell, 1977; Purcell and Kitting, 1982). The reversal from fighting tentacles is also possible, being correlated with isolation of individuals to areas with non-clonemates (Hand, 1956) or an animal's nutritional state (Williams, 1975; Hand, 1956). In both instances, a change in nematocyst content accompanies the transformation (Purcell, 1977; Watson and Mariscal, 1983a).

Despite finding holotrichs of two distinct morphologies in inner tentacles of specimens of Diadumene leucolena from Brazil (fig. 5H, I), these tentacles were not visibly differentiated into fighting tentacles. The holotrichs seen in tentacles of D. leucolena, however, are not the typical ones seen in fighting tentacles of Diadumene, but divided into two types of holotrichs: the first type (fig. 5H) similar to those described by Excoffon et al. (2004) as anisorhize haplonemes for feeding tentacles of D. lineata and by Östman et al. (2010a) as isorhizas for Metridium senile (Linneus, 1761) (Östman et al., 2010a) and a second type (fig. 5I) similar to the slender ones seen in fighting tentacles of Diadumene species (e.g., D. lineata, Kovtun et al., 2012: fig. 5e), including D. manezinha (fig. 9I). Unfortunately, we did not observe undischarged capsules of either type of holotrich and cannot further classify them into isorhizas or anisorhizas. In addition to having holotrichs, tentacles exhibited cnidae typical of feeding tentacles (i.e., spirocysts, basitrichs, and p-mastigophores B2a; fig. 5E–G, respectively), which is usually rare in fighting tentacles (Stephenson, 1935; Williams, 1975; Purcell, 1977; Watson and Mariscal, 1983a). This might suggest that feeding tentacles seen in specimens of D. leucolena were either in the early stages of transforming into fighting tentacles, given the predominance of small holotrichs over larger ones characteristic of later stages (table 1), or were in the process of reverting from fighting tentacles to feeding tentacles (see Watson and Mariscal, 1983b). Similarly, Carlgren (1950) found cnidae typical of fighting tentacles in the tips of tentacles of D. leucolena that were not visibly differentiated from other feeding tentacles. Fukui (1986) also noted that fighting tentacles of D. lineata were similar to feeding tentacles in coloration and shape when contracted, becoming differentiated only when expanded in the manner characteristic of these structures. Likewise, Larson and Daly (2015) hypothesized that a non-differentiated tentacle with holotrichs and rare spirocysts in one specimen of the actineoidean Epiactis ritteri Torrey, 1902, is a fighting tentacle. Alternatively, it is possible that contamination of other parts of the tentacle with cnidae typical of feeding tentacles occurred during sampling of the tip of tentacles in which holotrichs are concentrated in fighting tentacles (Watson and Mariscal, 1983a).

Given the transient nature and low frequency of fighting tentacles in populations of species that possess them (e.g., Hand, 1956; Williams, 1975), we agree with others (e.g., Hand, 1956; Riemann-Zürneck, 1975; Williams, 1975; Manuel, 1981; den Hartog and Ates, 2011) and recommend that presence of fighting tentacles be taken in combination with other characters to more effectively distinguish members of Diadumenidae from other metridioidean families. Nevertheless, we maintain the presence of fighting tentacles in the diagnosis of family Diadumenidae given its uniqueness within Actiniaria, as a way to help identify species that exhibit them. We infer that fighting tentacles are present in all species of Diadumene and not absent, but unobserved in populations of D. franciscana and D. lighti. The fact that for more than 75 years thousands of specimens from several populations from Japan, North America, and Europe of D. lineata were examined (e.g., Sephenson, 1925, 1935; Uchida, 1932; Hand, 1956) before Williams (1975) confirmed the presence of fighting tentacles in populations from Plymouth (England) and Misaki (Japan) suggests this could be the case for D. fransciscana and D. lighti. That fighting tentacles may be absent in D. lineata populations undergoing frequent pedal laceration while present in high abundance in other populations with frequent sexual reproduction (Fukui, 1986) might provide an avenue for exploration of the cause of their absence in other species and populations. In addition to the eight species of Diadumenidae, however, only nine species in five metridioidean families exhibit fighting tentacles with holotrichs: Metridium senile (Metridiidae) (Williams, 1975; Hand, 1956); Cereus pedunculatus (Pennant, 1777), Sagartia elegans (Dalyell, 1848), Sagartia troglodytes (Price in Johnston, 1847) (Williams, 1975), Verrillactis paguri (Verrill, 1869a), and Habrosanthus bathamae Cutress, 1961 (Sagartiidae Gosse, 1858); Flosmaris bathamae Hand, 1961(Andvakiidae Danielssen, 1890, sensu Rodríguez et. al., 2012); Sagartiomorphe carlgreni Kwietniewski, 1898 (Sagartiomorphidae Carlgren, 1934) (Carlgren, 1940); Tricnidactis errans Pires, 1987 (Haliplanellidae Hand, 1956). Despite the obvious polyphyly of Sagartiidae (Daly et al., 2008; Rodríguez et al., 2012, 2014), a close relationship among Metridiidae, Diadumenidae, and some sagartiids (e.g., V. paguri, S. troglodytes) with fighting tentacles has been recovered in phylogenetic analyses (Gusmão and Daly, 2010; Rodríguez et al., 2012, 2014), which suggests fighting tentacles have putatively independently evolved fewer times than suspected. In addition to metridioideans, tentacles with holotrichs have been found in actinioideans belonging to family Actiniidae Rafinesque, 1815: Epiactis japonica (Verrill, 1869a) (as Cnidopus japonicus: Sanamyan and Sanamyan, 1998; Larson and Daly, 2015), Pacific populations of Aulactinia stella (Verrill, 1864) (Sanamyan and Sanamyan, 1998), and Epiactis ritteri Torrey, 1902 (Larson and Daly, 2015). Earlier, Fautin and Chia (1986) found holotrichs in tentacles of Epiactis lisbethae Fautin and Chia, 1986, Epiactis fernaldi Fautin and Chia, 1986, and Epiactis prolifera Verrill, 1869b. Similarly, Sanamyan et al. (2013) found holotrichs on tips of outer tentacles of Aulactinia vancouverensis Sanamyan et al., 2013, which were hypothesized to be fighting tentacles. In addition, the actiniid Oulactis concinnata (Dayton in Dana, 1846) exhibits tentacles that do not possess holotrichs but have searching behavior and are morphologically differentiated from other feeding tentacles (e.g., longer, with different coloration and batteries of large b-mastigophores) (Häussermann, 2003). Unlike members of Metridioidea whose fighting tentacles develop from tentacles in inner cycles (often first, or first and second cycles; rarely also third cycle), all fighting tentacles of actinioideans develop from tentacles in outer cycles (Sanamyan et al., 2013). Although fighting tentacles of inner and outer cycles have similar histological and nematocyst content (i.e., holotrichs concentrated on the tip of tentacles: e.g., Williams, 1975; Sanamyan and Sanamyan, 1998), a detailed revision of this structure is necessary to establish homology between fighting tentacles of metridioideans and actinioideans. In addition, whether the tentacles of actiniids are similarly involved in agonistic behavior, as speculated by Häussermann (2003) for those in O. concinnata, needs further investigation (Fautin and Chia, 1986).

FIG. 10.

Maximum-likelihood tree resulting from combined analysis of 12S, 16S, and 18S data sets. Relative branch lengths are indicated. Numbers below branches are bootstrap resampling values expressed as percentages. Clade Metridina Rodríguez et al., 2012, indicated by dashed box; members of family Diadumenidae Stephenson, 1920, represented by shaded box. Asterisks indicate the two species examined in this study.

Nematocysts found in the acontia of Diadumene leucolena and D. manezinha corresponded to two distinct types that are easily identified from unexploded capsules: basitrichs and p-mastigophores B2a. Diadumene manezinha, however, has three distinct categories of p-mastigophores B2a, a characteristic shared with members of D. paranaensis, but whose smallest category does not overlap in size. Carlgren (1924a) recorded three types of nematocysts in the acontia of D. neozelanica, which we confirmed from examining syntypes of the species (ZMUC ANT-000076) to be basitrichs and two categories of p-mastigophores B2a. Hand (1956) transferred D. lineata to a newly erected family (Haliplaneliidae) and genus (Haliplanella Hand, 1956) due to a smaller category named microbasic amastigophores found in the acontia of eight populations of the species from California. After Cutress (1955) clarified that microbasic amastigophores also had a terminal tubule and should be regarded as p-mastigophores and Williams (1975) found fighting tentacles in populations of D. lineata from England and Japan, Manuel (1981) synonymized Haliplanellidae with Diadumenidae, but kept Haliplanella as a separate genus. To further distinguish these genera, Manuel (1981) redefined Diadumene to include species with only one category of p-mastigophores, as the nomenclature used in his study (i.e., Weill, 1934, modified by Carlgren, 1940) did not capture the distinction between the two categories of p-mastigophores present in their acontia. Manuel's (1981) diagnosis of Diadumene is problematic as it would exclude D. neozelanica, D. paranaensis, and D. manezinha from the genus, contradicting recent phylogenetic analyses (e.g., Grajales and Rodríguez, 2016) and the present study (fig. 10; see Discussion below).

Regardless of its familial or generic placement, the distinction between Diadumene lineata and other species became more evident after Pires (1988) described the haliplanelid Tricnidactis errans using the cnidae nomenclature of Schmidt (1969, 1972, 1974), which allowed differentiation of p-mastigophores in the acontia in two categories: p-mastigophores B2a and p-mastigophores B1. The presence of these two nematocysts in the acontia has since been confirmed for T. errans (Excoffon and Zamponi, 1993) and D. lineata (Excoffon et al., 2004; Acuña et al., 2004). The recovered position of D. lineata within Diadumenidae in recent phylogenetic analyses (e.g., Rodríguez et al., 2012, 2014; Lauretta et al., 2014; Grajales and Rodríguez, 2016) supports Manuel's (1981) hypothesis that an extra category of p-mastigophores did not warrant the erection of the family Haliplanellidae, but does not support the maintenance of D. lineata in a separate genus. Thus, the presence of a category of p-mastigophores B1 in the acontia of D. lineata is interpreted as a specific feature of the species. Given the low frequency of p-mastigophores B1 in the acontia of D. lineata and T. errans, however, it is possible that a revision of Diadumene species will reveal that this feature is more widespread within the genus. Assuming a correct specific identification of their material, many did not find p-mastigophores B1 of the smallest category in the acontia of D. lineata (Stephenson, 1935; Carlgren, 1945, 1952; Widersten, 1976). Alternatively, a detailed revision of species of Diadumene may show that this smaller category of nematocysts in the acontia corresponds instead to p-mastigophores B2a as seen in D. paranaensis and D. manezinha. Given the difficulties involved in the observation of the proximal shaft in small nematocysts (Östman et al., 2010a), distinguishing undischarged capsules of p-mastigophores B2a from p-mastigophores B1 in acontia of D. lineata is difficult and may lead to misclassifications. Observation of undischarged capsules of the small category in acontia of D. lineata from Brazil (São Sebastião, São Paulo) suggests that small p-mastigophores B1 could be p-mastigophores B2 (L.C.G., personal obs.). The fact that tentacles morphologically resembling fighting tentacles have also been observed in members of the family Aiptasiidae Carlgren, 1924a, albeit that confirmation based on cnidae was not possible (E.R., personal obs.), suggests that T. errans might belong within Aiptasiidae, a fact that could be supported by other morphological features (e.g., p-mastigophores B1 in acontia; presence of a weak mesogleal sphincter at the base of the tentacles).

Relevant notes on cnidae terminology: Regardless of whether only Diadumene lineata or more species of the genus possess p-mastigophores B1 in acontia and the reliable identification of nematocysts in this category, the choice of nomenclature used to differentiate p-mastigophores may reveal or obscure this feature. The terminology used by Sanamyan et al. (2012) to classify p-mastigophores (i.e., Schmidt, 1969, 1972, 1974, modified by den Hartog, 1995) identifies nematocysts without a “Faltstück” (i.e., without a divided shaft) but with bilateral symmetry as p-mastigophores B2a. By incorporating the modification of den Hartog (1995), differences between D. lineata and other species in the genus as well as among diadumenids and other metridioidean families with “true” p-mastigophores B2a with a Faltstück in the acontia gets blurred (e.g., den Hartog and Ates, 2011; Kovtun et al., 2012). For this reason, we chose to use Schmidt's (1969, 1972, 1974) nomenclature to differentiate p-mastigophores without the modification of den Hartog (1995) as opposed to Sanamyan et al. (2012); it is worth noticing that the latter authors do not use the terminology “B2c” suggested by den Hartog (1995) to differentiate p-mastigophores B1 with bilateral symmetric capsules from p-mastigophores B2a sensu Schmidt (1969, 1972, 1974). It is especially important to note that den Hartog (1995) corrected what he believed was a mistake made by Schmidt (1969) when he characterized p-mastigophores B1 of Telmatactis Gravier, 1916, as having a terminal tubule. Den Hartog (1995) differentiates p-mastigophores B2c as the only “true” amastigophores unlike p-mastigophores B2a of many species within Boloceroididae Carlgren, 1924b, Aiptasiidae, Diadumenidae, and Sagartiidae, which have a vestigial tubule left in the capsule after discharge.

Although we acknowledge that capsules of p-mastigophores B1 primarily found in filaments of sea anemones having a distinctive symmetrical ovate or teardrop shape (den Hartog, 1995; Reft, 2012) are different from other nematocyst types as confirmed by Reft ( 2012), the transfer of bilaterally symmetrical capsules of p-mastigophores B1 of andvakiids to category B2 is inconsistent with the more fundamental definition of that category as having a shaft divided in two parts (i.e., with Faltstück). While this might be interpreted as a semantic conflict, it is important to maintain internal consistency in these already poorly defined categories of cnidae and their conflicting or unparallel terminologies. That symmetry may help distinguish types of nematocysts has been emphasized by Reft (2012) when she created two categories for p-mastigophores B2a sensu Schmidt (1969, 1972, 1974) based on symmetrical (p-rhabdoids B2s) or asymmetrical capsules (p-rhabdoids B2d). Both categories clustered at opposite ends of a continuum with outliers crossing into each other's morphospace (Reft, 2012). Especially p-rhabdoids B2d exhibited a broad distribution with no obvious clustering, which suggests this category and its definition may have been misinterpreted (Reft, 2012). The difficulty in correctly assigning nematocysts to this category seen in the discriminant analysis by Reft (2012) further indicates that this category might not be appropriate, with misclassification scattered in several groups with no obvious patterns, including categories p-rhabdoids B2s and p-rhabdoids B1. Unfortunately, Reft (2012) included only p-mastigophores B1 typical of filaments of sea anemones (i.e., ovate/teardrop shaped) from four metridioidean genera in her study, leaving unanswered the question as to whether p-mastigophores B1 should be split into subcategories.

Den Hartog (1995) raised an issue regarding the possibility of misclassification of nematocysts due to their immaturity (Reft, 2012). Östman et al. (2010a) interpreted capsules of microbasic amastigophores as immature when they exhibited one or a combination of the following: long and/or broad capsule, long proximal shaft with V-shaped notch closer to the distal capsule may form loops or undulations, and no internal capsular matrix is visible. Likewise, we interpret the p-rhabdoids C with undulating shaft found in tentacles of Diadumene lineata and Tricnidactis errans, and with looped proximal shaft in acontia of Bellactis ilkalyseae Dube, 1983, and Exaiptasia pallida (Agassiz in Verrill, 1864) recorded by Pinto (2002) as immature p-mastigophores B2a. Our hypothesis is supported the larger size range of the supposedly p-mastigophores C compared to p-mastigophores B2a seen in the same structures. Schmidt (1969, 1972, 1974) differentiated p-rhabdoids B2b from p-rhabdoids C based on the length of the shaft (i.e., with one and a half longitudinal turns in p-rhabdoids B2b and fourfold turns in p-rhabdoids C) and the folding of the proximal part of the shaft (i.e., more densely folded due to spine arrangement in p-rhabdoids B2b, which explains the similar undischarged shaft length in both types). The nematocysts with undulating or looped shafts classified by Pinto (2002) are not p-rhabdoids C under these definitions. In addition, shafts in nematocysts belonging to the medium category of p-mastigophores B2a from the acontia of specimens of D. manezinha that loop (fig. 11A, B) or undulate (fig. 11C, D) are not rare and often found together with nematocysts with straightened shafts (fig. 11E, F) in squash preparations. These morphologies are interpreted as different developmental stages of p-mastigophores B2a as suggested by Östman et al. (2010b) for p-mastigophores B2a seen in acontia (Östman et al., 2010b: fig. 6B, C, F) and p-mastigophores B2a and p-mastigophores B1 in mesenteries (Östman et al., 2010b: fig. 7B–E) of Metridium senile. Capsules of p-mastigophores B2a with looped, undulating, and straightened shafts have also been observed in other metridioideans (fig. 12). Although we cannot establish a time series for the distinct developmental stages of p-mastigophores B2a of D. leucolena (fig. 11) and other metridiodeans (fig. 12), we interpret nematocysts with looped (figs. 11A, 12A, C, H, M, N) or undulating shafts (figs. 11B, C, 12B, D, G, I, J, K, O) as immature stages whose capsules become straighter and whose shaft becomes shorter as the helices tighten (figs. 11E, F, 12E, F, L, P, Q), especially in the proximal region, with a visible central rod-shaped structure on the proximal shaft (figs. 11E, 12E, K, I–K, P) and later without it (figs. 11F, 12F, L, Q) (see Östman et al., 2010b, 2010c).

FIG. 11.

Differentiation of undischarged medium p-mastigophores B2a from acontia of D. manezinha, sp. nov. A, immature nematocyst with looped proximal part of shaft; B, C, D, immature nematocyst with undulating proximal shaft; note increased distance between V-shaped notch of shaft and distal part of capsule, straightening of the shaft, and central rod-shaped structure visible on proximal shaft (arrow); E, immature nematocyst with straightened shaft and central rod-shaped structure visible on proximal part of shaft; F, nematocyst late in development with limit between proximal and main shaft indistinct, central rod-shaped structure indistinct, and internal capsular matrix visible close to V-shaped notch (note shorter shaft with increased distance between V-shaped notch of shaft and distal part of capsule). Abbreviations: cr, central rod-shaped structure; in, internal capsular matrix. Scale bars: 10 µm.

The two categories of p-mastigophores B2a in the acontia of specimens of Diadumene leucolena found together in slide preparations of all six specimens examined exhibited a continuous range distribution with overlap in the middle of the range. These two categories may correspond to either two distinct developmental stages of p-mastigophores B2 or two distinct size categories of p-mastigophores B2a. Mature and immature capsules of p-mastigophores B2a found in tentacles of Metridium senile were differentiated by Östman et al. (2010a: figs. 1, 2) based on the presence of a rod-shaped structure in immature capsules and the absence of this structure in mature capsules with no clear distinction between the main and proximal parts of the shaft. The categories of p-mastigophores B2a found in the acontia of D. leucolena may, thus, correspond to immature (fig. 5T) and mature nematocysts (fig. 5S). Additionally, as described by Östman et al. (2010a) for p-mastigophores, a shorter basal tubule with a corresponding increased distance between the V-shaped notch and the basal capsule wall was observed in presumed mature capsules of p-mastigophores B2a (fig. 5S), as opposed to immature ones (fig. 5T) in acontia of D. leucolena. Similarly, Carlgren (1924a) found larger, opaque, and curved p-mastigophores in the acontia of a large specimen of D. neozelanica that he considered a developmental stage of the larger category of p-mastigophores. Capsules of p-mastigophore B2a recorded in the actinopharynx of D. leucolena and D. manezinha may correspond to mature (figs. 5L, 9M) and immature ones (figs. 5M, 9N), respectively. For this reason, mature and immature measurements in actinopharynx were combined to give length and width ranges in table 2. That the two categories of p-mastigophores B2a recorded in the acontia of D. paranaensis are developmental stages of the same type of nematocyst (Beneti et al., 2015: fig. 4Q, S) is also hypothesized here. The fact that presumed immature capsules of p-mastigophores B2a seen in acontia of D. leucolena are more abundant than mature ones in the examined specimens may be related to the supply and demand of this nematocyst in acontia (Robson, 1988). Even if the two categories of p-mastigophores B2a in the acontia of D. leucolena are not distinct developmental stages of the same type of nematocyst but rather two distinct types of nematocysts, this character, combined with other anatomical characteristics of the species (e.g., lack of marginal sphincter, holotrichs in tentacles, number and pairs of mesenteries), confirm D. leucolena's classification within Diadumenidae.

FIG. 12.

Undischarged p-mastigophores B2a of acontia in different species and various degrees of development. Sagartiidae sp.: A, immature nematocyst with broad capsule and looped proximal and main shaft; B, immature nematocyst with broad capsule and undulating proximal and main shafts; C, immature nematocyst with looped proximal shaft; D, immature nematocyst with undulating proximal shaft and distinct limit between proximal and main shaft; E, nematocyst late in development showing shorter, straighter proximal and main shaft with visible central rod-shaped rod on proximal shaft; F, mature nematocyst with slender capsule and internal capsular matrix visible, straight shaft with indistinct central rod-shaped structure on proximal shaft and indistinct limit between proximal and main shaft. Scale bars: A–F, 10 µm.

(continued).

Diadumene lineata (Verrill, 1869a): G, immature nematocyst with coiled proximal part of shaft and undulating main shaft with undifferentiated V-shaped notch (arrow); H, immature nematocyst with curved capsule and distinct limit between proximal and main shaft forming loop; I, immature nematocyst with undulating proximal and main shaft, difference between proximal and main shaft distinct and visible central rod-shaped structure on proximal shaft (arrow); J, immature nematocyst with shorter and straighter undulating shaft, difference between proximal and main shaft distinct and central rod-shaped structure visible on proximal shaft (arrow); K, nematocyst in late development with straightened main shaft but with visible central rod-shaped structure on proximal shaft (arrow); L, fully mature nematocyst with straightened, shorter shaft with internal capsular matrix visible and no distinction between proximal and main shaft. Scale bars: G, 20 µm; H-L, 10 µm.

(continued).

Exaiptasia pallida (Agassiz in Verrill, 1864): M, N, immature nematocysts showing looped proximal shaft and undulating main shaft with undifferentiated V-shaped notch (arrow); note the capsule shortening and straightening as it develops; small basitrich seen close to distal part of p-mastigophore B2a in M; O, immature nematocyst with differentiated V-shaped notch (arrow), straightened proximal shaft distinctly differentiated from main shaft; P, nematocyst late in development with distinct limit between proximal and main shaft and distinct central rod-shaped structure; Q, mature nematocyst with shorter and straighter shaft, indistinct limit between proximal and main shaft, and visible intracellular capsular matrix. Abbreviations: cr, central rod-shaped structure, in, internal capsular matrix. Scale bars: H, M, 20 µm; N, 25 µm; O, 30 µm; P, Q, 10 µm.

Differential diagnosis from other Diadumene species: Members of Diadumene are uniform regarding many anatomical (e.g., presence of cinclides on scapus, six pairs of perfect mesenteries) and microanatomical characters (e.g., all species have retractors diffuse to restricted, parietobasilar and basilar musculature weak), and are distinguished by a mosaic of features related to the column (e.g., distribution of cinclides in column, presence of nematocyst aggregations), mesenteries (e.g., number of cycles, irregularity due to asexual reproduction), cnidae, and geographic distribution. Although the genus Diadumene is one of the most diverse within Actiniaria (Carlgren, 1945) in terms of the types of nematocysts, cnidae are at the same time quite uniform across species, making differences potentially informative at the species level (Carlgren, 1940).

The arrangement of cinclides in longitudinal rows seen in the column of Diadumene leucolena and D. manezinha differentiates them from species with a concentrated band of cinclides on distal scapus (D. fransciscana) and those with cinclides scattered throughout the column (D. schillerianna, D. cincta, D. neozelanica, D. lighti, D. lineata). Unfortunately, D. crocata could not be included in the comparison of Diadumene species, given the paucity of information available in the literature on its morphology and cnidae as well as the lack of type material for this species. From the remaining three species with cinclides arranged in longitudinal rows in the column, D. leucolena can be differentiated from D. kameruniensis by the number of tentacles and mesenteries (91–95 tentacles and up to 5 cycles of mesenteries in D. leucolena; up to 192 tentacles and 6 cycles of mesenteries in D. kameruniensis) and by the presence of anatomical irregularities due to asexual reproduction in D. kameruniensis that are absent in D. leucolena. Similarly, D. leucolena can be distinguished from D. neozelanica by the presence of anatomical irregularities due to asexual reproduction in the latter, as well as by cinclide morphology (cinclides inconspicuous and not on raised projections in D. neozelanica; cinclides conspicuous and on top of raised projections in D. leucolena), fertility pattern (some mesenteries of the third cycle fertile in D. neozelanica; mesenteries of third cycle always sterile in D. leucolena), and details of the cnidae (e.g., p-mastigophores B1 and large category of p-mastigophores B2a present in acontia and p-mastigophores A present in actinopharynx of D. neozelanica are all absent from D. leucolena). In addition to the anatomical irregularities resulting from asexual reproduction seen in D. paranaensis and absent in D. leucolena, cinclide morphology (cinclides inconspicuous and not on raised projections in D. paranaensis; cinclides conspicuous and on top of raised projections in D. leucolena), and cnidae details distinguish D. leucolena from D. paranaensis (e.g., p-mastigophores B2a with looped basal tubule in tentacles of D. paranaensis are absent from D. leucolena; two categories of non-overlapping p-mastigophores B2a in acontia of D. paranaensis are not found in D. leucolena). Specimens of D. leucolena examined here closely agree with previous descriptions of the species (e.g., Carlgren, 1950; Hand, 1956), including the Brazilian material identified as Diadumene sp. by Pinto (2002). Two categories of p-mastigophores B2a in the acontia recorded in all specimens of D. leucolena examined in this study have never been described for the species and were not found in the syntypes examined (YPM 665); the cnidae measurements from syntypes closely agree with those given for the Brazilian specimens (table 1), except holotrichs I were not recorded in the syntypes. We hypothesize that the two categories of p-mastigophores B2a are distinct developmental stages of the same type of cnidae (see relevant remarks on cnidae terminology above). Until the relationship between development and morphology of nematocysts is fully understood, we identify the specimens examined in this study as D. leucolena, as no other morphological difference exists between these specimens and previous descriptions of the species. This is the first record of D. leucolena for the southern hemisphere.

Similarly to Diadumene leucolena, D. manezinha can be differentiated from D. kameruniensis based on the number of mesenteries and tentacles (89–92 tentacles and up to 5 cycles of mesenteries in D. manezinha; up to 192 tentacles and 6 cycles of mesenteries in D. kameruniensis) and the presence of anatomical irregularities due to asexual reproduction found in D. kameruniensis and absent in D. manezinha. Unlike D. manezinha, whose mesenteries of the third cycle are always sterile and regularly arranged due to absence of asexual reproduction, D. neozelanica has some mesenteries of the third cycle fertile and always displays anatomical irregularities due to asexual reproduction. In addition, details of the cnidae, in particular nematocysts in the acontia, clearly differentiate D. manezinha and D. neozelanica: one category of p-mastigophores B2a is found in the acontia of D. neozelanica while three categories of p-mastigophores B2a are found in D. manezinha.

Diadumene manezinha can be easily distinguished from the three species of Diadumene recorded from Brazil (i.e., D. paranaensis, D. leucolena, D. lineata). The presence of anatomical irregularities due to asexual reproduction in D. paranaensis (absent in D. manezinha) and differences in nematocyst content of all tissues (e.g., extra category of p-mastigophores B2a in acontia, holotrichs in column, and p-mastigophores A in the actinopharynx in D. manezinha all absent from D. paranaensis; p-mastigophores B2b with looped basal tubule in the tentacles of D. paranaensis absent from D. manezinha) clearly differentiates these two species. Among other features, cinclide morphology (inconspicuous and not on raised projections in D. manezinhav; conspicuous and on top of raised projections in D. leucolena) and cnidae of acontia differentiate D. manezinha from D. leucolena (two size categories of p-mastigophores B2a in the acontia of D. leucolena; three nonoverlapping size categories p-mastigophores B2a in D. manezinha). Diadumene manezinha and D. lineata are differentiated by cinclide arrangement in the column (scattered in D. lineata; arranged in longitudinal rows in D. manezinha), details of cnidae (especially presence of p-mastigophores B1 in acontia of D. lineata), and anatomical irregularities in number of tentacles and mesenteries in D. lineata (often observed in populations of D. lineata outside Japan; Stephenson, 1935), which were never observed in D. manezinha. Similar to specimens of D. lineata from the Black Sea (Kovtun et al., 2012), D. manezinha has p-mastigophores A in the actinopharynx although they belong to a single category (vs. two categories in D. lineata) and are much more slender in D. manezinha than in D. lineata. Similarly, syntypes of D. neozelanica (ZMUC ANT-000076) exhibited p-mastigophores A not only in the actinopharynx, but also in the filaments (table 2). In both structures, p-mastigophores A of D. neozelanica were slender and small, being more similar to those of D. manezinha than those of D. lineata. Diadumene manezinha is the 11th species described for the genus and the fourth Diadumene recorded for Brazil.

Phylogenetic analysis: The phylogenetic relationships and clades recovered in this study agree with earlier broad-scale analyses of sea anemones (e.g., Daly et al., 2008; Gusmão and Daly, 2010; Rodríguez et al., 2012, 2014). The close relationship found between Metridiidae and Diadumenidae has been consistently recovered by most other phylogenetic analyses (e.g., Daly et al., 2008; Gusmão and Daly, 2010; Rodríguez et al., 2012, 2014; Grajales and Rodríguez, 2016; Larson, 2016) and named Clade Metridina by Rodríguez et al. (2012). While this result highlights the morphological similarity between members of Metriididae and Diadumenidae, the placement of Acricoactinidae as sister to Metridiidae in agreement with Larson (2016) presents a new hypothesis for the evolution of the marginal sphincter musculature within this clade. While previous studies predating the description of Acricoactinidae (e.g., Rodríguez et al., 2012, 2014) indicated that this character was lost in Diadumenidae, this new phylogenetic hypothesis suggests that the most parsimonious scenario (at the Metridina clade level) is a single loss of the marginal sphincter muscle, followed by a reappearance in Metridiidae. This would constitute the first instance of a novel evolution of this character within Metridioidea in a similar scenario to the independent evolution of a marginal sphincter in Halcuriidae Carlgren, 1918 (Rodríguez et al., 2014). Another possibility, which has been observed in several lineages within Actiniaria, is the repeated loss of the marginal sphincter muscle in Diadumenidae and Acricoactinidae, a less parsimonious scenario, but consistent with other instances of marginal sphincter loss in correlation to a reduction in size or degeneration and complete loss of acontia (Rodríguez et al., 2014).

The phylogenetic position of the three species of Diadumene found in Brazil shows a complex diversification scenario within the genus. While D. leucolena and D. manezinha exhibit morphological differences consistent with their being two distinct species, they appear as sister taxa in our phylogenetic tree (fig. 10), whereas the close geographical distribution of D. paranaensis and D. manezinha would suggest that these species would be more closely related. Instead, D. paranaensis is nested within a clade composed of species distributed in the northern hemisphere, a result also supported by morphological similarities between D. paranaensis and D. cincta suggested by Beneti et al. (2015). These two species also share p-mastigophores B2 with looped basal tubule in the outer tentacles, a character present in D. lighti (Hand, 1956). This author suggested that a new genus within Diadumenidae should be established for species presenting this character, as it differs from the condition found in D. schilleriana, the type species of Diadumene. A comprehensive revision of the cnidae seen in outer tentacles in other species of the genus as well as their phylogenetic position will be required to make decisive conclusions about the validity of the genus and whether a new genus should be erected.

Currently, molecular data are available for only five of the 11 valid species within the genus, but the inclusion of taxa from other biogeographic regions (e.g., Diadumene neozelanica, D. schilleriana) will undoubtedly contribute to our understanding of the diversification process within the group. Under the current scenario, it is clear that at least two nonrelated lineages of Diadumene are present in the southern hemisphere. Finally, the presence of D. leucolena in Brazil further confirms that some species within the genus have extensive distribution ranges, as it is the case of D. lineata, long considered a cosmopolitan species (Minasian, 1982; Podbielski et al., 2016). Due to the variability and the limited sampling within each species, conclusions about how these species disperse (e.g., natural process vs. human intervention) are beyond the scope of this study.

Utility of microcomputed CT scanning for the taxonomy of Actiniaria: In this study we evaluate the utility of micro-CT imaging for the taxonomy and systematics of sea anemones, which has been possible only as a result of recent protocol modifications of contrast enhancement in soft-bodied invertebrates (e.g., Metscher, 2009a, 2009b; Faulwetter et al., 2013a, 2013b; Descamps et al., 2014; Paterson et al., 2014; Fernández et al., 2014; Tessler et al., 2016; Holst et al., 2016). The appeal of micro-CT scanning for sea anemone studies is its potential to facilitate and improve characterization of morphological characters used in the taxonomy of the group especially in regard to the tridimensional anatomical representations based on of micro-CT images. Currently, morphological studies in Actiniaria combine complementary techniques: gross dissections provide information on internal features in three dimensions with low resolution whereas histology provides resolution at the cellular level in two dimensions. Hence, micro-CT methods combine qualities of both methods (i.e., high resolution with tridimensional qualities) with comparable cost (Chandler and Volz, 2004) while avoiding some of their shortcomings (e.g., slowness, sample preparation, small volumes, artifacts) (Shearer et al., 2016). When compared to dissections and histology, micro-CT scanning is also presumed to be nondestructive, easy, and fast. Thus, sea anemone taxonomy would highly benefit from incorporation of micro-CT data given the paucity of morphological characters whose number remain essentially unchanged since the beginning of modern studies (i.e., Hertwig, 1882, sensu Danielssen, 1890) and the difficulties involved in the establishment of primary homologies in the group. This often obscures our understanding of evolutionary patterns and morphological convergence found in molecular phylogenetic studies (e.g., Gusmão and Daly, 2010; Rodríguez et al., 2012, 2014). Hence, methods that can provide insights into details of traditionally used characters are imperative to advance our understanding of sea anemone systematics and evolution.

The protocol employed in this study (adapted from Tessler et al., 2016) using osmium tetroxide involved successfully staining the material so external and internal features were readily recognized in micro-CT scans (figs. 4, 8) as well as in resulting 3D renderings (figs. 4, 8). Staining is a crucial stage of micro-CT studies and is directly related to the final resolution of images of other soft-bodied organisms successfully stained with osmium tetroxide as a contrast enhancer (Handschuh et al., 2013; Fernández et al., 2014; Holst et al., 2016; Tessler et al., 2016), including other soft-bodied cnidarians (i.e., staurozoans, Holst et al., 2016). The core of Diadumene manezinha (fig. 8B, E, F), however, showed imperfect staining, which is probably a result of the self-limiting properties of osmium tetroxide due to large size and thickness of this specimen. This means that osmium tetroxide may not be be able to penetrate the tissues of large species of sea anemones, such as deep-sea ones, or it may penetrate too slowly to be practical. Likewise, phosphotungstic acid (PTA), known to stain muscle tissues more intensely (Metscher, 2009a), also presents depth limitations with a diffusion rate of approximately 1 mm per week (Fernández et al., 2014). Although PTA is far less toxic and simpler to use and dispose of compared with osmium tetroxide, it works best with alcohol-fixed specimens, rarely the case for freshly collected sea anemones, which are usually fixed in formalin for histological purposes. Perhaps for this reason, staining of sea anemones fixed in formalin and stained with 0.3% PTA in 70% ethanol did not lead to improvement in the resolution of details of the musculature in the material examined even when staining was successful (results not shown; L.C.G., personal obs.). Although iodine has been efficiently used to stain a variety of soft tissues (Metscher, 2009a, 2009b), the penetration of iodine was unsatisfactory during tests (results not shown; A.G., personal obs.), resulting in images with poor contrast and low resolution. We recommend the use of osmium tetroxide for chemical staining of sea anemones, but emphasize that differences in size, thickness, consistency, and texture of different species may require adaptation of the protocol used in this study depending on their physical properties.

The high-contrast micro-CT images obtained (figs. 4, 8) ensured that all relevant external and internal anatomical features of sea anemones were satisfactorily characterized. Micro-CT images were confirmed in observation of whole specimens and the traditionally used dissections and histology. These characters included hard-to-observe traits such as columnar cinclides (fig. 4A–E), small mesenteries without retractors (figs. 4F, G, 8E–G), basilar musculature (figs. 4L, 8J) and fertility patterns (fig. 8F), all of which are critical for correct identification of certain genera and species of actiniarians. Unprecedented for sea anemone taxonomy, reliable representation of the scanned specimens in 3D reconstructions was remarkable and corroborated the findings seen in the 2D micro-CT scans, but was particularly important in the establishment of mesentery growth patterns (figs. 4I, 8G) and comparison of the number of mesenteries proximally and distally (figs. 4G–I, 8E–G). This was a result of the ability to observe characters without having to section whole specimens, which is extremely laborious and timeconsuming to the point of being impractical using histology (Shearer et al., 2016); high-contrast micro-CT offered us the possibility of examining a specimen from multiple angles that does not have to be fixed a priori, as is the case for histology (Dinley et al., 2010; Berquist et al., 2012), and of manipulating a region of interest to depict a single character without obscuring others, something difficult to achieve in practice in dissections (Faulwetter et al., 2013a; Fernández et al., 2014; Tessler et al., 2016). Accuracy in the definition of these characters (e.g., growth patterns are used to distinguish Phelliactis Simon, 1892, and Paraphelliactis Carlgren, 1928; fertility patterns are important in defining Chondrophellia Carlgren, 1925, Amphianthus Hertwig, 1882, Stephanauge Verrill, 1899) and whether they contain any phylogenetic signal can be evaluated only when their presence and variability is accurately determined.

A limitation of the incorporation of micro-CT in taxonomic studies of sea anemones, however, pertains to determination of microanatomical musculature details: although it is possible to discern retractor (figs. 4G–H, J, 8E–F) and basilar musculature (figs. 4L, 8J) and their general morphology, the resolution obtained in the images did not permit observation of musculature details. Although microdetails in musculature are not used to distinguish Diadumene species, this shows that application of micro-CT may not be appropriate to study groups in which these microanatomical features (especially of musculature) are necessary for proper taxonomic descriptions. Micro-CT scanning may not be appropriate to study taxa for which the number of muscle processes in retractor or parietal musculature (e.g., Edwardsiidae de Quatrefages, 1842), observation of small sphincter musculature (e.g., Aiptasiidae or Haliplanellidae), or tentacle musculature (e.g., Actinostolidae Carlgren, 1893) are relevant. In addition, although tissue layers (i.e., epidermis, mesoglea and gastrodermis) exhibited varying attenuation levels in D. leucolena (fig. 4B–F) and D. manezinha (fig. 8B–D), tentacle musculature was not visible in scans of either species (figs. 4F, 8D). One drawback of micro-CT is that small marginal sphincter musculature cannot be determined from the scans (L.C.G. and A.G., personal obs.), as the resolution achieved in our studies was far coarser compared to that of histological sections, but this musculature can be observed in species that possess large mesogleal or endodermal marginal sphincters (results not shown). For the present study, the distinction is moot, since members of Diadumene do not possess marginal sphincter musculature. Given technical properties of the scanner, resolution is a trade-off between size and scanning time with higher resolution achieved faster in smaller specimens (Paterson et al., 2014). The muscle fibers of retractors, parietal or marginal sphincter musculature, for example, are probably at the resolution limit of the micro-CT scanner and cannot be portrayed with an appropriate resolution for proper observation. We agree with Holst et al. (2016) in that resolving issues of species complexes and cryptic species is not currently possible using micro-CT scanning and 3D renderings due to the method's low resolution compared with traditional techniques. Focusing on a small region of interest by dissecting the specimen can potentially increase image resolution up to the technical limit of the scanner, but tridimensional information is lost in the process.

The micro-CT scanning of the two species of Diadumene shows that this imaging technique can be a valuable tool to characterize taxonomic characters and maximize the potential of anatomical studies in Actiniaria. It is also possible that a different set of technical characteristics of the scanner (e.g., larger scanner detector) might improve the final resolution of the images in order to overcome the limitations observed for microanatomy. The speed with which 2D micro-CT images were acquired (i.e., on the order of hours) is a clear advantage of this method over histology, which can take several days and require specialized equipment and training (Shearer et al., 2016). This is only partly true, as manipulation of rendered images to display and isolate specific features was very time-consuming. Although micro-CT scanning speeds up the stage of obtaining data, unless the access to a micro-CT is unlimited (which was not the case for this study), the number of specimens scanned (i.e., low; one specimen in this study) prevents detection of variability and differences at the species level. Similarly, the nondestructive nature of micro-CT scanning is often proclaimed as a clear advantage of this method over dissections, and particularly over histology (Metscher, 2009a, 2009b; Dinley et al., 2010; Paterson et al., 2014; Nesteruk and Wiśniewski, 2015;). In our case, however, osmium tetroxide irreversibly changes tissue characteristics (Faulwetter et al., 2013a) and cannot be washed off after scanning (Fernández et al., 2014), rendering the specimens unusable for any other purpose. The high toxicity of osmium tetroxide makes its use as a staining agent a clear problem for valuable material.

The use of micro-CT would be especially advantageous when studying anemones that exhibit highly modified morphologies, which present a challenge for histology (e.g., members of Adamsia Forbes, 1840, Amphianthus, Peronanthus Hiles, 1899, Monactis (Gravier, 1918)). In cases in which micro-CT may not provide sufficient resolution, the combination of this technique with traditional ones (i.e., histology) is advisable. For sea anemones, this might be particularly important given that the microanatomical data on tissue derivation, shape, and development of musculature as well as reproductive structures and details of other structures provided by histology are essential for the identification to the species level (Spano and Flores, 2013). In addition, the necessity of squash preparations and examination of unstained samples (which provides size ranges and distribution of cnidae in a specimen) prevents the complete replacement of standard techniques by micro-CT alone. For this reason, virtual cybertypes (see Faulwetter et al., 2013a; Akkari et al., 2015) may never be appropriate for Actiniaria, but we agree with others (e.g., Faulwetter et al., 2013a; Fernández et al., 2014) that virtual representation can be a viable way to transfer data as well as being a potential tool for teaching (Berquist et al., 2012). Finally, given differences in size, consistency, texture, and thickness seen in different groups of sea anemones, the protocol provided in this study should be modified to the requirements and properties of each group. If combined with techniques traditionally used in sea anemone taxonomic studies, micro-CT scanning has the potential to solve morphological ambiguity, especially regarding characters for which tridimensionality is highly beneficial, thus helping resolve existing taxonomic issues in Actiniaria.