Gorgocephalidae (Digenea: Lepocreadioidea) in the Indo-West Pacific: new species, life-cycle data and perspectives on species delineation over geographic range Author Huston, Daniel C. Author Cutmore, Scott C. Author Miller, Terrence L. Author Sasal, Pierre Author Smit, Nico J. Author Cribb, Thomas H. text Zoological Journal of the Linnean Society 2021 2021-03-21 193 4 1416 1455 https://doi.org/10.1093/zoolinnean/zlab002 journal article 3108 10.1093/zoolinnean/zlab002 f769f705-8e4e-4c74-be78-ee5ce40f482c 0024-4082 5761768 AAA956A8-14F7-49E4-888F-072FAC7D3826 Gorgocephalus yaaji This species was described by Bray & Cribb (2005) based on adult specimens obtained from Kyphosus vaigiensis , collected from off Lizard Island, GBR, Australia . Gorgocephalus yaaji is easily distinguished from all other species of the family, largely in having a more robust, dorsoventrally flattened body shape and in having the vitellarium extend into the forebody. Previously, Huston et al. (2016) generated ITS2 and 28S rDNA gene sequences from adult and intramolluscan specimens of G . yaaji from the type locality. In the present study, additional adult gorgocephalids morphologically consistent with G . yaaji were obtained from the type locality and from Rangiroa Atoll in French Polynesia and Sodwana Bay in South Africa ( Fig. 1 ). Despite extensive investigation, including traditional morphometrics, PCA analyses and SEM imaging, we were unable to find any significant morphological differences between specimens from these three localities. Intramolluscan gorgocephalids from three Echinolittorina cinerea collected from Rangiroa Atoll, French Polynesia were molecularly matched to adults consistent with G . yaaji collected from the same locality. Furthermore, intramolluscan gorgocephalids have previously been collected from Austrolittorina unifasciata , from multiple locations along the coast of New South Wales , Australia ( O’Dwyer et al. , 2015 ; Huston et al. , 2016 ), but they had not previously been connected to an adult form. BLAST analyses showed that the sequences generated from intramolluscan gorgocephalids collected from Kioloa, NSW, matched sequences of ‘ Gorgocephalus sp. Aus’ from the study of O’Dwyer et al. (2015) with identity scores of 98–100%, demonstrating that gorgocephalids collected from A . unifasciata in the studies of O’Dwyer et al. (2015) and Huston et al. (2016) are conspecific. The present phylogenetic analyses ( Figs 3–6 ) demonstrate that these infections are representative of G . yaaji . Table 4. Morphometric data for two new species of Gorgocephalus expressed as a range and mean in micrometres or as percentages. Number of specimens measured presented as total (whole mounts + hologenophores). A dash (-) indicates distance between two features. Abbreviations : Bd, body; L, length; B, breadth; FBd, forebody; OS, oral sucker; ant, measurement taken at anterior; post, measurement taken at posterior; VS, ventral sucker; PPh, prepharynx; Ph, pharynx; Oe, oesophagus; Cae, caecum; PCae, post-caecal space; Csac, cirrus-sac; AT, anterior testis; PT, posterior testis; Ptest, post-testicular space; OV, ovary; Previt, previtteline region; Postvit, postvitteline region; VitOcc, vitellarium occupies. Egg measurements represent the mean obtained from multiple subsamples per specimen. An asterisk (*) next to a feature indicates it was included in the PCA

Species	Gorgocephalus euryaleae	Gorgocephalus euryaleae	Gorgocephalus graboides
Locality	Point Peron and Rottnest	Sodwana Bay, KZN, South Africa Lizard Island, QLD,
Island, Western Australia	Australia
Host(s)	Kyphosus gladius ;	Kyphosus cinerascens	Kyphosus cinerascens
Kyphosus sydneyanus
No. Measured	15 (10 + 5)	10 (8 + 2)	15 (12 + 3)
BdL*	1071–1555 (1242)	920–1463 (1168)	1109–1768 (1326)
BdB*	129–210 (155)	134–213 (180)	100–201 (136)
BdL / BdB	7.17–9.62 (8.19)	5.97–7.91 (6.83)	9.02–12.59 (10.65)
BdB % BdL	10–14 (12)	13–17 (15)	8–11 (9)
FBdL*	352–463 (399)	300–400 (359)	271–387 (311)
FBdL % BdL	27–34 (32)	27–33 (31)	21–26 (24)
OSL*	141–184 (167)	134–161 (147)	140–194 (159)
OSB ant*	108–143 (128)	91–106 (100)	97–130 (111)
OSB post*	66–83 (75)	49–65 (58)	48–69 (58)
OSL % BdL	11–16 (13)	11–15 (13)	10–13 (12)
OSL / OSB ant	1.10–1.44 (1.31)	1.40–1.61 (1.48)	1.24–1.66 (1.44)
OSL / OSB post	1.98–2.46 (2.23)	2.25–2.98 (2.56)	2.25–3.12 (2.76)
VSL*	54–72 (61)	53–68 (60)	50–78 (61)
VSB*	52–70 (62)	51–66 (59)	49–83 (62)
VSL % BdL	4.3–5.5 (4.9)	4.3–5.8 (5.1)	4.1–4.9 (4.5)
VSL / VSB	0.88–1.1 (0.99)	0.95–1.11 (1.02)	0.88–1.14 (0.99)
VSB / OSB ant	0.42–0.53 (0.48)	0.54–0.64 (0.59)	0.46–0.68 (0.56)
VSB / OSB post	0.72–0.89 (0.82)	0.96–1.24 (1.03)	0.83–1.32 (1.07)
VSL / OSL	0.30–0.42 (0.37)	0.37–0.44 (0.41)	0.32–0.47 (0.38)
PPhL	up to 74 (54)	up to 64 (39)	up to 72 (44)
PhL*	44–71 (56)	48–64 (55)	38–67 (50)
PhB*	58–83 (67)	44–59 (51)	49–78 (56)
PhL % BdL	3.8–5.8 (4.6)	3.7–5.5 (4.6)	3.0–4.3 (3.6)
PhL / PhB	0.63–1.22 (0.84)	0.81–1.24 (1.09)	0.73–1.17 (0.89)
PhL / OSL	0.26–0.46 (0.34)	0.34–0.40 (0.37)	0.27–0.35 (0.31)
PhB / OSB ant	0.41–0.61 (0.53)	0.46–0.58 (0.51)	0.42–0.67 (0.50)
PhB / OSB post	0.74–1.04 (0.90)	0.78–1.06 (0.88)	0.83–1.20 (0.96)
OesL	15–87 (61)	37–66 (52)	19–93 (51)
CaeL	652–1131 (854)	595–1069 (809)	749–1323 (1006)
CaeB	38–72 (52)	37–62 (47)	34–63 (42)
CaeL % BdL	60–73 (68)	65–73 (69)	66–76 (73)
CaeL / CaeB	11.2–20.9 (15.8)	15.3–23.8 (18.8)	19.7–30.9 (24.2)
PCae	83–165 (118)	74–161 (109)	79–147 (103)
PCae % BdL	7.1–12.7 (9.2)	6.4–12.5 (9.2)	6.2–9.2 (7.6)
CsacL*	261–412 (333)	221–325 (287)	279–467 (382)
CsacB*	51–95 (65)	35–58 (48)	41–82 (57)
CsacL % BdL	22–30 (26)	19–28 (24)	24–31 (28)
CsacL / CsacB	4.05–6.22 (5.32)	4.85–6.72 (5.97)	5.24–8.44 (6.84)
ATL*	84–125 (102)	77–129 (101)	66–127 (98)

Table 4. Continued

Species	Gorgocephalus euryaleae	Gorgocephalus euryaleae	Gorgocephalus graboides
ATB*	74–111 (89)	70–95 (83)	64–105 (81)
ATL % BdL	6.9–9.2 (8.3)	7.4–9.6 (8.6)	5.4–8.5 (7.3)
AT-VS*	355–560 (439)	312–508 (391)	412–771 (558)
AT-VS % BdL	33–37 (35)	31–36 (33)	36–44 (41)
PTL*	86–132 (105)	87–152 (113)	77–141 (102)
PTB*	77–110 (92)	70–100 (83)	67–101 (81)
PTL % BdL	6.9–9.4 (8.5)	8.1–13.1 (9.7)	5.9–8.9 (7.7)
Ptes*	140–263 (177)	142–200 (166)	158–315 (216)
Ptes % BdL	12–17 (14)	12–16 (14)	13–18 (16)
OVL*	58–88 (72)	45–81 (60)	50–80 (64)
OVB*	50–72 (59)	38–58 (47)	39–59 (48)
OVL % BdL	5.1–6.5 (5.8)	4.0–6.3 (5.2)	3.5–6.4 (4.7)
OV-VS*	253–424 (318)	179–398 (285)	303–591 (421)
OV-VS % BdL	23–30 (25)	19–27 (24)	27–33 (30)
OV-AT*	29–97 (56)	34–64 (45)	45–146 (91)
OV-AT % BdL	2.1–7.8 (4.7)	3.1–4.4 (3.9)	4.0–8.6 (6.5)
OS-VS*	176–262 (217)	171–234 (202)	93–201 (145)
OS-VS % BdL	15–21 (17)	15–19 (17)	8–14 (11)
OS-OV*	499–737 (582)	415–692 (553)	496–851 (627)
OS-OV % BdL	43–54 (47)	45–51 (47)	40–49 (45)
OS-Vit*	318–445 (378)	291–424 (351)	359–611 (455)
OS-Vit % BdL	27–35 (30)	27–32 (29)	29–38 (33)
Previt	501–624 (552)	437–577 (504)	506–784 (615)
Previt % Bdl	40–48 (45)	39–48 (43)	40–49 (45)
Postvit	26–55 (36)	34–60 (44)	21–49 (34)
Postvit % BdL	2.2–3.6 (2.9)	3.0–5.5 (3.8)	1.8–3.5 (2.6)
VitOcc % BdL	48–56 (53)	47–57 (53)	48–59 (52)
EggL	56–77 (64)	56–68 (62)	53–72 (63)
EggB	29–42 (34)	26–39 (31)	28–41 (34)

Despite adult gorgocephalids consistent with Gorgocephalus yaaji collected from across the IWP being morphologically indistinguishable, phylogenetic analyses of the ITS2 and 28S rDNA single-gene datasets did not recover a monophyletic G . yaaji clade ( Figs 4 , 5 ). In the ITS2 single-gene tree ( Fig. 4 ), specimens morphologically consistent with G . yaaji were paraphyletic and formed three well-supported clades: specimens from Lizard Island (adults and intramolluscan stages), specimens from French Polynesia (adults and intramolluscan stages) + those from Kioloa, NSW, Australia (intramolluscan stages only), and those from South Africa (adults only). The first two of these clades were recovered in polytomy with the third + G . euryaleae + G . kyphosi , with G . graboides sister to all of these clades together. All ITS2 sequences for G . yaaji were identical for specimens within each locality and differed by 0–5 bp (0.0–1.1%) between localities ( Table 5 ). In the 28S rDNA single-gene tree ( Fig. 5 ), G . yaaji was also recovered as paraphyletic, but with the South African representatives resolving as basal to all clades. The 28S rDNA sequences of G . yaaji differed by 0–6 bp (0.0–0.6%) between localities. In contrast to analyses of ITS2 and 28S rDNA, those for the COI mtDNA single-gene dataset ( Fig. 3 ) resolved all specimens morphologically consistent with Gorgocephalus yaaji as a monophyletic group, sister to the remaining lineages of the Gorgocephalidae , albeit without support in ML analysis. Sequences differed by only 0–3 bp (0.0–0.6%) within individual localities but differed by 12–62 bp (~3.0–13%) between localities. The largest difference (62 bp) was between specimens from French Polynesia and South Africa , representing opposite sides of the IWP marine region, although the South African specimens were also relatively divergent from those collected in Australia (50–58 bp or ~11–12%; Supporting Information, Table S5 ). The concatenated COI + ITS2 + 28S analyses ( Fig. 6 ) also resolved representatives of G . yaaji in a monophyletic clade, with lower BI posterior probability support but higher ML bootstrap support. Figure 3. Bayesian majority-rule consensus tree of the COI mtDNA single-gene alignment. Bayesian inference (BI) posterior probabilities and maximum likelihood (ML) bootstrap support shown at nodes. A ‘-‘ symbol indicates the node was not recovered in ML analysis. The scale-bar indicates the number of substitutions per site. Figure 4. Bayesian majority-rule consensus tree of the ITS2 single-gene alignment. Bayesian inference (BI) posterior probabilities and maximum likelihood (ML) bootstrap support shown at nodes. An asterisk indicates different topology recovered in ML analysis. The scale-bar indicates the number of substitutions per site. Figure 5. Bayesian majority-rule consensus tree of the 28S rDNA single-gene alignment. Bayesian inference (BI) posterior probabilities and maximum likelihood (ML) bootstrap support shown at nodes. A ‘-‘ symbol indicates the node was not recovered in ML analysis. The scale-bar indicates the number of substitutions per site. Similar to the pattern observed for Gorgocephalus kyphosi , COI and ITS 2 sequences of G . yaaji , generated from one Australian locality (Kioloa, NSW) were more similar to those from French Polynesia than to those from another Australian locality, Lizard Island, GBR (Supporting Information, Table S5 ). Such molecular variation coupled with the lack of monophyly in the ITS2 and 28S rDNA trees suggests the possibility of multiple species. However, in the ITS2 and 28S trees, sequences associated with specimens morphologically indistinguishable from G . yaaji , result in a paraphyletic, rather than a polyphyletic, G . yaaji (i.e. sequences are more, or at least, as related to one another as they are to other species represented). There are also no hostlevel distinctions to delineate additional species within the G . yaaji concept; the species appears to have a broad definitive and intermediate host range (within the confines of the Kyphosidae and Littorinidae ). The issues with paraphyly in the ITS2 and 28S trees might be alleviated with sequences from additional specimens collected from localities between Australia and South Africa . However, without further evidence there appears no justification for splitting the G . yaaji clade into multiple morphologically cryptic species. Sequences of G . yaaji differ from the others species of the Gorgocephalidae recognized here by 66–88 bp (14–19%), 5–16 bp (1.1–3.5%) and 12–21 bp (1.2–2.1%) in the COI , ITS2 and 28S gene regions, respectively.