Pegasus tetrabelos Osterhage, Pogonoski, Appleyard and White, 2016

Osterhage, Deborah, Pogonoski, John J., Appleyard, Sharon A. & White, William T., 2016, Integrαted Tαxonomy Reveαls Hidden Diversity in Northern Austrαliαn Fishes: A New Species of Seαmoth (Genus Pegasus), PLoS ONE (e 0149415) 11 (3), pp. 1-26 : 7-19

Pegasus tetrabelos Osterhage, Pogonoski, Appleyard and White sp. nov. urn:lsid:zoobank.org:act:359804F0-30D0-4ECC-B355-D60D45556018 .

( Figs 2 , 3a , 4a , 5a , 6a , 7a , 8–10 ; Table 1)

Material. Holotype. CSIRO H 6553–03 , female 110 mm PCL, northeast of Dungeness Island , Torres Strait, Australia, 09°46’9” S, 143°09’19” E, 19 m depth, 24 Jan 2004.

Paratypes. Northern Territory, Australia: NTM S. 10604–008, 32 mm PCL, near North Oxley Island , Oxley Islands, 11° S, 132°49’1” E, 10 m depth, 19 Oct 1982 ; NTM S. 13283–003, 115 mm PCL, northeast of Charles Point , Beagle Gulf, 12°15’ S, 130°40’58” E, 26 m depth, 2 Sep 1992 ; NTM S. 13284–003 (2 specimens), 99–119 mm PCL, northeast of Charles Point , Beagle Gulf, 12°16’58” S, 130°40’ E, 24 m depth, 2 Sep 1992 ; WAM P. 29139–002, 109+ mm PCL (rostrum tip damaged), ~ 84 km southwest of Darwin , 12°50’ S, 130°10’ E, 34–45 m depth, 10 Sep 1965 .

Queensland, Australia: AMS IA. 6761 (2 specimens), 57–60 mm PCL, Lindeman Island , 20° 27’ S, 149°02’ E , 1936; AMS I. 20771–097 (6 specimens), 109–120 mm PCL, 1–9 mile east of Captain Billy Creek , Cape York, 11°37’ S, 142°56’ E, 16–18 m depth, 18 Feb 1979 ; AMS I. 34375–001 (9 specimens), 75–92 mm PCL, 5 km southwest of Leicester Island , Shoalwater Bay, 22°18’54” S, 150°22’44” E, 16 m depth, 24 Oct 1993 ; CSIRO H 6520–02 (2 males, 1 female), 73–102 mm PCL, north of Broad Sound , 21°47’48” S, 149°32’12” E, 10 m depth, 13 Nov 2005 ; CSIRO H 6543–03 , 107 mm PCL, CSIRO H 6543–04 , male 109 mm PCL, CSIRO H 6543–05 , male 97 mm PCL, east of Banks Island , Torres Strait, 10°07’46” S, 142°45’51” E, 19 m depth, 12 Jan 2004 ; CSIRO H 6547–02 (1 male, 1 female), 100–123 mm PCL, east of Newcastle Bay , 10° 45’27” S, 142°47’21” E, 26 m depth, 25 Sep 2004 ; CSIRO H 6555–02 , male 102 mm PCL, northeast of Mackay , 21°01’34” S, 149°21’26” E, 25 m depth, 29 Apr 2004 ; CSIRO H 6667–03 , 114 mm PCL, north of Dungeness Island , Torres Strait, 9°26’57” S, 142°51’12” E, 12 m depth, 23 Jan 2004 ; CSIRO H 6688–02 , male 110 mm PCL, west of Mulgrave Island , Torres Strait, 10° 10’33” S, 141°58’39” E, 12 m depth, 17 Jan 2004 ; CSIRO H 6738–06 (3 specimens), 114–120 mm PCL, CSIRO H 6738–07 , female 110 mm PCL, southeast of Mackay , 21°17’58” S, 149° 34’11” E, 21 m depth, 29 Apr 2004 ; CSIRO H 6793–02 , male 115 mm PCL, east of Banks Island , Torres Strait, 10°16’41” S, 142°42’31” E, 19 m depth, 13 Jan 2004 ; CSIRO H 6795–03 (2 specimens), 101–106 mm PCL, west of Prince of Wales Island, Torres Strait, 10°41’38” S, 141°53’36” E, 14 m depth, 18 Jan 2004 ; CSIRO H 6912–06 , female 112 mm PCL, east of Saibai Island , Torres Strait, 9°18’34” S, 142°55’04” E, 11 m depth, 23 Jan 2004 ; CSIRO H 6914–04 , female 114 mm PCL, west of Mulgrave Island , Torres Strait, 10°02’43” S, 141°36’32” E, 16 m depth , 16 Jan

2004; CSIRO H 6918–04 (2 specimens), 91–92 mm PCL, east of Bowling Green Bay , 19°26’48” S, 147°34’ E, 16 m depth, 2 Dec 2003 ; CSIRO H 7090–02 (15 specimens), 88–121 mm PCL, CSIRO H 7090–03 , 103 mm PCL, CSIRO H 7090–04 , female 107 mm PCL, CSIRO H 7090–05 (dry), ~ 109 mm PCL, CSIRO H 7090–06 , 88 mm PCL, north of Broad Sound , 21°37’51” S, 149°36’03” E, 15 m depth, 28 Apr 2004 ; CSIRO H 7491–03 , male 109 mm PCL, west of Mulgrave Island, Torres Strait , 10°02’55” S, 141°39’37” E, 14 m depth, 16 Jan 2004 ; CSIRO H 7502–03 (2 females, rostra damaged), 86+ and 95+ mm PCL, south of Whitsunday Island , 20° 29’32” S, 148°59’21” E, 31 m depth, 5 Dec 2003 ; CSIRO H 7660–03 (5 specimens), 102–115 mm PCL, east of Northumberland Islands , 21°37’22” S, 150°07’47” E, 34 m depth, 28 Nov 2005 ; CSIRO H 7665–01 , 111 mm PCL, CSIRO H 7665–03 , female 110+ mm PCL (rostrum tip damaged), north of Dalrymple Island, Torres Strait , 9°21’55” S, 143°24’18” E, 20 m depth, 25 Jan 2004 ; CSIRO H 7666–01 (3 specimens), 105–120 mm PCL, west of Hook Island , 20°09’46” S, 148°46’43” E, 27 m depth, 4 Dec 2003 ; CSIRO H 7667–01 , 74 mm PCL, CSIRO H 7667–02 , female 103 mm PCL, CSIRO H 7667–03 , 102 mm PCL, south of Saibai Island, Torres Strait , 9° 28’6” S, 142°43’35” E, 8 m depth, 23 Jan 2004 ; CSIRO H 7668–01 (3 specimens), 64–74 mm PCL, north of Broad Sound , 21°52’58” S, 149°30’56” E, 10 m depth, 28 Apr 2004 ; CSIRO H 7673–02 (2 females; 1 male), 102–116 mm PCL, east of Banks Island, Torres Strait , 10°06’44” S, 142°39’38” E, 17 m depth, 12 Jan 2004 ; CSIRO H 7674–01 (2 specimens), 89–99 mm PCL, east of Flinders Group , 14°10’48” S, 144°11’38” E, 15 m depth, 21 Sep 2004 ; CSIRO H 7675–01 (2 specimens), both 71 mm PCL, CSIRO H 7675–02 (1 male, 3 females), 66–73 mm PCL, Broad Sound , 22°01’ S, 149°32’56” E, 11 m depth, 28 Apr 2004 ; CSIRO H 7676–01 (3 specimens), 98– 107 mm PCL, CSIRO H 7676–02 (3 females), 98–108 mm PCL, CSIRO H 7676–03 , female 102 mm PCL, west of Northumberland Islands , 21°35’36” S, 149°42’15” E, 21 m depth, 28 Apr 2004 ; CSIRO H 7677–01 , 108 mm PCL, west of Double Cone Island , 20°06’33” S, 148°41’10” E, 30 m depth, 4 Dec 2003 ; CSIRO H 7678–01 , male 94 mm PCL, CSIRO H 7678–02 (2 female, 1 male), 90–94 mm PCL, northeast of Broad Sound , 21°53’35” S, 150°04’48” E, 22 m depth, 9 Dec 2003 ; CSIRO H 7679–01 (10 specimens), 86–105 mm PCL, north of Broad Sound , 21°44’17” S, 149°36’11” E, 14 m depth, 12 Nov 2005 ; CSIRO H 7683–01 , female 108 mm PCL, north of Ince Bay , 21°24’57” S, 149°25’36” E, 10 m depth, 29 Apr 2004 ; CSIRO H 7684–01 , female 108+ mm PCL (rostrum tip damaged), south of Scawfell Island, Cumberland Island group, 20°55’32” S, 149°38’22” E, 40 m depth, 29 Apr 2004 ; CSIRO H 7685–01 (2 females), 110–115 mm PCL, west of Curlew Island, Northumberland Island Group , 21°41’18” S, 149°41’38” E, 18 m depth, 28 Apr 2004 ; CSIRO H 7686–01 , male 114 mm PCL, northeast of Newcastle Bay, Torres Strait , 10°34’23” S, 142°58’22” E, 22 m depth, 9 Jan 2004 ; CSIRO H 7687–01 , female 115 mm PCL, west of Mulgrave Island, Torres Strait , 10°09’48” S, 141°38’36” E, 15 m depth, 16 Jan 2004 ; CSIRO H 7688–01 , male 118 mm PCL, northwest of Long Island, Torres Strait , 9°58’51” S, 142°45’53” E, 18 m depth, 20 Jan 2004 ; CSIRO H 7689–01 , male 111 mm PCL, northeast of Dungeness Island, Torres Strait , 9°39’29” S, 142°47’36” E, 10 m depth, 23 Jan 2004 ; CSIRO H 7690–01 , female 109 mm PCL, northeast of Broad Sound , 21°54’52” S, 150°01’58” E, 21 m depth, 13 Nov 2005 ; CSIRO H 7691–01 , male 91 mm PCL, Princess Charlotte Bay , 14°19’19” S, 143°54’44” E, 8 m depth, 3 Mar 2006 ; NRM 66669, 108 mm PCL, north of Broad Sound , 21°37’51” S, 149°36’03” E, 15 m depth, 28 Apr 2004 ; QM I. 36186 (3 specimens), 91–103 mm PCL, northwest of Osborne Island , 22°13’ S, 150°13’ E, 21 m depth, 10 May 2004 ; QM I. 36040 (3 specimens), 104–110 mm PCL, east of Repulse Islands , 20°36’5” S, 148°55’59” E, 24 m depth, 30 Nov 2003 ; WAM P. 34325–001, 116 mm PCL, east of Newcastle Bay, Torres Strait , 10°50’49” S, 142°52’43” E, 25 m depth, 25 Sep 2004 ; USNM 434842, 113 mm PCL, south of Dalrymple Island, Torres Strait , 9°41’05” S, 143°21’41” E, 25 m depth, 24 Jan 2004 .

Other specimens. AMS IA. 6803, 11 mm PCL, Lindeman Island , Queensland, Australia, 20° 27’ S, 149°02’ E , 1936; NTM S. 13721–009, 12 mm PCL, south of Mitchell Creek , Darwin Harbour, 12°30’46” S, 130°56’23” E, 7 m depth, 16 Jul 1993 .

Diagnosis. Tail rings 12, anteriormost 9 mobile, articulating laterally, remaining 3 fused together, attenuated and dorsoventrally flattened; terminodorsal-lateral (tdl) and terminoventral-lateral (tvl) plates each with an anteriorly and posteriorly directed spine; terminal-lateral plates (tl) absent; interpectoral plate (ip) present; single ventral preopercular notch present; rostrum spatulate; carapace with three small posteriorly directed tubercles along each dorsal ridge, one at the centre of each dorsal plate; scales not present on orbit; pectoral fin composed of 9–10 (usually 10) soft rays, 5 th ray stouter than other rays; abdominal centra 7, caudal centra 14, total centra 21; tail with 4 dark saddles, no dark saddle on tail ring XI.

Description. Body depressed, carapace depth 8.3 in holotype (6.8–9.5 in paratypes)% SL, carapace depth generally constant; interpectoral width 17.3 (14.1–17.6)% SL; carapace width 16.5 (13.5–18.1)% SL; body width gradually tapering from mid-trunk to tail. Rostrum long, length 25.4 (22.5–29.5)% SL, tapering from anterior orbit to anterior subrostral chamber then about constant throughout its length; slightly club-shaped anteriorly, tip rounded (straight

to club-shaped, tip rounded to truncate in paratypes), width at tip 3.5 (2.0–3.6)% SL; rostral spines present from rostrum tip to anterior subrostral chamber (sometimes covered with skin), 35 each side in holotype (often damaged in paratypes: 33–41 on left side [n = 7], 30–38 [n = 8] on right side). Orbit subcircular; length 6.1 (5.5–7.2)% SL, height 4.6 (4.1–5.5)% SL; interorbital width 5.0 (4.5–5.5)% SL, narrowest at posterior pupil; shallow interorbital depression; suborbital spines absent; parietals and epioccipitals with small tubercles. Carapace length 34.6 (31.3–36.8)% SL; low, paired dorsal ridges starting posterior to orbit and bisecting dorsal plates, parallel but widening slightly at carapace base, broadening onto anterior tail rings to form dorsolateral margin of tail; 3 small posteriorly directed tubercles on each dorsal ridge, one at the centre of each dorsal plate ( Fig 3A ); weak transverse ridges interconnecting dorsal plate tubercles; shallow depressions formed by lattice of dorsal ridges and transverse ridges. Plates vl 3–5 fold over plates dl 2–4, forming a grooved lateral carapace ridge between pectoral fin insertion and lateral junction of tail ring I; dorsolateral carapace plates each with a weak posteriorly directed tubercle; ventrolateral plates II to V with weak tubercles on lateral edges (weak to distinct in paratypes). A single ventral preopercular notch present where anterior point of vl 1 slots into a single notch at outer junction of vl 1 and preopercular plate ( Fig 4A ). Body, rostrum, head, and tail covered in tiny denticles; denticles in longitudinal rows on rostrum; scales absent.

Tail with 11 paired caudodorsal plates (cd 1–11), 11 paired caudoventral plates (cv 1–11), an unpaired termino-dorsal plate (td), an unpaired termino-ventral plate (tv), and terminodorsal-lateral (tdl) and terminoventral-lateral (tvl) plates each with an anteriorly and posteriorly directed spine; terminal-lateral plates (tl) absent; 6 paired caudolateral plates (cl 1–6), cl 1 is on posterolateral side of tail ring I and does not overlap tail ring II; cl 2 overlaps tail rings II–III, cl 3 overlaps tail rings III to IV, cl 4 overlaps tail rings IV to V, cl 5 overlaps tail rings V to VI, and cl 6 overlaps tail ring VI to VII; lateral plates are scutella-like anteriorly and keel-like posteriorly; keel on cl 1 is largest, approximately ¾ visible tail ring length, elevated compared to remaining keels and broadly rounded; keels on remaining caudolateral plates posteriorly directed ( Fig 5A ) and roughly diamond-shaped. Tail rings I–VIII rectangular in cross-section, with dorsal, ventral, and lateral faces joined at dorsolateral and ventrolateral margins; tail rings sequentially tapering to form a dorsoventrally flattened posterior section. Small tubercle on centre of each caudodorsal plate, most prominent anteriorly, and decreasing in size and barely visible beyond tail ring IX. Tail rings I–VIII with weak tubercles/keels at ventrolateral margin. Tail rings VIII– XII with translucent, lateral ridges that extend length of tail ring; each lateral ridge with fine denticulations along its length except where ending in a spine tip; tail ring IX ridge terminating in small single posteriorly-directed spine (spines sometimes encased within skin); tail ring X and XI ridges starting and ending with small single anterior and posterior spines; tail ring XI also with superior ridge starting mid-tail ring and terminating in an additional posterior spine; terminodorsal-lateral (tdl) and terminoventral-lateral (tvl) plates each with an anteriorly and posteriorly directed spine, plates diverge posteriorly with distance between posterior spines twice the distance between anterior spines ( Fig 7A ).

Pectoral fin with 10 soft rays (9 on both sides of paratype CSIRO H 6543–04), 5 th ray stouter than other rays. Pelvic fin with 1 spine and 2 rays; 2 nd ray reduced to a single element, very small in size and connected by thin membrane to 1 st ray. Dorsal fin with 5 rays; anal fin with 5 rays; length of dorsal and anal fin rays not equal, anterior ray longest, posterior ray shortest. Caudal fin with 8 rays, truncate to slightly convex.

Colour. Holotype (female, Fig 2 ): Prior to preservation (post-thawing, specimen frozen for 10 years). Dorsal and lateral body surfaces cream to medium brown, overlaid with dark brown to black spots, blotches and saddles. Trunk medium brown with small dark brown to black spots; a dark brown to black transverse bar across plate dl 2; dorsal ridges cream; plate dl 4 with black border, thickest anteriorly at margin with plate dl 3. Head cream to reddish-brown with pale brown to black spots; a triangular pattern of dark brown spots between posterior orbits and anterior junction of plates dl 1, with a small, dark V-shaped marking posteriorly; three prominent dark brown to black spots about half pupil width, first below anterior eye (only visible laterally), second below posterior pupil, and third anterior of 3 rd circumorbital (second and third spots visible dorsally, laterally and ventrally). Rostrum translucent, in dorsal view from posterior to anterior: small dark brown spots between orbit and above subrostral chamber, a pair of large pupil-sized spots wrapping around lateral edges in line with anterior subrostral chamber, followed by a single dark brown bar and 5 roughly pupil-sized dark brown spots that alternate along the rostrum edges, ending just before the rostral tip; in lateral view: 3 horizontal blotches between anterior orbit and level of anterior subrostral chamber; in ventral view: no additional pigmentation, but dorsal and lateral markings visible through translucent section of rostrum. Pectoral-fin base cream to pale-brown with small reddish-brown spots. Pectoral fins translucent with small dark brown spots on fin membranes and fin rays; two sets of large, dark brown spots on fin rays, first at about their mid-length and second closer to outer membrane; large spots in pairs on 4 th –8 th fin rays; remaining rays with single spots; no large spots centrally on the stout 5 th pectoral fin ray; dark brown spots basally on 3 rd –5 th pectoral rays. Tail cream dorsally and laterally with small reddish-brown spots; dark brown blotch centrally on anterior part of tail ring I; clusters of larger spots or blotches forming 4 dark saddles, first on tail rings III– V at about position of dorsal-fin base, second on tail rings VII–VIII, third on tail ring IX–X, and last on tail ring X; some less distinct saddles or groupings of spots present. Dark brown spots present on lateral side of caudoventral plates 1–3 and 6–7. Dorsal and anal fins translucent; each dorsal-fin ray with a small dark brown spot near base and near tip; each anal-fin ray with a small dark spot distally. Caudal fin translucent with dark brown blotch at base and dark brown spots on rays, most evident distally. Pelvic rays cream with 2 faint brown blotches toward tip. Ventral surfaces mostly pale cream and whitish, mediumbrown at pectoral-fin base and anterior half of head, including mouth; some dorsal saddles visible ventrally through tail rings, especially posteriorly.

In preservative: Reddish brown and cream fade to shades of greyish brown and yellow, respectively. On carapace, pale dorsal ridges less distinct and blending into background colouration; transverse bar less distinct; dorsal lateral plates finely outlined in black. Dorsal plate margins, ventral plate margins posterior to gular plate, and ventral tail margins outlined in light grey. Pectoral and ventral plates darkened to yellowish brown; ventral base of pectoral fins coloured yellow to yellowish brown.

Paratype ( CSIRO H 7491–03 , male): Prior to preservation (post-thawing, specimen frozen for 10 years). Similar to holotype, but bands of spots on pectoral fins are usually single spots (not paired), except midway along on 4 th and 7 th rays which are paired. Each dorsal-fin ray with an additional medial small dark brown spot; each anal-fin ray with two small dark spots distally .

Colour variation amongst measured paratypes and rostrum markings from sexed specimens: Some paratypes without rostral markings, and/or single dark brown bar either absent or replaced by parallel spots, and/or when present, remaining markings appear as 4–8 alternating spots along rostrum edges or 3–4 bars. Large spots on pectoral fins occur either paired or singly. Dark brown to black spots below anterior eye and/or below posterior pupil may be absent. Sometimes with 3 pelvic fin spots.

Freshly caught specimen (paratypes CSIRO H 6543–04 and CSIRO H 7678–01 , both males): Carapace including supraorbital tinged olive to dark green; pectoral fins and dorsal tail rings with small orange-brown spots; single orange-brown spot on base of 1 st dorsal ray visible in dorsal view; single orange-brown spots anteriorly on tail rings I and II along both dorsal tail ridges .

Size

Type specimens ranged from 32 to 123 mm PCL (morphometric types ranged from 79 to 98 mm SL, 94 to 116 mm PCL). Two non-type specimens of 11 and 12 mm PCL are probably this species .

Distribution

Pegasus tetrabelos is known from the east coast of Queensland and Torres Strait between latitudes 9°15’ S and 22°01’ S, and in the Northern Territory from the Beagle Gulf to off Darwin ( Fig 8 ). Specimens collected during the Torres Strait and Great Barrier Reef trawl surveys [ 27, 28] formed three distinct clusters ( Fig 9 ). The first cluster was located in the Torres Strait between latitudes 9°15’ S and 10°51’ S; the second cluster in Princess Charlotte Bay (~ 14°15’ S); and the third from Bowling Green Bay (19°26’ S) south to Broad Sound (22°01’ S). Despite intensive trawling, no specimens of the new species (or P. volitans ) were caught between these clusters in the trawl survey. This likely reflects specific habitat preferences. There are currently no records of this species from the Gulf of Carpentaria or Western Australia.

Specimens of P. tetrabelos were collected at depths of 8–45 m, mostly in depths less than 30

m. Although the trawling surveys of the Great Barrier Reef and Torres Strait were conducted to

depths of 100 m, P. tetrabelos was not collected in depths greater than 40 m.

Etymology

The species name tetrabelos is a combination of the Greek ‘ tetra’ meaning four and ‘ belos ’ meaning dart or arrow in allusion to the four backward pointing spines on the terminal tail ring (two on each side). The name is treated as a noun in apposition.

Vernacular names

Short-spined Seamoth.

Genetic analyses

The genetic identity of the Pegasus specimens was based here on their 16S and COI sequence

information. Each of the 24 samples sequenced successfully for both mtDNA fragments; aver-

age COI sequence was 583 base pairs, while the average 16S sequence was 546 base pairs. The

14 P. volitans samples were all highly matched (99.7% pairwise identity) to Accession No.

AP005984 ( P. volitans mtDNA complete genome, [ 31]) while the nine P. tetrabelos samples

matched the same Accession (No. AP005984) at a lower level of 97.3% pairwise identity.

Average 16S nucleotide composition across the P. volitans specimens consisted of 23.6%

Thymine, 23.5% Cytosine, 30.9% Adenine and 22.0% Guanine residues. This was essentially

the same across the P. tetrabelos specimens with the exception of a higher Cytosine content

(24.2%). For COI, the residue content varied slightly more between the species ( P. volitans :

27.4% T, 27.8% C, 26.1% A, 18.7% G; P. tetrabelos : 27.6% T, 27.9% C, 25.0% A, 19.5% G).

Evolutionary divergence over pairs of P. volitans samples (i.e. average genetic distances/

diversity from the number of base substitutions per site, ± SE) was 0.001 (±0.001) for 16S and

0.011 (±0.003) for COI. In P. tetrabelos , the 16S genetic diversity among specimens was also

low at 0.001 (±0.000), with a slightly higher divergence of 0.004 (±0.001) for COI. The K2

divergence between the two species at the 16S gene was 0.015 (±0.004), while the COI gene

reflected a much higher divergence of 0.081 (±0.011). This concordant, yet higher sequence

divergence between the two species in the two mtDNA genes was observed in the longer

branch lengths of the ML trees for COI as compared to 16S (see Fig 10a and 10b ). As Fig 10

shows, there was clear delineation of the species. The P. tetrabelos sequences clustered together,

and specimens of the two Pegasus species formed separate groups (with external branch lengths

well supported by high bootstrap values). Both species were definitively separated from the out-

group specimen, E. draconis (Accession No. AP005983). The NJ and Bayesian trees while not

shown here, displayed the same topology.

Figures

Fig 2. Holotype of Pegasus tetrabelos (CSIRO H 6553–03, 110 mm PCL). (A) dorsal; (B) lateral; and (C) ventral views. doi:10.1371/journal.pone.0149415.g002

Fig 3. Lateral view of dorsal ridge. (A) Pegasus tetrabelos (CSIRO H 7665–01); (B) Pegasus volitans (CSIRO H 6649–02). doi:10.1371/journal.pone.0149415.g003

Fig 4. Ventral view of preopercular plate. (A) Pegasus tetrabelos (CSIRO H 7665–01), arrow indicates single ventral preopercular notch; (B) Pegasus volitans (CSIRO H 6649–02), arrows indicate double ventral preopercular notches. doi:10.1371/journal.pone.0149415.g004

Fig 5. Ventral view of tail from mid-tail ring IV to mid tail ring VII. Caudolateral plate keels at intersects of tail rings. (A) Pegasus tetrabelos (CSIRO H 7665–01); (B) Pegasus volitans (CSIRO H 6649–02). doi:10.1371/journal.pone.0149415.g005

Fig 6. Dorsal view of posterior tail from mid-tail ring X to tail ring XII. (A) Pegasus tetrabelos (CSIRO H 7665–01); (B) Pegasus volitans (CSIRO H 6649– 02). doi:10.1371/journal.pone.0149415.g006

Fig 7. Lateral view of tail ring XII. (A) Pegasus tetrabelos (CSIRO H 7665–01) showing terminodorsal-lateral and terminoventral-lateral plates each with an anteriorly and posteriorly directed spine; (B) Pegasus volitans (CSIRO H 6649–02) showing terminal-lateral plate with an anteriorly and posteriorly directed spine. doi:10.1371/journal.pone.0149415.g007

Fig 8. Map showing the collection locations of the material examined. Pegasus volitans specimens indicated by yellow dots and Pegasus tetrabelos specimens indicated by black (holotype) and red (paratypes) dots. Map generated in QGIS using Natural Earth 1:10,000,000 data (www.naturalearthdata.com). doi:10.1371/journal.pone.0149415.g008

Fig 9. Trawl sites from the Torres Strait and Great Barrier Reef surveys. Sites where Pegasus tetrabelos (red dots) and Pegasus volitans (yellow dots) were recorded; black dots refer to those sites where Pegasus specimens were not captured. Map generated in QGIS using Natural Earth 1:10,000,000 data (www. naturalearthdata.com). doi:10.1371/journal.pone.0149415.g009

Fig 10. Molecular species identification of Pegasus species using Genetic treeML trees. (A) sequences from the 16S gene; (B) sequences from the COI gene. Trees are based on the K2 evolutionary distance model and are shown here with mined Pegasus and Eurypegasus sequences from GenBank. The trees are shown here with an E. draconis outgroup. Bootstrap support values (following 1000 replicates) are shown above the nodes. doi:10.1371/journal.pone.0149415.g010