New Species and New Records of Sponge-Inhabiting Barnacles (Cirripedia, Balanidae, Acastinae) from Australia

The subfamily Acastinae contains a diverse group of barnacles that are obligate symbionts of sponges and alcyonacean and antipatharian corals. Integrating morphological and genetic (COI) data to compare against known species, this paper reports on nine species of sponge-inhabiting barnacles of the subfamily Acastinae, including three undescribed species (Acasta caveata sp. nov., Euacasta acutaflava sp. nov., and E. excoriatrix sp. nov.) and three species previously not recorded in Australian waters (A. sandwichi, Pectinoacasta cancellorum, and P. sculpturata). The new species are distinguished from similar species by a suite of morphological characters as well as genetic distances. A lectotype for Pectinoacasta cancellorum is designated. Sponge hosts were identified for all specimens where possible and are represented by 19 species from eight families and five orders.


Introduction
Barnacles can be found as obligate commensals with a variety of taxa, but those in symbiosis with sponges are proving to be particularly diverse [1][2][3][4]. The majority of sponge symbionts are classified under the subfamily Acastinae [5], with approximately 90 described species, and this number is expected to at least double [1,4]. Historically, this group had been arranged within informal groups based on characters of the fourth cirrus by Broch [6] and opercular plates by Hiro [7] under the genus Acasta sensu Leach, 1817 [8]. Neither author took this further, largely due to the close affinity with Conopea Say, 1812 [9], and Membranobalanus Hoek, 1913 [10], whose members are obligate commensals with cnidarians and sponges, respectively, but also Armatobalanus [10], which contained a mixture of free-living and commensal species. This caution has proven to be warranted, as a number of species have been transferred between these genera by subsequent workers [11]. Further to this, phylogenetic studies have also shown that the subfamily and family level arrangement is also unsupported within the Balanoidea [12,13]; as a result, the family Archaeobalanidae was recently synonymised with the Balanidae [14]. Kolbasov [5], in his revision of Acasta, was the first to evaluate host usage in the Acasta and how that was reflected in his newly proposed phylogeny and generic arrangement, which revealed some loose trends, but was hampered by a distinct lack of previously published host data.
Since then, more studies on sponge-inhabiting barnacles have been placing emphasis on host identification [1][2][3]15,16] to build a better understanding of host usage. Not only have the evolutionary relationships of the hosts begun to be looked at, but also the morphology of the host has been examined to test larval barnacle biology [17]. Integrating modern techniques such as genetic sequencing and computed tomography [18,19] has also provided greater precision when evaluating inter-and intra-specific variation by shedding light on character evolution.
The waters surrounding the Australian coast are inhabited by a highly diverse sponge fauna [20][21][22], leading to an expectation of a correspondingly diverse community of sponge-inhabiting barnacles. This paper reports on a collection of sponge-inhabiting barnacles from across Australian territorial waters as part of a broader study on the biodiversity of commensal barnacles.

Sampling and Morphological Examination
Specimens of both the barnacles and the sponges examined in this study are deposited in the following institutions: Western Australian Museum, Perth (WAM), Queensland Museum, Brisbane (QM), University of Copenhagen Zoological Museum, Denmark (ZMUC), and Seto Marine Biological Laboratory, Japan (SMBL). For direct morphological examination of barnacle shell plates and arthropodal characters, the body and associated soft tissues were removed from the shell. The remnants of the barnacle tissue and host sponge on the surfaces of the parietes, scutum and tergum, were removed using a brush and forceps. The shell was then immersed in 2% bleach for < 2 hrs hours to completely digest the organic tissue and was subsequently rinsed in water. Any remaining debris or contaminants were then removed by cleaning in an ultrasonic cleaner for < 5 s. The specimens were examined under a Leica M205 C (Leica, Germany) stereomicroscope and digital photographs produced with a Leica DMC4500. All images and figures were processed using GIMP 2.10 (www.gimp.org accessed on 20 June 2021) or Inkscape 0.92 (www.inkscape.org accessed on 20 June 2021). Barnacle size is measured in millimetres along the rostro-carinal (RC) axis at the basal rim. Setal terminology follows that of Chan et al [23].
Sponge identifications were partly facilitated by examining gross morphology, surface characteristics, and microscopic skeletal characteristics of specimens. Subsamples of sponge tissue were sectioned at right angles to the surface of the sponge and processed for microscopic examination using a Shandon Elliott tissue processor. This included ethanol dehydration, histolene to clear the tissue, and subsequent paraffin impregnation. Thick sections (~90 μm) were cut with a Leitz slide microtome from wax blocks. Sections were de-waxed in histolene and mounted using Shandon EZ-Mountant. Skeletal slides were examined with an Olympus BX50.

Molecular Analysis
Adductor or depressor muscle tissues of barnacles were subsampled from specimens, and genomic DNA was extracted using either a Bioline Isolate II or Qiagen DNeasy extraction kit following the manufacturer's instructions. Partial fragments of the cytochrome c oxidase I (COI) gene were amplified using the primers dgLCO1490 5'-GTCAACAAATCATAAAGAYATYGG-3' and dgHCO2198 5'-TAAACTTCAGGGTGACCAAARAAYCA-3' [24] in a 25 μL reaction volume and consisting of 2 μL DNA extract, 1 unit MyTaq DNA polymerase, MyTaq PCR buffer, and 0.1 μM of each primer. The following polymerase chain reaction conditions were used: 2 min at 95 °C for initial denaturing, then 35 cycles of 30 s at 95 °C, 30 s at 46 °C, 45 s at 72 °C, and a final extension for 7 min at 72 °C. The resulting amplicons were sequenced at the Australian Genome Research Facility, Perth, using the same primers via Sanger (cycle) sequencing.
The sequences were assembled and trimmed using Geneious Prime and submitted to GenBank (Table 1, https://www.ncbi.nlm.nih.gov/genbank/ accessed on 20 June 2021). Quality control of sequences involved trimming low-quality bases, inspecting for stop codons, verifying the reading frame, checking for matches on BLAST, and re-examining original specimens.
Representative COI sequences identified as belonging to the genera studied herein were downloaded from GenBank and BoLD (www.boldsystems.org) ( Table 1). Membranobalanus porphyrophilus Hosie and Jones, 2019 [18], was selected to serve as an outgroup in the phylogenetic analyses. The combined dataset was aligned in Geneious Prime 2020.2.5 (https://www.geneious.com) and checked manually. The Assemble Species by Automatic Partitioning (ASAP; [25]) software was used to compare the genetic data with the morphological identifications by delimiting putative species under three substitution models: Jukes-Cantor (JC69) [26],   [27], and uncorrected p-distance. Pairwise genetic distances were calculated using the software MEGA v10 [28]. Phylogenetic reconstructions using a maximum likelihood analysis were conducted using the IQ-TREE webserver [29]. Model selection was automatically assessed by the webserver, with the COI data partitioned by codon, and branch support was calculated by conducting 1000 ultra-fast bootstraps [30]. Bayesian inference analyses were conducted in MrBayes 3.2.6 [31] utilising the CIPRES webserver [32] with four heated chains, a General Time Reversible model with proportion of invariable sites and rate heterogeneity across sites (GTR+I+G), an MCMC of 11 million generations, and a burn-in of 10%. Diagnosis: Shell pinkish or yellow-brown, orifice edge toothed. Basis cup-shaped with crenate edge. Radii with creased edges. Externally, parietes rugged with several irregularly spaced calcareous projections; internally, sheath extending over half of parietes with vesicular structure, inner surface below sheath with longitudinal ribs. Scutum with feeble growth ridges and longitudinal striations, articular furrow shallow. Tergum beaked, apex tinged with red, spur truncated, width less than 0.5 of basal margin. Curved teeth on anterior ramus of cirrus IV feeble. Cirrus V protopod with vertical row of denticles on posterior edge. Distribution: Australia: Perth-Montebello Islands, WA. Taiwan [3]. 0-16 m. Hosts: Lissodendoryx (Acanthodoryx) KMB1 (Coelosphaeridae), and Hamigera PB1 (Hymedesmiidae). Previous records: Crella (Yvesia) spinulata (Hentschel, 1911) [35] (Crellidae), and Iotroata sp. [3] (Iotrochotidae).
Remarks: This species was originally identified inhabiting Iotroata sp. and Crella (Yvesia) spinulata from the central western coast of Australia and Taiwan, respectively. Here, we report two additional host species from the families Coelosphaeridae and Hymedesmiidae, demonstrating that A. aspera is a relative generalist, capable of colonising sponge species from several families within the order Poecilosclerida.
Acasta caveata sp. nov. (Figures 1-4 Diagnosis: Shell fenestrate; very large, subrectangular windows present between each parietal junction, occupying over half of parietal height, as wide as or wider than adjacent parietes at basal rim. Parietes with blunt, calcareous projections, spaced irregularly in longitudinal rows. Basis saucer-shaped, rim undulating, crenate where it interlocks with parietes, otherwise smooth. Cirrus IV protopod with single horizontal tooth, anterior and posterior rami with hooked teeth arranged in triangular shape. Cirrus V anterior ramus with hooked teeth. Penis basidorsal point prominent. Description: Shell ( Figure 1A-M) erect, slightly elongate vertically, plates semitransparent, white or tinged red or burgundy near apices. Externally, parietes with fine, horizontal growth lines and short, irregularly spaced spines; apices of parietes curved toward orifice. Radii and alae summits oblique, not reaching basal margin of corresponding parietes, leaving elongate, subrectangular membranous windows between parietes. With ontogeny, windows secondarily filled with calcareous shell, maintaining lower margin approximately level with basal margin of sheath. Internally, sheath adpressed with vesicular structure, faint horizontal growth lines, lower margin separated from inner lamina except on carina; inner lamina with weak longitudinal ribs extending to basal rim, lateral margins inflected or thickened.
Basis ( Figure 1H,M) saucer-shaped, making up approximately a quarter of total shell height, with six wide and shallow longitudinal furrows corresponding to the bases of parietes; rim undulating, crenate where interlocking with parietes, inter-parietal rim concave, smooth.
Scutum ( Figure 1N-P) semitransparent, triangular, approximately as high as wide, growth lines prominent with fine crenulations and fringed with short, inconspicuous setae; basitergal angle broadly rounded; occludent margin toothed. Internally smooth, adductor muscle pit weakly defined, lateral depressor muscle pit well defined; articular ridge low, parallel with tergal margin, with shallow articular furrow, extending approximately 0.75 length of tergal margin.
Tergum ( Figure 1Q-R) semitransparent, apex acute, tinged with red; external growth lines fringed with short inconspicuous setae; articular margin concave in apical half, carinal margin evenly arcuate, basal margin slightly sinuous on carinal side of spur; scutal side straight, but obtuse to, articular margin; spur length approximately twice width, occupying approximately half of basal margin, positioned approximately half own width from scutal margin; spur and furrow margins coincident, defined by abrupt change in growth lines and corresponding groove on articular side and gentle change on carinal side; distal margin convex, obliquely truncate. Internally smooth, crests for depressor muscles faint or absent.
Labrum (Figure 2A-B) bilobed, lobes separated by deep, medial, v-shaped notch, each rounded lobe with two or three marginal teeth and numerous fine setae.
Mandible ( Figure 2D-F) with five teeth, second to third teeth bifid, fourth and fifth teeth serrate of moloriform, inferior angle with denticles. Dense, short setae regularly spaced on inner and outer faces, longer, fine setae on inferior and superior margin.
Maxillule ( Figure 2G-H) cutting margin straight, with 11 cuspidate setae, upper and lower pairs longer than remaining setae, medial setae serrulate on inferior margin. Dense, short setae regularly spaced on inner and outer faces, longer, fine setae on inferior and superior margin.
Maxilla ( Figure 2I) bilobed, lobes ovate, distal lobe elongate, curved towards mouth, serrulate setae arranged on anterior margins, becoming more dense at apex, longer on distal lobe than those on basal lobe.
Cirral segment counts are given in Table 2.
Cirrus III ( Figure 3C-E) protopod anterior margin lined with long, plumose setae, posterior margin with pappose setae basally and plumose setae distally. Rami unequal, anterior ramus approximately 1.25 times longer than posterior ramus, setation lasiopod, segments with serrulate and simple setae on mesial face, less densely arranged than cirrus I and II. Anterior ramus with row of sharp, erect spines on anterodistal portion of all segments, except terminal segment, most prominent on medial segments. Posterior ramus similar but spines smaller.
Cirrus IV ( Figure 4A-E) protopod elongate, length 1.8 times width, with simple setae on anterior and posterior margins, tuft of simple setae at posterodistal angles of both segments; basis with single tooth at anterodistal angel. Rami subequal, setation ctenopod, segments with 3 pairs of serrulate setae on anterior margin. Anterior ramus with up to 5 hooked teeth arranged in a rough triangular formation on anterior margin of first 15 segments, anterodistal angle with group of erect spines. Posterior ramus with 1 or 2 hooked teeth on segments 10-21, anterodistal angle with group of erect spines.
Cirrus V ( Figure 4F-H) protopod elongate, length 1.38 times width, with simple setae on anterior and posterior margins, anterodistal angle unarmed, tuft of simple cuspidate setae at posterodistal angles of both segments. Rami subequal, setation ctenopod, anterior ramus with 3 and posterior ramus with 4 pairs of serrulate setae on anterior margin, posterior ramus with four and both rami of cirrus VI with 4 pairs of serrulate setae; posterodistal angles with tuft of simple setae. Anterior ramus with 1 or 2 hooked teeth on segments 13-21.
Cirrus VI ( Figure 4I) protopod elongate, length 1.4 times width simple setae on anterior and posterior margins, lacking denticles or erect spines on anterodistal angles, tuft of simple cuspidate setae at posterodistal angles of both segments. Rami subequal, setation ctenopod, with 4 pairs of serrulate setae; posterodistal angles with tuft of simple setae; anterior margins without teeth or erect spines.
Hosts: Gelliodes KMB1 (Niphatidae). Distribution: Western Australia: Exmouth Gulf. 23 m. Etymology: The specific epithet is derived from the Latin noun cavea (cage, hollow, cavity), in reference to the large membrane-covered windows that give the shell a cagelike appearance.
Remarks: This species, with its very conspicuous membranous windows, is externally similar to a number of species of Acasta. The most similar are A. sandwichi Yu et al. 2017 [2] and A. pertusa Kolbasov 1990 [36], which exhibit close morphological similarities in the form of the windows and opercular plates. The external shell and opercular plates are nearly identical, but with some potential differences in the relative widths of the windows and adjacent parietes. The windows of A. caveata sp. nov. occupy more than half the shell height (excluding basis) and are level with, or just above, the lower margin of the sheath, thus larger than in Acasta sandwichi and A. pertusa where the margin is distinctly lower than the sheath. In A. caveata, the free, lower margins of the parietes are parallel with the basis, giving the windows a more rectangular shape, whereas in A. pertusa and A. sandwichi, they are curved, giving them a more arched look. The key arthropodal character to distinguish these species is the armature of cirri IV and V. For A. caveata, both the anterior and posterior rami of cirrus IV and the anterior ramus of cirrus V bear strong hooked teeth, whereas only a single, straight "denticle" is found on each segment of the anterior ramus of cirrus IV in A. sandwichi, while such armature is completely absent in A. pertusa.
Of the other species with conspicuous windows (A. armata Gravier, 1921 [37], A. crucibasis Yu et al., 2017 [2], A. fenestrata Darwin, 1854 [38], A. foraminifera Broch, 1931 [6], and A. tzetlini Kolbasov, 1992 [39]), only A. tzetlini bears teeth, but only on cirrus IV anterior ramus, and it has much smaller windows with the parietes articulated with the basis over most of its circumference. In addition to lacking teeth on cirrus IV, A. armata and A. fenestrata lack longitudinal ribs on the inner lamina of the parietes; A. armata also has very distinct opercular plates, and the windows of A. fenestrata are significantly smaller. The carinolatus of A. foraminifera and A. crucibasis do not reach the basis at all but are instead suspended between the latus and the carina.      Diagnosis: Shell typically globuloconic, parietes with irregularly spaced spines, radii and alae oblique, membranous windows present between each shell plate, windows may reach half height of shell plates, maximum width approximately same as carinolatus at basal margin. Tergal spur truncate, basal margin concave with disto-articular angle projecting. Cirri III and IV anterior rami with row of sharp erect spines on anterodistal portion segments. Both rami of cirri IV-VI with specialised multicuspidate setae at posterodistal angles of basal-most segments.
Description: Shell ( Figure 5A-G) plates white or tinged red or burgundy, typically globuloconic, but may elongate to match growth of host. Externally, parietes with fine, horizontal growth lines and short, irregularly spaced spines; apices of parietes curved toward orifice. Radii and alae oblique, not reaching basal margin of corresponding parietes, leaving elongate membranous windows between parietes. With ontogeny, windows secondarily filled with calcareous shell to maintain apical margin with internal basal margin of sheath. Internally, smooth below sheath, without longitudinal ribs, lateral margins not inflected or thickened. Sheath adpressed.
Basis ( Figure 5G) cup-shaped, depth varying from nearly flat to making up more than twice total shell height ( Figure 5A); rim smooth.
Scutum ( Figure 5H-I) triangular, apex coloured dark red or purple, slightly higher than wide, growth lines prominent with fine crenulations and fringed with short, inconspicuous setae; basitergal angle broadly rounded; occludent margin strongly toothed.
Internally smooth with slight depressions for adductor and lateral depressor muscles; articular ridge prominent, with deep furrow, extending halfway along tergal margin.
Tergum ( Figure 5J-K) apex acute, tinged dark red or purple, growth lines fringed with short, inconspicuous setae; articular margin concave in apical half, carinal margin slightly convex almost straight, basal margin straight; spur length 0.5 times width, occupying approximately half of basal margin, positioned less than half own width from scutal margin; spur and furrow margins coincident, defined by abrupt change in growth lines and corresponding groove on articular side and gentle change on carinal side; spur truncate distal margin slightly concave with, disto-articular angle projecting ( Figure 5J-K). Internally smooth, crests for depressor muscles faint.
Labrum ( Figure 6A-B) bilobed, lobes separated by deep medial v-shaped notch, each rounded lobe with two or three marginal teeth and numerous fine setae.
Mandible ( Figure 6C-D) with five teeth, second to fourth teeth bifid, inferior angle with denticles. Dense, short setae regularly spaced on inner and outer faces, longer fine setae on inferior and superior margin.
Maxillule ( Figure 6E-G) cutting margin straight, with 9-11 cuspidate setae, upper and lower pairs longer than remaining setae, 3 medial setae serrulate on inferior margin. Dense, short setae regularly spaced on inner and outer faces, longer fine setae on inferior and superior margin.
Maxilla ( Figure 6H) bilobed, lobes ovate, serrulate setae arranged on anterior margins, becoming more dense at apex, longer on distal lobe than those on basal lobe.
Cirral segment counts are given in Table 2.
Cirrus I ( Figure 7A-B) protopod with pappose setae basally on protopod posterior margin. Rami unequal, anterior ramus twice length of posterior ramus, segments with densely arranged serrulate and simple setae on mesial face.
Cirrus III ( Figure 7D-E) protopod anterior margin lined with long plumose setae, posterior margin with pappose setae basally and plumose setae distally. Rami subequal, anterior ramus 1.15 length of posterior, segments with serrulate and simple setae on mesial face, less densely arranged than cirrus I and II. Anterior ramus with row of sharp, erect spines on anterodistal portion of all segments, except terminal segment, most prominent on medial segments.
Cirrus IV ( Figure 8A-D) protopod elongate, length 1.83-1.95 times width, with simple setae on anterior and posterior margins, lacking denticles or erect spines on anterodistal angles, tuft of simple, cuspidate setae at posterodistal angles of both segments. Rami subequal, setation ctenopod, segments with 3 pairs of serrulate setae on anterior margin; posterodistal angles of basal-most segments with specialised, multicuspidate setae with pair of denticles situated midway along setae. Anterior ramus with row of sharp, erect spines on anterodistal portion of all segments except terminal segment, most prominent on medial segments.
Cirrus V ( Figure 8E-G) and VI similar, protopod elongate length 1.32-1.38 and 1.21-1.31 times width, respectively, with simple setae on anterior and posterior margins, lacking denticles or erect spines on anterodistal angles, tuft of simple cuspidate setae at posterodistal angles of both segments. Rami subequal, setation ctenopod, cirrus V anterior ramus with 3 pairs of serrulate setae margin, posterior ramus and both rami of cirrus VI with 4 pairs of serrulate setae; posterodistal angles of basal-most segments with specialised multicuspidate setae with 1 pair of denticles situated midway along setae.
Penis longer than cirrus VI, annulated, sparsely setose, basidorsal point may be low and rounded or triangular and pointed.
Remarks: In the literature, descriptions attributed to this species show significant variations that lead us to conclude that more than one species has been confounded under the name A. fenestrata. Darwin [38] only described the shell and opercular plates, stating that the body was poorly preserved, but that as far as he could tell, the cirri and mouthparts were similar to Neoacasta glans (Lamarck, 1818) [57]. The first description of the mouthparts and cirri to be attributed to A. fenestrata was provided by Utinomi [51], who stated that cirrus IV had hooked teeth on the anterior ramus, a feature not found in N. glans. Another departure from Darwin's description is that the tergum, as figured, is much narrower in Utinomi's specimens with the spur rounded, rather than truncated, and situated confluent with the basiscutal angle. Rosell [52] noted several differences between his accounts and those by Darwin and Utinomi, but maintained the identification based on the similarity with Utinomi's description of the mouthparts and cirri. Key differences that Rosell highlighted were the presence of pliable, chitinous spines seated on calcareous "elevations" on the parietes, as opposed to the simple, short, blunt, calcareous spines described in other accounts, internal basal ribs on the parietes, and a crenated rim on the basis. The specimen figured by Foster and Buckeridge [53] has a generally globose appearance, with relatively broader radii, smaller windows, and hooked teeth on cirrus IV. Their records also extended the known depth range from 65 to 195 m.
These records contrast with the present specimens and the descriptions and illustrations given by Ren [45], Liu and Ren [47], Sulistiono et al. [49], and Wibowo et al. [48], where the examined specimens only have erect spines on the anterior margins of cirrus IV. Our material also presents a novel character not mentioned by previous authors: the multicuspidate setae on the posterodistal angles of cirri IV-VI ( Figures 8D,G). A re-examination of type specimens, and possibly type host, along with additional specimens from across the reported geographic and host range would provide clarity to this species concept.    Diagnosis: Shell fenestrate, windows present between each parietal junction, very large, occupying over half of shell height, as wide as or wider than adjacent parietes at basal rim. Parietes with blunt, calcareous projections, spaced irregularly in longitudinal rows. Basis shallow saucer-shaped, rim undulating, crenate where it interlocks with parietes, otherwise smooth. Cirrus IV anterior ramus with single denticle on anterior margin of segments.
This species has a morphologically and genetically close relationship to A. caveata sp. nov., and the morphological characters separating the two are dealt with under the treatment for that species. Distinguishing A. sandwichi from other Acasta with large windows was detailed by Yu et al. [2], and of these, the only A. fenestrata currently known is from Australia. This species can be distinguished by the relatively smaller windows, the lack of longitudinal ribs on the internal surface of the parietes, and the broader and more truncate tergal spur; the cirrus IV anterior ramus has erect spines on the anterodistal angles of segments and the stout multicuspidate setae are present on the posterodistal angles of cirri IV-VI. Further to this, there is no indication that either species inhabit the same hosts.

Euacasta Kolbasov, 1993 [5]
Euacasta acutaflava sp. nov. (Figures 9-12 Diagnosis: Shell squat, cuboid; parietes thick, strongly articulated, growth lines pronounced, inflected upwards at lateral margins, inner lamina with broad, prominent longitudinal ribs. Basis flat, square in outline with angular corners, diagonal grooves running from each corner. Opercular plates with yellow-orange, chitinous cuticle in apical section, particularly prominent on tergum; scutum with growth lines scalloped by longitudinal ridges. Cirrus IV with 2-4 hooked teeth on basis of protopod; anterior ramus with up to 3 teeth on basal segments. Description: The shell ( Figure 9A-Q) is white and squat with maximum diameter greater than shell height and square in outline. The parietes are thick and strongly articulated; externally having variably pronounced and irregularly spaced longitudinal ribs, with horizontal growth lines inflected upward on raised lateral margins; the parietes are slightly curved toward the orifice, the latter only slightly smaller than the diameter of the shell. The opercular plates are exposed, with the apices extending beyond the apices of the parietes. The radii and alae reach the basal margin of the corresponding parietes, and the summits are oblique and broad with horizontal striations. Rostrum, laterals, and carina are approximately equal in width; carinolateral is exceptionally narrow, the parietal area is represented by a beaded strip, and radii and alae are similar to other plates. Internally, the sheath has conspicuous horizontal growth lines, and the lower margin is separated from the inner lamina; below the sheath, broad, longitudinal, parietal ribs are very prominent.
The basis ( Figure 9H-I) is flat and quadrangular with distinct angular corners; the concentric growth lines are conspicuous; the basal rim is strongly crenate, interlocking with internal ribs on the parietes.
The scutum ( Figure 9R-S) is triangular, with height 1.35 times its width, and growth lines scalloped by longitudinal ridges; the ridges are increasing in number basally; the basitergal angle is rounded, and the tergal section is narrow; the occludent margin is strongly toothed. Internally smooth, the apex has a yellow chitinous cuticle, and adductor and lateral depressor muscle pits are present; the articular groove is deep, bounded by a distinct articular ridge, the latter extending halfway along the tergal margin.
The tergum ( Figure 9T-U) apex is acute and beaked, with calcification giving way to a yellow-orange chitinous point, and the apex projecting beyond scutum when articulated; growth lines are prominent, fringed with short, inconspicuous setae; the yellow chitinous cuticle extends onto the carinal half; and the articular margin is concave in the apical half. The carinal margin is evenly concave; the basal margin is straight; the spur is truncate, the length is 0.5 times the width, occupying approximately half the basal margin, positioned less than half its own width from the scutal margin; the spur and furrow margins are coincident, the spur furrow is defined by an abrupt change in growth lines and a corresponding groove on the articular side, and a gentle change on the carinal side; internally smooth, the chitinous cuticle covers the upper half, and the crests for depressor muscles are faint. The articular furrow is broad, the articular ridge is low and rounded, occupying 0.25 of the scutal margin.
The labrum ( Figure 10A-B) is bilobed, divided by a deep, medial, v-shaped notch, and each rounded lobe has two or three marginal teeth and numerous fine setae.
The mandibular palp ( Figure 10A,C) is rhomboid, distal, and obliquely truncate, the anterior margin is concave, and the posterior margin is straight; the setae are heavily serrulate, becoming longer and denser distally; the distal angle has long, simple setae.
The mandible ( Figure 10D-G) cutting margin has four distinct teeth, the second to fourth teeth are bifid, and the fifth tooth is congruent with molariform or spinose inferior angle; the superior and inferior margins have a row of long, fine, simple setae, with dense short setae covering the inner and outer faces.
The maxillule ( Figure H) cutting margin is straight with 9-10 cuspidate setae, the upper and lower pairs are larger and more robust than the remaining setae, and the inferior angle has a tuft of shorter cuspidate setae. Dense, short setae are regularly spaced on the inner and outer faces, with longer fine setae on the inferior and superior margin.
The maxilla ( Figure 10I) is bilobed, the lobes are ovate, and serrulate setae are arranged on the anterior margins, becoming denser at the apex and longer on the distal lobe than those on the basal lobe.
Cirral segment counts are given in Table 2.
The cirrus I ( Figure 11A) protopod has pappose setae basally on the posterior margin. The rami are unequal, with anterior ramus 3.9 times the length of the posterior ramus and lasiopod setation, and segments have densely arranged serrulate and simple setae on the mesial face.
The cirrus II ( Figure 11B-C) protopod anterior margin is lined with long plumose setae, posterior margin with pappose setae basally and plumose setae distally. The rami are unequal, the anterior ramus is 1.25 times longer than the posterior ramus, and segments have densely arranged serrulate and simple setae on the mesial faces.
The cirrus III ( Figure 11D-F) protopod anterior margin is lined with long plumose setae, the posterior margin with pappose setae basally and plumose setae distally. The rami are subequal with anterior ramus 1.1 times the length of the posterior ramus, and segments have serrulate and simple setae on the mesial face, less densely arranged than cirrus I and II. The anterior ramus has a row of ctenoid scales on the anterodistal portion of all segments, most prominent on medial segments.
The cirrus IV ( Figure 12A-F) protopod has simple setae on the anterior and posterior margins, and a tuft of simple, short setae at the posterodistal angles of both segments; the basis is elongate, with length 3.1-3.4 times the width, and the distal quarter of the anterior margin has a row of 2-4 hooked teeth increasing in size distally. The rami are subequal with ctenopod setation. The anterior ramus has 1-3 hooked teeth on the first nine segments, basal segments have one pair of serrulate setae at the anterodistal angle, and intermediate segments have two pairs. The posterior ramus with three pairs of setae on intermediate segments.
The cirrus V ( Figure 12G-I) and VI ( Figure 12J-K) protopods both have simple setae on the anterior and posterior margins and a tuft of simple, short setae at the posterodistal angles of both segments; the basis is less elongate than cirrus IV, with length 1.7-2 and 1.5-1.7 times width, respectively, without hooked teeth; the rami are subequal with intermediate segments having three or four pairs of setae. The anterior rami lack hooked teeth or erect spines.
The penis ( Figure 12L) is longer than cirrus VI, annulated, and sparsely setose. The basidorsal point is low and rounded.
Etymology: The species epithet is derived from the Latin acuta (point, sharp) and flava (yellow) in reference to the yellow-orange chitinous tips of the terga.
Remarks: This species presents interesting variability in the details of the parietes. The longitudinal ribbing may be very faint, as it is in the holotype, to very pronounced (compare Figure 9A-E with Figure 9F-G). In some specimens, the basal margins are markedly convex, making the basal perimeter scalloped. The junctions between the rostrum, carina, and latera in these specimens cause the basal plate to form acute angles that are drawn up from the basal plane. This in turn causes grooves to run diagonally across the basis. In small specimens (~2 mm diameter), the horizontal growth lines appear denticulate and fringed with chitinous setae. The growth lines are especially pronounced at the lateral margin, where they inflect upwards and seem almost spine-like.
The chitinous apex to the opercular plates is also a character unique to the E. acutaflava sp. nov. among the Euacasta. Further distinguishing features include the hooked teeth of cirrus IV protopod, which are restricted to the distal-most portion, whereas E. excoriatrix sp. nov., E. ctenodentia, E. dofleini, E. porata, and E. zuiho all have numerous teeth lining at least half of the basis (but see Yu et al. [68]). Both E. abnormis and E. tabachniki completely lack hooked teeth on cirrus IV, while E. sporillus, as figured by Kolbasov [5] and Yu [68], has three well-spaced teeth.    Other material: WAM C58573, same data as for holotype. Diagnosis: Shell white, parietes studded with irregularly spaced, short calcareous spines. Basis concave, quadrangular, drawn up into angular corners at junctions of parietes, diagonal grooves crossing at the centre. Scutum elongate, prominent growth ridges crossed with longitudinal ridges, giving a beaded appearance. Tergum narrow, falcate apex beaked, spur broad, occupying over half of basal margin. Mandibular palp with distal corner produced into long narrow projection. Cirrus I posterior ramus ~5 times length of anterior ramus. Cirrus III anterior ramus with small, erect spines on anterodistal angle. Cirrus IV protopod with row of hooked teeth on 0.75 of anterior margin and group of erect spines on anterodistal angle of basis; anterior ramus with 2-3 hooked teeth on anterior margin and group of erect spines on anterodistal angle. Cirrus V with or without hooked teeth on protopod, anterior ramus with single tooth on medial segments.
Description: Shell ( Figure 13A-O) white, slightly globose, parietes divergent, only slightly curved toward orifice, diameter of orifice equal to or greater than basis. Parietes externally with irregularly spaced, short, blunt calcareous projections, horizontal growth lines faint, basal margins convex. Radii and alae reaching basal margin of corresponding parietes, summits oblique, broad with fine horizontal and oblique striations. Rostrum and carina broadest plates, approximately equal in width; carinolateral exceptionally narrow, parietal area represented by thin strip, radii and alae similar to those on other plates. Internally, sheath occupying one third of inner lamina, with conspicuously raised, horizontal, growth ridges, lower margin separated from inner lamina, below sheath, narrow, longitudinal, parietal ribs extending to basal margin.
Basis ( Figure 13J,O) shallow, subrectangular in outline, with rounded corners and convex sides; corners drawn up into junctions between parietes; conspicuous grooves running diagonally from each corner in "x"-shape; concentric growth lines conspicuous; basal rim finely crenate, interlocking with internal ribs on parietes.
Tergum ( Figure 13R-S) narrow, apex tinged with red, acute, strongly beaked, projecting beyond scutum when articulated; growth lines fine, well-spaced, fringed with short, inconspicuous setae; carinal margin evenly concave; basal margin straight; spur distally truncate, broad, length 0.3 times width, occupying approximately 0.6 of basal margin, positioned approximately 0.15 own width from scutal margin; spur and furrow margins coincident, spur furrow defined by abrupt change in growth lines on both sides; internally smooth, crests for depressor muscles absent; articular furrow narrow, articular ridge low, rounded occupying approximately 0.3 scutal margin.
Mandible ( Figure 14D-E) cutting margin with five distinct teeth, second and third teeth bifid, fourth tooth becoming molariform, fifth tooth congruent with molariform or spinose inferior angle; superior and inferior margins with row of long, fine simple setae; dense, short setae covering inner and outer faces.
Maxillule ( Figure 14F-G) cutting margin straight with 10-12 cuspidate setae, upper and lower pairs larger and more robust than remaining setae; inferior angle with tuft of shorter cuspidate setae. Dense, short setae regularly spaced on inner and outer faces, longer fine setae on inferior and superior margin.
Maxilla ( Figure 14H) bilobed, lobes ovate, serrulate setae arranged on anterior margins, becoming more dense at apex, longer on distal lobe than those on basal lobe.
Cirral segment counts are given in Table 2.
Cirrus II (Figure 15B-C) protopod anterior margin lined with long plumose setae, posterior margin with pappose setae basally, plumose setae distally. Rami unequal, anterior ramus 1.33 times longer that posterior ramus, segments with densely arranged serrulate and simple setae on mesial face, transverse row of ctenoid scales on distal margin of each segment.
Cirrus III ( Figure 15D-E) protopod anterior margin lined with long plumose setae, posterior margin with pappose setae basally, plumose setae distally. Rami subequal, anterior ramus 1.1 times longer that posterior ramus segments with serrulate and simple setae on mesial face, less densely arranged than on cirrus I and II. Anterior ramus with row of small, erect spines and ctenoid scales on anterodistal portion of all segments, except terminal segment, most prominent on medial segments.
Cirrus IV ( Figure 16A-G) protopod with simple setae on anterior and posterior margins, tuft of simple, short setae at posterodistal angles of both segments; basis elongate, length 3.1-3.4 times width, anterior margin with row of 9-12 hooked teeth extending 0.75 length, evenly sized or increasing in size distally, anterodistal angle with additional upward turned teeth and erect spines. Rami subequal, setation ctenopod. Anterior ramus with 1-3 hooked teeth on first 10-11 segments, anterodistal angle with erect spines and ctenoid scales; basal segments with 1 pair of serrulate setae at anterodistal angle, intermediate segments with 2 pairs. Posterior ramus without hooked teeth, anterodistal angle with small erect spines, ctenoid scales on distal margin, 3 pairs of setae on intermediate segments.
Cirrus V ( Figure 16H-J) protopod with simple setae on anterior and posterior margins, tuft of simple, short setae at posterodistal angles of both segments; basis less elongate than cirrus IV, length 2.2-2.5 times width, up to 3 teeth distally on anterior margin or teeth absent. Rami subequal, intermediate segments with 3 or 4 pairs of setae. Anterior ramus with 1 hooked tooth on 11 th -18 th segments; posterior ramus lacking teeth.
Cirrus VI ( Figure 16L-M) similar, protopod with simple setae on anterior and posterior margins, tuft of simple, short setae at posterodistal angles of both segments; basis less elongate than cirrus IV, length 1.6-2.1 times width, respectively, without hooked teeth. Rami subequal, intermediate segments with 3 or 4 pairs of setae. Anterior rami lacking hooked teeth or short erect spinules along distal margins.
Etymology: The species epithet translates as "the flayer" and is derived from the Latin excorior (to flay, to skin), this is in reference to the long whip-like cirri and the strong hooked teeth on cirrus IV and V that are used to scrape away encroaching host tissues to prevent being overgrown.
Remarks: This species is characterised by several features that separate it from the remainder of the genus. In particular, the beaked tergum, the relative lengths and armature of the cirri, and mandible palp clearly define the morphological boundaries of this species. The upwardly directed teeth on the anterodistal angle of the protopod of cirrus IV also appear to be unique within the genus, as the other species have only been documented with teeth that are horizontal or hooked downward. Both E. abnormis and E. tabachniki have a tergum with a similarly beaked apex, but these species lack teeth on cirrus IV and V; the mandibular palps are not elongated; in E. abnormis, there are no internal parietal ribs; and E. tabachniki has long spines on the parietes [61,65]. In external appearance, this species is most similar to E. zuiho (sensu Hiro 1936 [45]), but this species has a typically truncated mandibular palp, shorter cirri (segment counts can be up to half of those in the present specimens), and the anterior ramus of cirrus I is approximately 2.5 times longer than the posterior, compared with 5 in E. excoriatrix [66].  Figure  14, Supplementary Figures S10, 12) show a prominently beaked tergum, and, in the case of Ren, a mandible with a slightly produced distal angle. Their accounts still differ from the present species, having relatively short cirri and no erect spines on the protopod basis of cirrus IV. Further to this, the ribs on the inner lamina of the parietes are restricted to the basal margin, and the basidorsal point of the penis is absent in Ren's specimens. Lastly, in Yu et al., the tergal spur is distinctly separated from the basi-scutal angle. These records show some differences with those of the original description [66] and potentially also represent different species.
The armature of cirrus IV is rather constant, with only small changes in the number of teeth on the protopod and segments, but the variation seen in the protopod of cirrus V is peculiar and not symmetrical. One of the paratypes (WAM C61475) bears two small, upturned teeth on the left and three hooked teeth on the right, while the holotype bears no trace of teeth.     Kolbasov, 1993 [5] Pectinoacasta cancellorum (Hiro, 1931) [69] (Figures 17-20 [7].

Pectinoacasta
Pectinoacasta cancellorum Kolbasov 1993: p. 411 [5]. Diagnosis: Shell globose, parietes permeated with narrow longitudinal incisions extending from basal rim and small membranous windows at parietal junctions; externally surfaces studded with short sharp calcareous spines; internally longitudinal ribs extend below sheath and interlock with crenate basal rim. Scutum with longitudinal ridges crossing growth ridges. Tergum with setose, yellow chitinous cuticle covering external surface of carinal side of spur furrow, growth ridges crossed with longitudinal ridges giving a latticed appearance. Cirrus IV protopod with row of 6-7 teeth on anterior margin, anterior ramus basal segments with 1-2 recurved teeth on anterior margins.
Description: Shell ( Figure 17A-D, J-K), white or grey; orifice small relative to shell diameter, rim bluntly toothed. Parietes with longitudinal incisions extending from basal rim to over half parietal height, creating narrow, membrane-covered slits, incisions secondarily calcified in upper portions of parietes; external surfaces with numerous short, sharp, calcareous projections arranged irregularly, or in longitudinal rows where confined by incisions; parietes strongly curved toward orifice. Radii and alae not reaching basal rim, leaving small membranous windows between parietes; broad with striations perpendicular to oblique summits. Rostrum widest shell plate; internally, sheath with conspicuous horizontal growth lines; lower margin separated from internal parietal wall, extending to just above apical extent of parietal incisions.
Basis circular, shallow to deeply cupped, not more than half total shell height; concentric growth lines conspicuous; basal rim irregularly crenate, interlocking with internal ribs of parietes.
Scutum ( Figure 17L-M) triangular, as wide as or slightly wider than high, growth ridges strongly crenate with raised points and short setae, longitudinal ridges variably pronounced; basitergal angle rounded, tergal section indistinctly separated; occludent margin strongly toothed. Internally smooth, apex with yellow chitinous cuticle, adductor and lateral depressor muscle pits present; articular ridge short, occupying 0.3 length of tergal margin, prominently protruding beyond tergal margin, with deep articular furrow, extending entire length of tergal margin.
Tergum ( Figure 17N-O) apex blunt, external surface covered by yellow, setose, chitinous cuticle on carinal side of spur furrow; growth lines prominent, crossed by strong longitudinal ridges giving a lattice-like or cancellated appearance, fringed with short inconspicuous setae; articular margin concave in apical half. Carinal margin almost straight, only curved near apex; basal margin straight; spur length approximately 1.25 times width, occupying approximately 0.25 basal margin, positioned less than half own width from scutal margin; spur furrow defined by abrupt change in growth lines, margins infolded, furrow open in basal half; distal margin rounded or truncate. Internally roughened with raised tubercles in apical section, chitinous cuticle on carinal and basal margins, crests for depressor muscles prominent, particularly in larger specimens. Articular furrow broad, articular ridge low, rounded occupying 0.25 scutal margin.
Mandibular palp (Figure 18A,C) rhomboid, distal end obliquely truncate, anterior margin concave, posterior margin straight; setae heavily serrulate, becoming longer and more dense distally; distal angle with long, simple setae Mandible ( Figure 18D-F) cutting margin with 4 distinct teeth, second to fourth tooth bifid, fifth tooth congruent with molariform or spinose inferior angle; superior and inferior margins with row of long, fine, simple setae, dense short setae covering inner and outer faces.
Maxillule ( Figure 18G-H) cutting margin straight, with small notch between second and third setae; 10-11 cuspidate setae, upper and lower pair larger and more robust than remaining setae, 3-4 medial setae serrate on inferior margin. Dense, short setae regularly spaced on inner and outer faces, longer fine setae on inferior and superior margin.
Maxilla ( Figure 18I) bilobed, lobes ovate, distal lobe elongate, length more than twice width, serrulate setae arranged on anterior margins, becoming more dense at apex, setae longer on distal lobe than on basal lobe.
Cirral segment counts are given in Table 2.
Cirrus I ( Figure 19A-B) protopod with pappose setae on basal segment posterior margin, basis with tuft of setae at posterodistal angle. Rami unequal, anterior ramus 3 times length of posterior ramus, setation lasiopod, segments with densely arranged serrulate and simple setae on mesial face, tuft of simple setae on posterodistal angle.
Cirrus II ( Figure 19C) protopod lined with long serrulate setae on anterior margin and pappose or plumose setae on posterior margin on basal segment; basis with long serrulate setae on anterior margin. Rami unequal, anterior ramus 1.25 times longer than posterior ramus, setation lasiopod, segments with densely arranged serrulate and simple setae on mesial face.
Cirrus III ( Figure 19D-F) protopod lined with long serrulate setae on anterior margin and pappose or plumose setae on posterior margin on basal segment. Rami unequal, anterior ramus 1.25 times length of posterior ramus; setation lasiopod, segments with serrulate and simple setae on mesial face, less densely arranged than on cirrus I and II. Anterior ramus with transverse row of distally directed spines or ctenoid scales at anterodistal angle on all but distal-most segments, spines most prominent on medial segments; posterodistal angle with transverse row of ctenoid scales. Posterior ramus with similar spination but much reduced in size.
Cirrus IV ( Figure 20A-F) protopod basal segment posterior margin with few plumose setae, tuft of simple, short setae at posterodistal angle; anterior margin with widely spaced plumose setae; basis elongate, length 2.09-2.15 width, with tuft of simple, long setae at posterodistal angle; anterior margin lined with up to 7 well-spaced, hooked teeth, lower third lacking teeth. Rami subequal in length, setation ctenopod. Anterior ramus with 1-2 hooked teeth on anterior margins of first 14 segments, anterodistal angles with transverse row of erect spines on all but distal-most segments; 3 pairs of serrulate setae on medial segments. Posterior ramus with similar setation as anterior ramus, with erect spines on anterodistal angles, with or without hooked teeth.
Cirrus V ( Figure 20G) protopod with well-spaced, long setae and dense row of short spinules on anterior margins; posterior margin lacking setae; basis elongate, length 1.7-1.75 width, without hooked teeth, row of long simple setae on anterior margin and tuft of setae at posterodistal angle. Rami subequal, setation ctenopod, 3 pairs of serrulate setae on anterior margins; without teeth or erect spines.
Cirrus VI ( Figure 20H-K) protopod and rami setation as in cirrus V; protopod basis elongate, length 1.78-1.97 width, without hooked teeth, longitudinal row of small spinules on anterior and posterior margins of basal segment. Anterior ramus without erect spines or teeth on anterior margins; both rami with 3 pairs of serrulate setae on medial segments. Penis ( Figure 20H) shorter than cirrus VI, annulated, sparsely setose; basidorsal point rounded, inconspicuous.
Distribution: Australia: oceanic coral reefs and shoals off northern WA. Southern Japan [69].
Hosts: Agelas KMB1, Agelas cf. mauritiana (Carter, 1883) [70] (Agelasidae). Remarks: The presence of longitudinal slits in the parietes immediately distinguishes P. cancellorum from all other members of the genus except P. sculpturata [6], with which it also shares the chitinous cuticle on the opercular plates. The two species share an obvious morphological affinity, as recognised by Hiro [7], although what he considered to be "remarkable differences between… the sculpture of the opercular valves" of the two species was presumably in reference to the presence of the yellow cuticle on the tergum, which was omitted from Broch's description of P. sculpturata [6]. This leaves a finer morphological distinction between the two species than at first conceived. Key differences include the presence of hooked teeth on the protopod of cirrus IV of P. cancellorum (absent in P. sculpturata), the basis of which is more elongate (height/width ratio >2.0 in P. cancellorum, <2.0 in P. sculpturata), and the basidorsal point, which is rounded and inconspicuous (triangular and prominent in P. sculpturata). Furthermore, the calcareous spines on the parietes appear smaller and more numerous in P. cancellorum compared to the stout, blunt spines on P. sculpturata. This morphological affinity also corresponds with closely related host usage and a close genetic relationship (see Section 3.2).
There are no slide-mounted specimens among the extant cotypes with which to compare to our material. Only the specimen selected to serve as lectotype still has the prosoma present, which has at some point dried out. This has distorted the prosoma and makes dissection exceptionally difficult without risking further damage. However, some key characters are still observable, e.g., the teeth on cirrus IV are arranged in a similar fashion. There is some variability in the armature of cirrus IV within this species. Hiro describes the posterior ramus of cirrus IV as typically, but not always, bearing a single hooked tooth on the segments; in the present material, only one specimen was found to have this character. The number and development of the teeth on the protopod are also variable, with some specimens having few teeth restricted to the upper portion of the protopod and others having irregular spacing, and the left and right cirri do not necessarily have the same arrangement.
Description: Shell ( Figure 21A-H) globose, ranging from white to orange or pink; orifice small relative to shell diameter, rim toothed. Parietes externally with variously pronounced longitudinal ribs studded with small, sharp points; parietes strongly curved over opercular plates, creating narrow orifice. Radii and alae not reaching basal rim, leaving small membranous slits between parietes; summits oblique, broad with horizontal striations. Rostrum widest shell plate. Internally, sheath with conspicuous horizontal growth lines, vesicular structure where sheath adjoins parietes; lower margin separated from internal parietal wall, below sheath longitudinal ribs prominent.
Basis ( Figure 21H) circular, shallow to deeply cupped, making up to half total shell height; concentric growth lines conspicuous; basal rim strongly crenate, interlocking with internal ribs on parietes.
Scutum ( Figure 21I-J) triangular, transversely elongate, height 0.6 times width, growth lines marked by longitudinal ridges, ridges increasing in number basally; basitergal angle angular, tergal section narrow; occludent margin strongly toothed. Internally smooth, apex with yellow chitinous cuticle, adductor and lateral depressor muscle pits present; articular ridge short, occupying 0.3 length of tergal margin, prominently protruding beyond tergal margin with deep articular furrow, extending entire length of tergal margin.
Tergum ( Figure 21K-L) apex blunt, growth lines prominent, crossed by strong longitudinal ridges giving a lattice-like or cancellated appearance, fringed with short, inconspicuous setae; articular margin concave in apical half. Carinal margin straight, only curved near apex; basal margin straight; spur length 0.3 times width, occupying approximately 0.4 of basal margin, positioned less than half own width from scutal margin; spur furrow defined by abrupt change in growth lines, margins infolded, furrow open in basal half; distal margin concave with disto-articular angle projecting. Internally smooth, crests for depressor muscles faint. Articular furrow broad, articular ridge low, rounded occupying 0.25 scutal margin.
Mandible ( Figure 22D-F) cutting margin straight with 4 distinct teeth, second to fourth tooth bifid, fifth tooth congruent with molariform or spinose inferior angle; superior and inferior margins with row of long, fine simple setae, dense short setae covering inner and outer faces.
Maxillule ( Figure 22G-H) cutting margin with 11 cuspidate setae, upper and lower pair larger, more robust than remaining setae. Dense, short setae regularly spaced on inner and outer faces, longer fine setae on inferior and superior margin.
Maxilla ( Figure 22I) bilobed, lobes ovate, serrulate setae arranged on anterior margins, becoming more dense at apex, longer on distal lobe than those on basal lobe.
Cirral segment counts are given in Table 2.
Cirrus III ( Figure 23E-F) protopod anterior margin lined with long plumose setae, posterior margin with pappose setae basally, plumose setae distally. Rami subequal, setation lasiopod, segments with serrulate and simple setae on mesial face, less densely arranged than on cirrus I and II. Anterior ramus with row of ctenoid scales on anterodistal portion of all segments, except terminal segment, most prominent on medial segments.
Cirrus IV ( Figure 24A-E) posterior margin of protopod basal segment margin lined with plumose setae, tuft of simple, short setae at posterodistal angle; basis elongate, length 2.09-2.15 times width, with tuft of simple, short setae at posterodistal angle; anterior margin lined with 14-15 closely-spaced, hooked teeth. Rami subequal, setation ctenopod. Anterior ramus with 1-4 hooked teeth on first 8 segments, distal segments with up to 3 erect spines on anterior margins; long serrulate setae arranged in row at anterodistal angle. Posterior ramus lacking hooked teeth or erect spines on anterior margins; 3-6 pairs of long, serrulate setae arranged in row at anterodistal angle.
Penis long, annulated, with sparse, short setae; basidorsal point low, rounded. Distribution: Australia, from Perth north to the Kimberley region, south to New South Wales and Victoria. South Africa, Andaman Islands, Java Sea, Sulu Archipelago, Malay Archipelago, South China Sea, Hong Kong, China, Philippines, Japan. Depth: 0-170 m.
Hosts: Trachytedania L1, Trachytedania L2, Trachytedania MM1, and Tedania sp.1 (all Tedaniidae). Previous records: Halichondria (Halichondria) okadai (Kadota, 1922) [80], H. (H.) japonica (Kadota, 1922) [7,69,80]; Tedania sp. [81]; and Halichondriidae [5]; Remarks: This distinctive species has been reported across the Indo-Pacific, but host identifications have been rare. Morphological variation has been reported in the armature of cirri IV and V with regards to the number of teeth present on the protopodite and anterior ramus, as well as the degree of the sculpturing on the shell plates, including the scutum [7,69]. Given the vast geographic range reported for this species, this raises the potential for more than one species to be present. These records provide the first COI sequences for this species. Coupling these with the description and host identifications will greatly aid further workers in identifying this species and distinguishing any potential cryptic species.
The remaining species currently assigned to the genus Pectinoacasta can be readily separated from P. pectinipes. Firstly, P. cancellorum and P. sculpturata may be considered fenestrate, but in addition to the small membranous windows between parietes are longitudinal slits within parietal sections of the wall. These two species also have a yellow chitinous cuticle on the external surface of the tergum, and their respective tergal spurs are also distinctly more elongate. Pectinoacasta angusticalcar (Broch, 1931) [6] does not have fenestra between the parietes, the tergal spur is elongate, and the furrow is completely closed along its length. Lastly, in P. zevinae (Kolbasov, 1991) [61], the orifice is large, the parietes do not curve over the opercular plates, exposing the opercular plates from above, the tergum lacks a distinct spur or spur furrow, and the armature of the posterior cirri is reduced to small erect spines, lacking the hooked teeth of P. pectinipes.    Pectinoacasta sculpturata (Broch, 1931) [6] (Figures 25-28) Acasta sculpturata Broch, 1931: p. 101, Figure 35 [6]. Rosell 1991: p. 50 [82]. Acasta sculptura: Newman and Ross 1976: p. 54 [44]. Pectinoacasta sculpturata: Kolbasov 1993: p 411 [5]. Jones  Diagnosis: Shell globose, parietes permeated with narrow, longitudinal incisions extending from basal rim and small windows at parietal junctions; externally surfaces studded with short, blunt, calcareous projections; internally longitudinal ribs extending below sheath and interlock with crenate basal rim. Scutum with longitudinal ridges crossing growth ridges. Tergum with setose, yellow, chitinous cuticle, growth ridges crossed with longitudinal ridges, giving a latticed appearance. Cirrus IV protopod lacking curved teeth, anterior margin with longitudinal row of small spinules, anterior ramus basal segments with 1-2 recurved teeth on anterior margins.
Description: Shell ( Figure 25A-L) globose, white or grey; orifice small relative to shell diameter, rim toothed. Parietes with longitudinal incisions extending from basal rim to over half parietal height, creating narrow, membranous slits, incisions secondarily calcified in upper portions of parietes; external surfaces with short, blunt calcareous projections arranged irregularly, or in longitudinal rows where confined by incisions; yellow, chitinous cuticle on basal sections; parietes strongly curved toward orifice. Radii and alae not reaching basal rim, leaving small membranous slits between parietes; broad with striations perpendicular to oblique summits. Rostrum widest shell plate. Internally, sheath with conspicuous horizontal growth lines; lower margin adpressed to inner lamina, extending to just above apical extent of parietal incisions; longitudinal ribs below sheath formed either side of incisions.
Basis circular, shallow to deeply cupped; concentric growth lines conspicuous; basal rim irregularly crenate, interlocking with internal ribs of parietes.
Scutum ( Figure 25M-N) triangular, approximately as high as wide, growth lines marked by longitudinal ridges, ridges increasing in number basally; basitergal angle rounded, tergal section narrow; occludent margin strongly toothed. Internally smooth, apex with yellow chitinous cuticle, adductor and lateral depressor muscle pits present; articular ridge short, occupying 0.5 length of tergal margin, prominently protruding beyond tergal margin with deep articular furrow extending entire length of tergal margin.
Tergum ( Figure 25O-P) apex blunt, external surface covered by yellow, setose, chitinous cuticle; growth lines prominent, crossed by strong longitudinal ridges, giving a latticed or cancellated appearance, fringed with short, inconspicuous setae; articular margin concave in apical half. Carinal margin straight, only curved near apex; basal margin straight; spur length 2 times width, occupying approximately 0.3 basal margin, positioned less than half own width from scutal margin; spur furrow defined by abrupt change in growth lines, margins infolded, furrow open in basal half; distal margin rounded.
Mandible ( Figure 26C-D) with five teeth, second to fourth teeth bifid, fifth tooth confluent with molariform inferior angle; inferior angle with denticles. Regularly spaced, dense, short setae on inner and outer faces, longer fine setae on inferior and superior margin.
Maxillule ( Figure 26E-G) cutting margin straight, with 10 cuspidate setae, upper and lower pairs longer than remaining setae; three medial, shorter setae serrulate on inferior margin; tuft of short setae at basal angle. Dense, short setae regularly spaced on inner and outer faces, longer fine setae on inferior and superior margin.
Maxilla ( Figure 26H) bilobed, lobes ovate, serrulate setae arranged on anterior margins, becoming more dense at apex, longer on distal lobe than those on basal lobe.
Cirral segment counts are given in Table 2.
Cirrus I ( Figure 27A) protopod with pappose setae on basal segment posterior margin, basis with tuft of setae at posterodistal angle. Rami unequal, anterior ramus 3.5 times length of posterior ramus, setation lasiopod, segments with densely arranged serrulate and simple setae on mesial face, tuft of simple setae on posterodistal angle.
Cirrus II ( Figure 27B) protopod lined with long serrulate setae on anterior margin, with pappose or plumose setae on posterior margin on basal segment; basis with long serrulate setae on anterior margin. Rami unequal, anterior ramus 1.25 times longer that posterior ramus, setation lasiopod, segments with densely arranged serrulate and simple setae on mesial face.
Cirrus III ( Figure 27C-E) protopod lined with long, serrulate setae on anterior margin, pappose or plumose setae on posterior margin of basal segment. Rami subequal, anterior ramus 1.1 times length of posterior ramus, setation lasiopod, segments with serrulate and simple setae on mesial face, less densely arranged than on cirrus I and II. Anterior ramus with transverse row of distally directed spines at anterodistal angle on all but distal-most segments, spines most prominent on medial segments; posterodistal angle with transverse row of ctenoid scales. Posterior ramus with similar spination but much reduced in size.
Cirrus IV ( Figure 28A-F) protopod basal segment anterior and posterior margins with well-spaced row of short, simple setae; tuft of simple, short setae at posterodistal angle; dense row of short, sharp spinules on anterior margin; basis elongate, length 1.80-1.91 times width, with tuft of simple, long setae at posterodistal angle; anterior margin lined with short, simple and short, sharp spinules. Rami subequal in length, setation ctenopod. Anterior ramus with 1-2 hooked teeth on anterior margins of first 10 segments, anterodistal angles with transverse row of erect spines on all but distal-most segments; 3 pairs of serrulate setae on medial segments. Posterior ramus with similar setation to anterior ramus, with erect spines on anterodistal angles, but lacking hooked teeth.
Cirrus V ( Figure 28G) and VI ( Figure 28H,J) similar, protopod with well-spaced, long setae and dense row of short spinules on anterior margins; posterior margin lacking setae; basis elongate, cirrus V length 1.76-1.82 width, cirrus VI length 1.71-1.75 width, hooked teeth absent, row of long simple setae on anterior margin, tuft of setae at posterodistal angle. Rami subequal, setation ctenopod; 3 pairs of serrulate setae on distal portion of anterior margins of each segment; rami without teeth or erect spines.
Remarks: Re-examination of the type series of this species was key in determining the identity of the Australian material, as the original description failed to mention the chitinous cuticle, a rather prominent feature of the species. The type series of this species includes mounted slide material, disarticulated shell plates, extracted whole specimens, and specimens still embedded in the host. Unfortunately, it is not possible to connect the slides to the dissected shell plates. It is also not clear which, if any, of these specimens is the one reportedly extracted from an alcyonacean. The specimen designated as "type" in Broch's figure 35a is clearly still extant and whole. It is this specimen we treat as being the holotype.
Distinguishing P. sculpturata from P. cancellorum and P. pectinipes has been summarised under the treatment of these latter species. The remaining species of Pectinoacasta, P. angusticalcar and P. zevinae, both lack the incisions in the parietes seen in P. sculpturata. Further to this, P. zevinae lacks a distinct tergal spur or teeth on the protopod or rami of cirrus IV. While in P. angusticalcar, the spur furrow is fully closed, the scutum is distinctly wider than high, and the protopod of cirrus IV has a row of teeth. Given Broch's omission of the chitinous cuticle in , it is not clear if it was similarly omitted from his description of P. angusticalcar in the same paper [6].
Examination of the type-host reveals it to be a species of Agelas, albeit distinct from the species inhabited by the Australian specimens, as well as the Agelas inhabited by P. cancellorum. The addition of A. sulphurea as a host broadens the generic range but affirms this species' affinity for sponges of the Agelasidae.

Molecular Results
The top-ranked partitions produced by the ASAP analyses suggested between 26 or 28 putative species. The three substitution models provided similar results and the first two highest-ranked partitions received close scores. Under the JC69 model and p-distance partitions of 26 species and 28 species were received an ASAP score of 2.0 and 2.50, respectively, at a distance threshold of 7% or 4%. The difference between these partitions was the lumping or splitting of A. huangi and A. sp.2, and A. sandwichi and A. caveata sp. nov. The K2P model ranked partitioning the dataset into 26 or 27 species with scores of 1.0 and 3.0, respectively, at a distance threshold of 2.9% or 3.5%. The difference between these two partitions is the lumping or splitting of A. sandwichi and A. caveata sp. nov.
IQTree selected the general time-reversible model with a proportion of invariant sites and gamma-distributed rate heterogeneity (GTR+I+G) as this was ranked highest under the Bayesian information criterion [85] and this was utilised in the BI analysis as well. The phylogenetic tree of sponge barnacles, with ML and BI support values, exhibited very similar topologies and statistical support ( Figure 29). All new species concepts presented herein formed reciprocally monophyletic clades under all analyses; however, none of the genera were recovered as monophyletic. Although there was little support for the branching patterns observed except at nodes separating closely related species such as the nodes between P. cancellorum and P. sculpturata and the clade containing A. undulaterga, A. radenta, A. huangi, and A. sp. 2 MCY-2020.

Discussion
The present study documents an additional six species of the Australian Acastinae; however, there are still many more species to be formally described, and the total number of species is likely to be well over 60 [4]. Hosts for most of the previously reported barnacle species are still unknown (e.g. [86,87]), and given how integral this is to understanding barnacle biodiversity and patterns of speciation [1,4,17], it is the other key gap in our knowledge.
Most of the Australian specimens documented in this study were collected from a restricted host range, typically inhabiting a single or few sponge genera. Only A. aspera appears to have a broad host range, with hosts from multiple families, but all from the same sponge order (Poecilosclerida). Species such as P. pectinipes and A. fenestrata have been documented from multiple host orders in other parts of their geographic range. However, once more material of A. fenestrata is sequenced and type specimens examined, our expectation is for a more restricted range of hosts.
In the case of P. sculpturata, molecular data proved that specimens reported from Taiwan [17] are conspecific with the Australian specimens, while identifying the type-host shows that this species appears to specialise in species within the genera of the Agelasidae: Agelas and Amphinomia (this latter genus is monotypic). However, the report of P. sculpturata inhabiting an alcyonacean coral [6] requires further investigation, as it seems unlikely for a species to specialise in one family of sponges but also embed in hosts from a different phylum. Pectinoacasta cancellorum was recovered as sister to P. sculpturata and is not only morphologically similar, but also inhabits species of Agelas, albeit different species. Both the genetic results and host used show these two species to be rather distantly related to P. pectinipes, suggesting that the morphological similarities are superficial.
The proposed new species, A. caveata sp. nov., E. acutaflava sp. nov., and E. excoriatrix sp. nov., were each identified from single host genera, but it is too soon to tell if they are host specialists, as they have so far been identified from only a few host individuals. The factors involved in determining the range of hosts used by individual barnacle species are unknown and would be a valuable avenue of study.
It was already known that A. aspera was distributed in Australia and Taiwan with no discernible genetic differentiation [3]. In an additional three species, specimens from Taiwanese waters were nested within clades of Western Australian specimens: A. sandwichi, A. fenestrata and P. sculpturata. Interestingly, the single sequence of A. fenestrata from Queensland was sister to the clade formed by the WA and Taiwanese specimens. This result suggests that there is potentially greater connectivity between WA and Taiwan than with the east coast of Australia, for this species at least, but further specimens and sequences will be needed to test this hypothesis.
The types of armature found on the fourth cirrus have long been seen as critical characters upon which to base taxonomic decisions at the species level or above [5,6]. The key morphological distinction between A. sandwichi and A. caveata sp. nov. is that the former bears a single, straight, and sharp "denticle" on each segment [2] compared to the multiple, robust, and hooked teeth of the latter. The close, but distinct, genetic relationship of these apparent sister species indicates that the evolution, or loss, of these character states may occur independently in different lineages. The ASAP analysis did not provide unequivocal support for separating these species. However, this analysis is looking for a partitioning threshold that can give predictive power to molecular identification as a starting point for assessing biological or adaptive significance [25]. The interspecific genetic distance between these two species (0.60) is still much higher than the greatest intraspecific distance (0.19) observed in this dataset and supports the hypothesis that the specimens represent separate species. Therefore, while there is more work needed on the intraspecific variation of the fourth cirrus in the Acastinae, it is considered a reliable character for delimiting species and a key support for erecting this new species and is believed to be a functional adaptation to the host [5,16]. Further evidence that supports this decision includes the separate host families (Niphatidae and Petrosiidae) suggesting ecological separation and that the Australian specimens identified as A. sandwichi are indistinguishable morphologically and genetically from those from Taiwan but are easily separated from those of A. caveata sp. nov.
Supplementary Materials: The following are available online at www.mdpi.com/article/10.3390/en14133862/s1, Table S1: genetic distances.  Institutional Review Board Statement: Ethical review and approval were waived for this study, as the animals (marine sponges and barnacles) used in this study are invertebrates that are not subject to animal ethics regulations in Australia.

Data Availability Statement:
The data presented in this study are available in article or supplementary material.