Placement of Sternaspidae within Annelida
Combined analyses of 18S, 16S and COI found Sternaspidae as sister to Scalibregmatidae with weak support and this sister pair in turn sister to Cirratuliformia (Acrocirridae, Flabelligeridae and Cirratulidae) with moderate support (Fig. 4). The position of Fauveliopsidae is often reported as sister to Sternaspidae in phylogenetic analyses (e.g. Rousset et al. 2007; Struck et al. 2007; Struck et al. 2008) and more recently in a phylogenomic analysis that utilized thousands of genes, again finding strong support for this sister pair (Andrade et al. 2015). The position of Fauveliopsidae in this study was unresolved—however, this is likely due to a lack of data, as the analysis was limited to three genes and only three fauveliopsid sequences were available for use, two 18S sequences and one 16S sequence (Online Resource 3), with one of the 18S sequences (Fauveliopsis sp.) shown to be particularly long branched in relation to other sequences in the dataset. Furthermore, basal support values across the tree were poorly resolved, limiting interpretation of inter-familial relationships.
Sternaspidae Carus, 1863
Sternaspis affinisStimpson, 1864
Material examined: All examined material loaned from the Scripps Institution of Oceanography Benthic Invertebrate Collection (SIO-BIC). Specimens SIO-BIC A5918 and SIO-BIC A6281, collected from East Sound, WA, USA (depth 25 m), and off Santa Barbara, CA, USA (depth 100 m), respectively (see Table 1; Online Resource 2). See also Online Resource 1 for footage of live specimen SIO-BIC-A5918.
Description: Preserved specimen SIO-BIC A5918 with a completely inverted introvert (Fig. 5a); SIO-BIC A6281 with introvert fully everted (Fig. 5c); Full body length of SIO-BIC A6281 11.7 mm, width 5.1 mm. Body light tan to beige in colour and covered with fine papillae; papillae are densest and largest on segments 7–9, becoming more widely spaced on the preceding six segments, and more fine on subsequent segments; body papillae are often encrusted with a fine sandy sediment.
Prostomium hemispherical, browner in colour than surrounding tissue and slightly opalescent; eyespots not visible; peristomium rounded, without obvious papillae; mouth region damaged in specimen SIO-BIC A6281 (Fig. 5c), but long papillae apparent on sections of mouth still visible.
First three chaetigers bearing bundles of 12–15 thick, bronze-coloured slightly falcate introvert hooks that become darker at the tips (Fig. 5c, d). One pair of long, digitate gonopodial lobes present on the ventral side between segments 7 and 8 (Fig. 5c). Abdomen consists of seven segments, with fine papillae that become denser, longer and more filament-like on the dorsum than on the ventral body wall, particularly towards the shield region.
Ventro-caudal shield ranging from brick red to orange in colour, with distinct concentric lines, particularly towards the shield margins; ribbing present throughout shield of SIO-BIC A6281 (Fig. 5c) but less distinct in the shield interior of SIO-BIC A5918 (Fig. 5a); anterior margins rounded, anterior keels slightly visible; anterior depression ranging from deep and triangular (Fig. 5c) to relatively more shallow and rounded (Fig. 5a). Suture visible throughout shield. Lateral margins slightly rounded, relatively straight. Posterio-lateral corners well developed in both specimens, demarked by a particularly large diagonal rib; posterior fan slightly expanded beyond corners; posterior margin distinctly crenulated in SIO-BIC A6281 (Fig. 5c), but more smooth in SIO-BIC A5918 (Fig. 5a); both specimens with distinct median and lateral notches; notches notably deep in SIO-BIC A6281 (Fig. 5c).
Marginal chaetal fascicles surround the shield, with 10 ovally arranged fascicles on either side of lateral margins (Fig. 5c) and five linearly arranged posterior fascicles per shield plate. Numerous thick, coiled branchiae protrude from branchial plates, interspersed with fine, long and filamentous papillae.
Remarks: These specimens match the neotype description of Sternaspis affinis in Sendall and Salazar-Vallejo (2013), with variation between the two specimens within the intraspecific variation reported in a case study of S. affinis morphology (also Sendall and Salazar-Vallejo 2013) and within general patterns of ontological variation reported for sternaspids. Furthermore, SIO-BIC A5918 was collected from East Sound, WA, USA—roughly 60 km from the type locality of S. affinis, off Vancouver, BC, Canada.
Genetic data (see sections on within-Sternaspidae Phylogenetics and Population genetics) revealed relatively low intraspecific variation between SIO-BIC A5918 and SIO-BIC A6281 in both 16S and COI analyses, despite a geographic distance of approximately 1800 km between Washington and California collection sites. Furthermore, these specimens fell into a clade with GenBank COI sequence data identified as Sternaspis fossor Stimpson, 1854 collected from off Bamfield, BC, Canada, approximately 250 km from the SIO-BIC A5918 collection site (see Fig. 17 in later results section). The geographical location of the 16S S. fossor GenBank sequence is unknown.
Sternaspis affinis and S. fossor are morphologically similar species; until its neotype description and reinstatement in 2013, S. affinis was considered to be a junior synonym of S. fossor (Sendall and Salazar-Vallejo 2013). Sternaspis fossor is now known to differ from S. affinis primarily in that posterior corners and ribbing of the ventro-caudal shield are poorly developed and that it is reported from the northwestern Atlantic from along the East Coast of Canada and the USA, whereas S. affinis is reported from the eastern Pacific, from Alaska to California (Sendall and Salazar-Vallejo 2013). The GenBank S. fossor sequences were published in 2011, i.e. before S. affinis was reinstated as a species in 2013; it is thus likely that these sequences are currently misidentified and are in fact S. affinis, based both on geographic proximity to the type locality of S. affinis and the phylogenetic and population genetic relationships revealed in this study (see Figs. 17 and 18a).
Sternaspiscf.annenkovaeSalazar-Vallejo & Buzhinskaja, 2013
Material examined: Specimens IN2017_V03-040-138 and IN2017_V03-040-139, collected off southeastern Australia, 2500 m (see Table 1; Online Resource 2).
Description: Two specimens, both with introvert everted (Fig. 6a, b)—the introvert is fully inflated and bulbous in specimen IN2017_V03-040-139, with segments between the introvert and the rest of the body appearing cinched (Fig. 6b). Body slightly bi-coloured in both specimens, with abdomen opaque and light tan in colour, and the introvert slightly darker and orange. Body finely papillose, with a fine silty sediment adhering to papillae in both specimens and with papillae largest and densest on segments 7 and 8 and; body papillae more eroded on IN2017_V03-040-139. Both specimens similar in size, with a body length and width of 11.7 mm and 5.2 mm for IN2017_V03-040-138 and 11.4 mm and 4.6 mm for IN2017_V03-040-139.
Prostomium projected, rounded and slightly conical, similar in colour to surrounding tissue; eyespots not observed; peristomium rounded; mouth rounded, heavily papillose and particularly dark in specimen IN2017_V03-040-138 (Fig. 6c)—it is unclear if this is due to dark coloured sediment adhering to the mouth, in contrast to the pale sediment that encrusts the body, or whether this is true pigmentation.
Introvert chaetigers bearing bundles of 10–12 brassy, slightly falcate hooks that are darker at the distal tips (Fig. 6c, d). Gonopodial lobes not clearly observable and could either be eroded or hidden between segmental folds of segments 7 and 8.
Abdomen with around seven segments, papillose, with denser bands of papillae somewhat observable on segments close to the shield (Fig. 6a).
Ventro-caudal shield orange to brick red, with ribbing and fine concentric rings; inner rings at the anterior-most end of the shield form a plate that appears to be somewhat raised relative to the rest of the shield; suture visible throughout shield; anterior depression relatively shallow; anterior margins somewhat angular; lateral margins gently rounded; posterio-lateral corners not distinct; posterior margin straight, faintly crenulated; fan barely projected past posterior corners; lateral notches not distinct; fans are somewhat divergent, forming a distinct, deep and triangular median notch, displays some medial fusion between; posterior margins slightly narrower than anterior margins.
Marginal chaetal fascicles with 9–10 lateral bundles on either side of the shield, with chaetae arranged ovally, and 5–6 posterior fascicles per shield plate, with linearly arranged chaetae.
Branchiae mostly eroded, but where present range from thick to slender, and coiled to relatively straight, interspersed with dense filamentous branchial papillae; branchial plates narrow and rounded, slightly wider anteriorly and curved around the anus.
Remarks: These specimens somewhat resemble the deep-water Pacific species, Sternaspis annenkovae, in that they bear shields that are wider anteriorly, with divergent fans that form a deep median notch, and with indistinct lateral notches, poorly developed posterior corners, a straight posterior margin, ribbing and fine concentric lines. It is possible that these specimens also display some bi-coloration, a feature that is unique to S. annenkovae within the genus. However, the bi-colouration is subtle and not particularly distinct in these specimens when compared with S. annenkovae. Further, the anterior shield margins of these specimens are only slightly wider than posterior margins, whereas anterior margins are distinctly wider in S. annenkovae.
These specimens also bear similarities to two other deep-water pacific species, S. maureri Salazar-Vallejo & Buzhinskaja, 2013 and S. williamsae Salazar-Vallejo & Buzhinskaja, 2013 including bearing anteriorly expanded shields with truncate posterior margins. However, the shields of these specimens have more distinct concentric lines and are less anteriorly expanded than in S. maureri and S. williamsae. They also lack the lateral notches present in S. maureri and have more developed median notches than in S. williamsae—the median notch is barely developed in S. maureri. Further, the prostomium is not notably smaller than the mouth, as in S. maureri and S. williamsae.
It has been previously noted that S. annenkovae, S. maureri and S. willimasae all bear similarities (Salazar-Vallejo and Buzhinskaja 2013). Our Australian specimens mostly resemble S. annenkovae, closely followed by S. williamsae; however, it is difficult to definitively place the Australian specimens as one over the other, or as a morphologically distinct but separate species, particularly considering the fact that, with only two similar sized specimens available, both general and ontological intraspecific variation cannot be assessed. Due to this ambiguity, we identify these specimens as S. cf. annenkovae.
Interestingly, in terms of genetic data, these specimens appear to be closely related to other deep-water specimens identified in Kobayashi et al. (2018) as S. cf. williamsae from the northwestern Pacific, with relatively low interspecific variation between two clades (see section on within-Sternaspidae Phylogenetics), despite a geographic distance of over 9000 km. Without further sampling from sites between these disparate localities, it is again difficult to definitively identify these specimens. It is worth noting that the type locality of S. annenkovae off the northern Kuril islands is closer to S. cf. williamsae collection sites than S. cf. williamsae is to the type locality of S. williamsae, off Oregon, USA; conversely, the Australian specimens are closer to the type locality of S. williamsae than to S. annenkovae. Furthermore, Kobayashi et al. (2018) found considerable morphological variation within the S. cf. williamsae sample population, with some specimens resembling S. annenkovae more than S. williamsae.
Material examined: Twenty specimens (Figs. 7 and 8a–h) (identifier prefix P17_145) collected from the English Channel (depth 18 m), ranging in size from 5 to 20 mm in length, all with everted introverts (Fig. 7a–b).
One Mediterranean specimen SIO-BIC A5986, collected off Rovinij, Croatia (depth 25 m), small (2.1 mm long abdomen), introvert fully inverted (Fig. 8i). Specimen loaned from the Scripps Institution of Oceanography Benthic Invertebrate Collection (see Table 1; Online Resource 2).
Description: Body leathery, beige and opaque in large specimens, becoming thinner, paler and more translucent in smaller specimens. Introvert somewhat paler than abdomen in specimens of all sizes. Body finely papillated, more eroded on introvert than on abdomen; papillae more visible in smaller specimens when contrasted against pale, transluscent integument (Fig. 8i).
Prostomium hemispherical, opalescent; eyespots not observed; peristomium rounded; mouth circular, densely papillated (Fig. 7b). First three chaetigers bearing bundles of 8–10 widely separated, slightly falcate hooks (Fig. 7b); hooks range in colour from bronze in larger specimens to pale gold in smaller specimens, but tend to have darker subdistal tips in specimens of all sizes. Gonopodial lobes often eroded, but when present long and somewhat cirriform, projecting lateroventrally between segments 7 and 8. Abdomen with seven segments; the abdomen begins to bear longitudinal wrinkles in larger specimens, particularly as the integument becomes thicker and more leathery.
Ventro-caudal shield ranging in colour from orange and red in small specimens to blue-black in the largest specimens; shield covered by integument bearing minute filamentous papillae (Fig. 7c, d), with the integument thickest in the smallest specimens (Fig. 8i) and becoming heavily eroded in the largest specimens (Fig. 8a, b). All shields with concentric lines and ribbing, though the latter less pronounced in smaller specimens; in the smallest specimen, SIO-BIC_A5986, concentric lines are barely visible through thick, papillated integument (Fig. 8i); thin suture somewhat visible throughout all shields, tending to be more distinct in the anterior-most regions and becoming less defined posteriorly; anterior depression deep, triangular and becoming slightly shallower and rounded in smaller specimens; anterior margins truncate (Fig. 8c, e) to angular (Fig. 8d, f); introvert not exposed; lateral margins relatively straight across specimens of all sizes, though appearing slightly more rounded in smaller specimens (Fig. 8h, i); posterior corners well developed, demarked by distinct diagonal rib; posterior margins ranging from smooth (Fig. 8e) to slightly crenulated (Fig. 8f); posterior fan ranging from projecting beyond posterior corners with a shallow median notch (Fig. 8a, b, e, g) to relatively truncate, not projecting past corners, with an indistinct median notch (Fig. 8d, f, h).
Marginal chaetal fascicles (Fig. 7c) with 9–10 lateral bundles of ovally arranged chaetae on either side of the shield, and six posterior bundles per plate, with chaetae in a slightly curved arrangement and with bundles slightly offset and parallel to each other (see Sendall 2006). Peg chaetae often broken or hidden by chaetae and branchiae, but when present, appear to be as long as the first lateral chaetal fascicle (Fig. 7c); peg-associated capillary chaetae sometimes present (Fig. 7c).
Branchiae abundant, thick and coiled; interbranchial papillae long, filamentous and abundant; branchial plates diverge from anus, thick and ovoid in shape and densely papillated.
Remarks: Specimens from the English Channel more or less agreed with the neotype description of Sternaspis scutata in Sendall and Salazar-Vallejo (2013). Interestingly, there was some variation in a key diagnostic character, the projection of the posterior fan, which ranged from projected beyond posterior corners and notched medially, to truncate, not projecting past corners, and with no distinct median notch.
Notably, a truncate posterior fan not projecting past posterio-lateral corners is a diagnostic character of S. thalassemoides Otto, 1821, another Mediterranean sternaspid, sympatric with S. scutata. Sternaspis thalassemoides and S. scutata are two morphologically similar species; the former had previously been regarded as a junior synonym of the latter, until 2013 when S. thalassemoides was reinstated as a species, based on the character of a truncate fan not expanding past posterio-lateral corners (Sendall and Salazar-Vallejo 2013). A third species, S. assimilis (Malmgren, 1867), originally described from the northeastern Atlantic, off Île de Ré, France, was initially considered to be a junior synonym of S. scutata (Sendall, 2006), but was then found to be more similar to, and thus suggested to be a junior synonym of, S. thalassemoides, as the fan is only slightly projected in this species (Sendall and Salazar-Vallejo 2013).
Our English Channel specimens included individuals of similar sizes with both typical S. scutata posterior margins (Fig. 8c, e) and more truncate S. thalassemoides-like margins (Fig. 8d, f), in addition to specimens with more intermediate, slightly projected fans, perhaps similar to S. assimilis. Some of this variation may be due to ontogenic variation, with shield features such as the posterior fan and posterior corners poorly developed in smaller specimens. However, both types of diagnostic shield margin were observed in moderately sized adults (Fig. 8c–f), and there was variation in the degree of fan projection and depth of median notch even amongst the largest of specimens (Fig. 8a, b). Despite the morphological variation observed, molecular analyses (see section on within-Sternaspidae phylogenetics and population genetics) found very little genetic structure within the English Channel samples.
The single Mediterranean specimen, SIO-BIC A5986, collected off Croatia in the Adriatic Sea was one of the smallest specimens examined, possibly juvenile/sub-juvenile. Thus, full ontogenic development of shield characters could not be assessed. However, no character of SIO-BIC A5986 (Fig. 8i) strongly conflicted with the diagnostic characters of S. scutata, with slight median notch and fan projection evident. Notably, the specimen closely resembled English Channel specimens of a similar size.
In molecular analyses, these specimens, in addition to sequence data from specimens identified as S. scutata from the Bay of Biscay, northeastern Atlantic and another Mediterranean specimen identified as S. scutata, SIO-BIC A1012 (Fig. 9) collected off Banyuls France, formed a monophyletic clade (see Figs. 16 and 17). However, while SIO-BIC A5986 was found to be almost identical to SIO-BIC A1012 and the northeastern Atlantic sequence, differing by only one nucleotide mutation in COI analyses (see Fig. 18b), English Channel specimens displayed moderate genetic isolation from both northeastern Atlantic and Mediterranean sequences (see Figs. 16 and 17, Table 5). The type locality of S. scutata is within Aegean Sea, in the Eastern Mediterranean; the recent neotype description of the species included specimens from the original type locality, though it is worth noting that this description also incorporated a large quantity of additional material from other localities, including individuals collected from off Rovinij, Croatia (the same locality as SIO-BIC A5986) and others from across the Mediterranean, along with several northeastern Atlantic specimens collected off Portugal and France (Sendall and Salazar-Vallejo 2013).
Both morphology and molecular data support the identification of SIO-BIC A5986 as Sternaspis scutata. However, as the English Channel specimens exhibited variable morphology and were relatively genetically isolated from specimens close to the type locality of the species, we cannot with confidence identify them as S. scutata and instead tentatively identify them as S. cf. scutata for the time being. Wider sampling and further molecular and morphological investigations of sternaspids from the English Channel to the Mediterranean are necessary to definitively confirm presence of S. scutata in the English Channel—such investigations could potentially lead to the reinstatement of S. assimilis if the English Channel population is found to be a cryptic species. Further studies within the Mediterranean would also be useful in re-assessing the species status of S. thalassimoides.
Sternaspis sendalliSalazar-Vallejo, 2014
Sternaspis monroi Salazar-Vallejo, 2014 syn. n.
Material examined: Sixty-five individuals from new Southern Ocean material were examined, with a partial or wholly everted introvert observed in only 12 specimens (Table 1; Online Resource 2; see Table 3 for locality information and body size metrics).
Type materials from Natural History Museum London collections were also examined: S. sendalli holotype (NHM 1918.104.22.1682–2400), paratypes (NHM 1922.214.171.1242–2400p), S. monroi holotype (NHM 19126.96.36.1992–2400R), paratypes (NHM 19188.8.131.522–2400Rp).
Description: Body opaque, yellow to pale beige in colour (Figs. 10a, 11a, 12 and 13), with paler and more translucent body walls observed in very small specimens (Fig. 13); body papillae are significantly or partially eroded in many specimens, though when present are fine and evenly spaced, often encrusted with sand particles, and tending to become longer and denser on and around segments 7 and 8, and then sparser on the introvert; gonopodial lobes are often eroded, but observed as short, small and digitate if present (Fig. 11c).
Where visible, the prostomium is observed as pale, hemispherical and projected, with no discernable eyespots (Fig. 10a, b); peristomium round, with sparse fine papillae; mouth round, densely papillated (Figs. 10b and 11c).
The first three chaetigers bearing bundles of 6–7 introvert hooks (Figs. 10a, b and 11a, c); hooks slightly falcate, range in colour from brassy to dark brown in colour, though darker at the tips in all cases; hook tips often broken or damaged, particularly in larger specimens, however when intact are observed to have tapered tips (Figs. 10a, b and 11a, c, d).
Ventral shield colour varies, ranging from orange (Figs. 12a and 14) and brick red (Figs. 10a and 12b–d, f, g) to dark maroon (Fig. 12e), with grey to grey-brown shields (Figs. 11a and 12h) observed in 12 individuals; an integument bearing fine filamentous papillae covers most shield surfaces (Fig. 10d), though this varies in thickness, and is eroded in some specimens (Fig. 12c); diagonal ribs often visible though occasionally indistinct where integument is thick and intact (Fig. 12h); concentric lines are primarily fine and poorly defined to not visible at all, but are moderately defined in some specimens (Fig. 12e); suture consistently distinct, wide and visible throughout shield, tending to expand posteriorly in most specimens; anterior margins rounded to sub-angular with a shallow and wide anterior depression; anterior keels vary in terms of exposure (e.g. Fig. 12b, not visible; Fig. 12g, visible); lateral margins rounded, tending to expand posteriorly in large specimens; posterior corners well developed; posterior fan ranges from projecting past posterior corners, notched medially and laterally (Figs. 10a and 12a, c, e, g), to truncate, not significantly expanding beyond posterior corners, and with poorly defined notches (Figs. 11a and 12b, d, f), in additional to specimens with intermediate characters (e.g. Fig. 12h, wide median notch, slightly projected fan, poorly developed lateral notches); posterior margins slightly crenulated (Fig. 12f, g) to smooth (Fig. 12c, d).
Marginal chaetal fascicles as nine lateral and five to six posterior chaetal bundles (Figs. 10 c and 11e), with all chaetae arranged in oblique rows; posterior chaetae often damaged; peg chaetae not observed, assumed to be damaged and/or hidden by sediment or interbranchial papillae; peg-associated capillary chaetae often present, though delicate and easily broken. SEM reveals that marginal chaetae are covered by a thick fibrous sheath (Fig. 10e, f) likely similar to the sheath and fibrous matrix that bind chaetal bundles to form peg chaetae, as observed using SEM by Zhadan et al. (2017).
Branchiae are often eroded and lost, though where present are abundant, long and coiled. Interbranchial papillae are long, dense and fine, often coated by sediment. Where branchiae are removed, branchial plates bordering the anal peduncle are observed to vary from narrow, curved and somewhat tapered anteriorly to anteriorly expanded and rounded (Figs. 11b and 13).
Juveniles: In small specimens and juveniles, the shield is rounded and smooth, with lateral margins expanded anteriorly and posterior corners indistinct (Fig. 14). With increasing body size, the lateral margins tend to expand posteriorly and the plates become somewhat less rounded, with corners, ribbing and margin crenulation tending to become more distinct and shield colour tending to darken (Figs. 12 and 14).
Type material: Type materials of Sternaspis monroi and S. sendalli were examined (Fig. 15) and found to agree with corresponding species descriptions outlined in Salazar-Vallejo (2014). Both species were described from material originally collected from the Scotia Sea off the South Orkney Islands, identified as S. scutata by Monro (1930). Both species are similar morphologically, differentiated primarily by characters of the ventro-caudal shield. According to the original description (Salazar-Vallejo 2014) barely defined concentric lines, fan projected past posterior corners, notched medially and laterally, and with a barely crenulated margin, characterize the shield of S. sendalli (Fig. 15a, c, e, g), whereas the shield of S. monroi is characterized by an absence of concentric lines, a truncate fan not projecting past posterior corners, a short narrow median notch without lateral notches and a crenulated posterior margin (Fig. 15b, d, f, h).
Remarks: The morphological variation observed amongst the Antarctic specimens examined included specimens that agreed with the morphological descriptions of either Sternaspis sendalli (Figs. 10a and 12a, c, e) or S. monroi (Fig. 11a and 12b, d, f), as well as specimens with intermediate morphologies (e.g. Fig. 12h), that could not be with confidence allocated to one species over the other. The size of the animal did not appear to influence the presence of key diagnostic characters. For example, extended and truncate posterior fans were observed both in large and small specimens (Fig. 12).
Despite morphological variation, molecular analyses revealed very little genetic variation (see Figs. 16, 17 and 19; Tables 4 and 5) with no genetic structure based on geography or shield morphology observed. We therefore consider S. monroi Salazar-Vallejo, 2014 a new synonym of S. sendalli Salazar-Vallejo, 2014. As both species were first described in the same paper (Salazar-Vallejo 2014), we prefer to use the name of a current researcher, Kelly Sendall, who participated in the revision of the family in 2013.
Phylogenetic relationships within Sternaspis
Of the three genera that constitute Sternaspidae, there was no obtainable molecular data for Petersenaspis Sendall & Salazar-Vallejo, 2013, and only two sequences from a single species, Caulleryaspis nuda Salazar-Vallejo & Buzhinskaja, 2013, were available for Caulleryaspis Sendall & Salazar-Vallejo, 2013. Furthermore, the Caulleryaspis sequences in question were previously found to be nested within Sternaspis (Kobayashi et al. 2018), which either raises questions surrounding the validity of C. nuda and generic diagnostic characters or suggests that these specimens may have been misidentified; addressing either of these is beyond the scope of this study. Therefore, the following analyses were primarily based on Sternaspis spp.
The taxa included differed between 16S and COI analyses due to a variable availability of sequences, a poorer sequencing success rate for COI and fewer 16S sequences on GenBank relative to COI (Table 1; Online Resource 3). Different taxa and low sampling relative to the current number of valid species mean that phylogenetic and biogeographic interpretations are limited. Furthermore, deeper branches were often poorly resolved with low support values. However, several consistent patterns of note are observed for both phylogenies (Figs. 16 and 17).
S. affinis and S. fossor—West Coast North American clade
Whole specimen vouchers A5198 and A6281, collected from off the coast of Washington and California states respectively, and which matched the morphological description of Sternaspis affinis (see the ‘Systematics’ section) fell into a clade with specimens identified on GenBank as Sternaspis fossor in both 16S and COI analyses (maximum within-clade uncorrected p distances of 0.005 and 0.009 for 16S and COI analyses respectively (Tables 4 and 5)) (Figs. 16 and 17). As discussed earlier (see the Remarks section for S. affinis in the ‘Systematics’ section), it is likely that GenBank S. fossor sequences are misidentified S. affinis specimens—the entire West Coast North American clade therefore can be seen to represent a single species, S. affinis.
Sternaspis cf. annenkovae, S. cf. williamsae and other Pacific clades
As with Kobayashi et al. (2018), we also found specimens identified as Caulleryaspis cf. nuda (Sea of Okhotsk) to be nested within Sternaspis rather than as a separate genus, close to S. cf. williamsae (off eastern Japan and the Kuril Islands), with a minimum uncorrected p distance of 0.005 and 0.019 between C. cf. nuda and S. cf. williamsae clades in the 16S and COI analyses respectively (Figs. 16 and 17; Tables 4 and 5). This, and considering that S. cf. williamsae displayed relatively high intra-clade variability (maximum uncorrected p distance of 0.01 in COI analysis (Table 5)) highlights a close affinity between these northwestern Pacific species, as discussed in Kobayashi et al. (2018).
In both analyses, deep-water specimens from off the southeastern coast of Australia identified as S. cf. annenkovae (see the ‘Systematics’ section) fell close to the C. cf. nuda + S. cf. williamsae clade (minimum uncorrected p distance between this clade and S. cf. annenkovae clade of 0.007 for 16S and 0.035–0.038 for COI (Tables 4 and 5)) (Figs. 16 and 17). This is despite a geographic distance of up to ~ 9600 km between specimens in these clades.
Other closely related clades despite large geographic distances include GenBank S. scutata sequences from the Bay of Bengal and the Bering Sea (minimum uncorrected p distance of 0.021 between clades in COI analysis (Table 5)) and specimens from the East Coast of Japan and a GenBank S. scutata sequence from Southern Chile (minimum uncorrected p distance of 0.023 in COI analysis (Table 5)) (Fig. 17).
Conversely, a number of genetically discrete specimens were also observed from within relatively small geographic ranges, for example from around the islands of Japan (several unidentified species off the East Coast of Japan and S. costata Marenzeller, 1879 from the Ariake Sea, southwestern Japan) (Figs. 16 and 17) and within the South China Sea (S. radiata Wu & Xu, 2017 and S. spinosa Sluiter, 1882) (Fig. 16). In the COI analysis, Sternaspis sp. 7 (GK612), one of the unidentified Japanese specimens, was nested within the S. costata clade (maximum uncorrected p value of 0.005 (Table 5)) (Fig. 17). The reported range of S. costata spans from Sakhalin Island, Russia, along the eastern and southwestern coastlines of the Japanese archipelago, to the Philippines, with the neotype described from the Boso Peninsula, off Chiba, eastern Japan (Sendall and Salazar-Vallejo 2013)—Sternaspis sp. 7 (GK612) was collected close to the neotype locality of S. costata (Table 1; Online Resource 2), and based on both molecular and geographic data, we suggest identifying it as such. No 16S sequences were available for other S. costata specimens; however, in the 16S analysis, Sternaspis sp. 7 (GK612) was found to be closely associated with S. chinensis Wu, Salazar-Vallejo, & Xu, 2015 (minimum uncorrected p distance of 0.012 between clades (Table 4)) (Fig. 16), in turn sister to the S. radiata clade, forming a broader clade of shallow water East Asian species, excluding S. spinosa.
The two Sternaspis scutata specimens collected from Mediterranean formed a monophyletic clade in both analyses (maximum uncorrected p value of 0.002 for both 16S and COI analyses (Tables 4 and 5)). In the COI analysis (Fig. 17; Table 5), only one out of five GenBank sequences identified as S. scutata fell close to the Mediterranean S. scutata clade. This sequence was collected from the North Eastern Atlantic coast (the Bay of Biscay), with a minimum uncorrected p distance between this clade and the Mediterranean clade of 0.002, the same as the within-clade uncorrected p distance for Mediterranean specimens. Notably, several other sequences recorded on GenBank as S. scutata from non-Atlantic localities did not fall near this clade.
Sternaspids identified as S. cf. scutata from the English Channel formed a clade with low intra-clade variation in both analyses (maximum uncorrected p values of 0.002 and 0.0 for 16S and COI analyses respectively) and fell close to the Mediterranean S. scutata clade both in the 16S analysis (minimum uncorrected p distance between clades 0.002) and the COI analysis (minimum uncorrected p value between English Channel clade and both Mediterranean and North East Atlantic clades of 0.026) (Figs. 16 and 17; Tables 4 and 5).
In the COI analysis, a phenomenon in which nodes were supported by high bootstrap but low posterior probability values was observed between the two Mediterranean specimens within the larger S. scutata clade and within a handful of other clades, such as the West Coast North America clade (Fig. 17). The phenomenon of recovering high posterior probability but moderate bootstrap values is widespread (see Lewis et al. 2005 and references therein), though the opposite can also occur, depending on the data (Cummings et al. 2003). However, Bayesian posterior probability and maximum likelihood bootstrap are not equivalent measures of support (Alfaro et al. 2003), and the exact relationship between the two for any given dataset is complex (Cummings et al. 2003). The incongruence between support values in this analysis was only observed at internal, intraspecific nodes within often well-supported clades. Within these clades, low variation between sequences and low sampling coverage relative to the population may have possibly resulted in too little phylogenetic information to resolve internal topologies more accurately.
Intra-clade variation in new Southern Ocean material
In both analyses, Antarctic specimens identified as S. sendalli formed a monophyletic clade with low intra-clade variation, with a maximum uncorrected p distance of 0.003 in both 16S and COI analyses (Figs. 16 and 17; Tables 4 and 5).
Population genetic analyses
Haplotype network analyses were performed for several clades highlighted in COI phylogenetic analyses. A haplotype network (Fig. 18a) constructed for the clade of North American West Coast specimens (see Fig. 17) revealed interesting patterns of connectivity. The site of collection for sequences identified as Sternaspis fossor on GenBank, collected off Bamfield, British Columbia, Canada (Online Resource 3), is located approximately 250 km from the site of collection for S. affinis SIO-BIC A5918 from East Sound, Washington, USA; both Washington and British Columbia specimens are in turn roughly 1800 km from the site of collection for S. affinis SIO-BIC A6281, off Santa Barbara, CA, USA. However, a single British Columbia sequence, ‘S. fossor’ 1 (accession number: HM473681), displayed fewer nucleotide mutations between it and S. affinis SIO-BIC A5918 (nucleotide substitutions, n = 4) and with S. affinis SIO-BIC A6281 (n = 7) than with the rest of the S. fossor sequences collected from the same site (n = 8). Likewise, S. affinis SIO-BIC A5918 is closer in terms of genetic structure to S. affinis SIO-BIC A6281 (n = 7) than to the majority of the British Columbia specimens (n = 8), despite being much closer geographically. As discussed earlier (see relevant sections in ‘Systematics’ and ‘Phylogenetic relationships within Sternaspis’), it is likely that these GenBank sequences have been misidentified and are in fact S. affinis. Though moderate intraspecific variation is present in this clade, for example relative to the Southern Ocean clade (see the next section), these results show genetic structure over a large geographic range that is not necessarily influenced by geographic proximity.
Conversely, in a COI haplotype network analysis of the clade consisting of the English Channel specimens identified as S. cf. scutata, and the North East Atlantic (Bay of Biscay) sequence and two Mediterranean sequences identified as S. scutata (Fig. 18b), the English Channel haplotype had a distance of n = 15 nucleotide mutations from the Bay of Biscay sequence, which in turn was separated by n = 1 nucleotide mutation from S. scutata SIO-BIC A5986 (collected off Rovinj, Croatia), itself n = 1 mutation away from S. scutata SIO-BIC A1012 (collected off Banyuls, France). This highlights a degree of genetic isolation between English Channel specimens and more southern Atlantic and Mediterranean specimens, which was also reflected in the uncorrected p values (Table 5) and appears to confirm the presence of non-Mediterranean S. scutata in the northeast Atlantic, as previously reported based on morphology (e.g. Sendall and Salazar-Vallejo 2013), while questioning the presence of the species further north in the English Channel.
It is worth noting that in this case, the Straits of Gibraltar may act as a geographic barrier to dispersal, in addition to the relatively large distances between the English Channel and Mediterranean collection sites (between roughly 3500–4500 km apart). However, the Bay of Biscay sequence also shares the same major geographic barrier and similar geographic distances to the Mediterranean sites, despite very low genetic distance from the Mediterranean specimens. Furthermore, the distance between English Channel S. cf. scutata and the Bay of Biscay sequences (roughly 800 km apart) is less than half the distance between those of S. affinis specimens from Washington and California, despite there being considerably less genetic distance between the North American specimens.
A 582-bp COI haplotype network analysis (figure not shown) of S. cf. williamsae and southwestern Pacific specimens identified as S. cf. annenkovae revealed the latter to be n = 20 nucleotide mutations from the network of S. cf. williamsae haplotypes, over a much greater distance of roughly 9000 km than in the above two analyses (see Kobayashi et al. 2018 for a COI Haplotype network analysis of S. cf. williamsae sequences from the northwestern Pacific).
Genetic structure in terms of geographic distribution and morphological variation
In total, 28 COI sequences were successfully obtained from the new Southern Ocean specimens, belonging to three haplotypes (haplotype a = 10 individuals, b = 17, c = 1) (Fig. 19). The three haplotypes differed by only two mutations, and no clear patterns in morphological characters, such as shield colour or posterior fan projection, were apparent for the different haplotypes. No pattern in relation to geographic distribution was observed either, with the two largest haplotypes (a = 10, b = 17) both including specimens from all major South Orkney Island localities, in addition to Adelaide Island localities—sites approximately 1400 km apart. The n = 10 haplotype is seen to be nested within the rest of the Antarctic clade observed in Fig. 17, though total intra-clade variation did not exceed 0.003 in terms of uncorrelated p value (Table 5). COI is a relatively fast-evolving gene (Hebert et al. 2003), and the lack of genetic variation over a large geographic area suggests a high degree of gene flow within the population and a lack of reproductive barriers.