Larval development of the Ambon damselfish Pomacentrus amboinensis, with a summary of
pomacentrid development
B. F. MURPHY*†, J. M. LEIS*‡ AND K. D. KAVANAGH§
*Ichthyology, Australian Museum, 6 College Street, Sydney, NSW, 2010, Australia,
†School of Biological Sciences, University of Sydney, Sydney 2006, NSW, Australia and
§Institute of Biotechnology, University of Helsinki, P. O. Box 56, Viikinkaari 9A, Helsinki, FIN-00014, Finland
(Received 4 October 2006, Accepted 15 March 2007)
Morphological development of the larvae of the Ambon damselfishPomacentrus amboinensis from the Great Barrier Reef, Australia, is described from 34 reared specimens (25–130 mm standard length), and two wild settlement-stage larvae (108–113 mm) captured in light traps.
Reared larvae emerged from demersal eggs at c. 25 mm, underwent notochord flexion at 40–45 mm, and settled at 11–13 mm at 19–21 days after hatching (within the known range for wild larvae). Reared larvae grew at 03 mm day1(preflexion) to 05 mm day1(postflexion).
Development was direct with few specializations for pelagic existence and was typical of pomacentrids. Head spination consisted of very weak spines on the preopercle, opercle, interopercle, subopercle and supracleithrum. Development of the olfactory system and retina are also described. A summary of published descriptions of early life-history stages of pomacentrid damselfishes is provided (n¼15% of known species). Larvae of several genera (e.g. Abudefduf, Chromis and Stegastes) are deeper-bodied and more ‘hunchbacked’ than P.
amboinensis. Larvae of other genera (e.g. Amblyglyphidodon, ChrysipteraandNeoglyphidodon) are similar to P. amboinensis. Larvae of only three other Pomacentrus species have been described, and they are similar toP. amboinensisin body morphology, pigmentation and head
spination. #2007 The Authors
Journal compilation#2007 The Fisheries Society of the British Isles
Key words: damselfish; development; growth; larva; Pomacentridae; sense organs.
INTRODUCTION
The perciform family Pomacentridae (damselfishes) contains c. 350 relatively small, site-attached, demersal species distributed among 28 genera of primarily coral-reef fishes (Allen, 1991; Randall, 2005). Pomacentrids are among the most conspicuous and abundant fishes in many reef communities, and have received a great deal of attention from reef-fish ecologists (Fishelson, 1998).
Indeed, a large proportion of current understanding of the ecology of coral-reef
‡Author to whom correspondence should be addressed. Tel.:þ61 2 9320 6242; fax:þ61 2 9320 6059;
email: [email protected]
doi:10.1111/j.1095-8649.2007.01524.x, available online at http://www.blackwell-synergy.com
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fishes is based on study of pomacentrids (Sale, 1991, 2002). Most species are trophically flexible, feeding on plankton, algae and small invertebrates (Allen, 1991). Except for the aquarium trade, pomacentrids are of little commercial importance, but their great diversity and abundance on coral reefs makes them ecologically important (Kavanagh, 2000; Kavanaghet al., 2000). The early life- history stages of only c. 15% of pomacentrid species have been described at any level (Table I), and the quality and extent of existing descriptions are vari- able. Many of the descriptions are in hard-to-access journals. The purpose of the present contribution was to summarize current knowledge of pomacentrid development and to describe the development of the pelagic larval stages of a prominent pomacentrid, the Ambon damselfish Pomacentrus amboinensis Bleeker.
Pomacentrus amboinensisis a very common pomacentrid that attainsc. 11 cm standard length (LS). It occurs in the western Pacific and eastern Indian Oceans, including the Andaman Sea, the Indo-Malayan Archipelago and the Melanesian Islands (except Fiji) (Allen, 1991). It is also found south to New South Wales in Australia and north to the Ryukyu Islands, the Marshall Islands, the Caroline Islands and the Mariana Islands (Allen, 1991). Adults inhabit sandy areas around coral heads and rocky outcrops in a depth range of 2–40 m (Allen, 1991; Randall et al., 1997). P. amboinensis has a somewhat oblong egg of 12–13 mm that hatches in 4 days (Kavanagh & Alford, 2003). Larvae emerge at 26–29 mm LS (Kavanagh, 1996; Kavanagh & Alford, 2003), undergo a pelagic larval duration (PLD) of 15–32 days, with time-vary- ing mean values of 18–23 days (Thresher et al., 1989; Wellington & Victor, 1989; Bay et al., 2006) and settle at 103–151 mm LS (Kerrigan, 1996). At set- tlement, P. amboinensis undergoes minimal change, transformation being lim- ited primarily to rapid changes in colour (McCormick et al., 2002). This species has received considerable attention in the ecological literature because of its abundance and suitability for field experimentation. All life-history stages have been studied, but, surprisingly, there has been no description of the larval development ofP. amboinensis. A description of the development of the pelagic larval stages of this high-profile damselfish is overdue.
MATERIALS AND METHODS
Morphological definitions, measurements and abbreviations follow Neiraet al.(1998) and Leis & Carson-Ewart (2000). Sizes of larvae areLS, which for preflexion and flexion- stage larvae are measured to the tip of the notochord. Principal caudal-fin rays are con- sidered those that are supported by the hypurals and parhypural (Moser et al., 1977;
Moser, 1996; Leis & Carson-Ewart, 2000). In most, if not all, pomacentrids, the equiv- alent of the 8th ventral principal ray in most other perciform fishes is supported by the haemal spine of the penultimate vertebral centrum, not the parhypural (Fujita, 1990), and thus does not constitute a principal caudal ray, resulting in a count of 9þ7. Lar- vae and juveniles examined were measured with an eye-piece graticule of a dissecting microscope at magnification of6 to50. Precision of measurements varied with mag- nification but ranged from 002 to 017 mm. Where morphometric values are expressed as a percentage, they are as a proportion ofLS unless otherwise specified. Preflexion, flexion and postflexion larval stages are as defined by Leis & Carson-Ewart (2000).
Drawings were prepared using either a camera lucida [Fig. 1 (a), (b)] or by tracing from digital photomicrographs [Fig. 1 (c)–(g)]. Figure 2 was taken with a Leica Digital
TABLEI.Pomacentridspecieswithdescribedorillustratedeggsorlarvae GenusSpecies
Life-historystagesdescribed ReferenceEggPre.Flex.Post.Setl. Abudefdufluridus(Cuvier)yynnnRe(1980) Abudefdufsaxatilis(Lacepe`de)yyyynAlshuthetal.(1998) Abudefdufseptemfasciatus(Cuvier)nynnnTanaka(1998) AbudefdufsordidusForsska˚ly*y*nynKinoshita(1988);Lobel&Schreiber(2004) Abudefduftaurus(M¨uller&Troschel)nnnnyParis-Limouzyetal.(2006) AbudefduftroschelliiGillnyyynWatson(1996) Abudefdufvaigiensis(Quoy&Gaimard)nnnynKinoshita(1988) Acanthochromispolyacanthus(Bleeker)y——nyKavanagh(2000) Amblyglyphidodoncuracao(Bloch)yynnnTanaka&Mori(1989) Amphiprionclarkii(Bennett)nnnynKinoshita(1988) Amphiprionpercula(Lacepe`de)nynnnDelsman(1930) Amphiprionpolymnus(L.)y*yy*y*nTanaka(1998);Chenetal.(2003)* Chromiscaeruleus(¼viridis)(Cuvier)nnnynKinoshita(1988) Chromischromis(L.)yyyynPadoa(1956) Chromiscyanea(Poey)nyyyyParis-Limouzyetal.(2006) ChromisdispilusGriffinyyyynKingsford(1985) ChromisenchrysuraJordan&GilbertnynynParis-Limouzyetal.(2006) Chromislimbata(Valenciennes)yynnyRe&Gomes(1982) ChromismargaritiferFowlernynnnTanaka,(1998) Chromismultilineata(Guichenot)yyyyyParis-Limouzyetal.(2006) ChromisnotataTemminck&SchlegelyyyynFujita(1957);Suzukietal.(1985a);Kinoshita(1988) Chromispunctipinnis(Cooper)yyyynWatson(1996) Chromisvanderbilti† (Fowler)nyyynKavanaghetal.(2000) Chrysipterahemicyanea(Weber)yyyyyTanaka&Yamada(2001) Chrysipteraleucopoma(Lesson)nynnnTanaka(1998) Chrysipteraparasema(Fowler)yyyyyTanaka&Nitta(1997b) Chrysipterarex(Snyder)yyyynTanaka&Fushimi(1998) Dascyllusaruanus(L.)yynnnTanaka(1999) DascyllusmelanurusBleekeryynnnTanaka(1999) Dascyllusreticulatus(Richardson)yynnnTanaka(1998);Tanaka(1999)
TABLEI.Continued GenusSpecies
Life-historystagesdescribed ReferenceEggPre.Flex.Post.Setl. Dascyllustrimaculatus(R¨uppell)yynnnTanaka(1999) Dischistodusmelanotus(Bleeker)nynnnTanaka(1998) Hypsypopsrubicundus(Girard)yyyynWatson(1996) Microspathodonchrysurus(Cuvier)yyyyyPotthoffetal.(1987);Paris-Limouzyetal.(2006) Neoglyphidodonmelas(Cuvier)yyyyyTanakaetal.(1996) Neoglyphidodonnigroris(Cuvier)yyyynTanaka&Takamiya(1999) Neopomacentrusviolasceus(Bleeker)yyyyyTanaka(1998);Tanakaetal.(2003) Plectroglyphidodonlacrymatus(Quoy&Gaimard)nynnnTanaka(1998) Plectroglyphidodonleucozonus(Bleeker)nnyynKinoshita(1988) PlectroglyphidodonjohnstonianusFowler&BallnnnnyKinoshita(1988) PomacentrusamboinensisBleekerny*nnnKavanagh&Alford(2003) PomacentruscoelestisJordan&StarksnyyyySuzukietal.(1985b);Kinoshita(1988);Tanaka&Nitta (1997b) PomacentrusnagasakiensisTanakayyyynKinoshita(1988);Tanakaetal.(2002) Pomacentruspavo(Bloch)yyyyyTanakaetal.(2004) PomacentrusphilippinusEvermann&SealenynnnTanaka(1998) PomacentrustaeniometoponBleekernnnnyKinoshita(1988) Pomachromisrichardsoni(Snyder)nynynKinoshita(1988);Tanaka(1998) PremnasbiaculeatusBlochnynnnTanaka(1998) Stegastesdiencaeus(Jordan&Rutter)yynynParis-Limouzyetal.(2006) Stegastesdorsopunicans(Poey)yyyyyParis-Limouzyetal.(2006) Stegastesleucostictus(M¨uller&Troschel)yyyyyLimouzy-Parisetal.(1994);Paris-Limouzyetal.(2006) Stegastesotophorus(Poey)nnnnyParis-Limouzyetal.(2006) Stegastespartitus(Poey)yyyyyParis-Limouzyetal.(2006) Stegastesrectifraenum(Gill)nyyynWatson(1996) NoteA.polyacanthushatchesatthepostflexionstage.y,yes;n,no;Pre,preflexionstage;Flex,flexionstage;Post,postflexionstage;Setl.,settlementstage. *Descriptionasaphotographonly. † Tentativeidentification.
photomicrograph system. Using an image analysis system, muscle area (AM, in mm2), which can be important in relating morphological development to swimming ability (Fisher et al., 2000), was measured by outlining the total lateral area excluding head, gut, yolk and fins. Eye differentiation was observed from histological sections, and
FIG. 1. Larval development of rearedPomacentrus amboinensis. Age is in days after hatch (DAH): (a) 250 mm standard length (LS) yolk-sac preflexion-stage larva, 0 DAH, AMS I. 38139-034. Note incompletely pigmented eye and apparently non-functional mouth. The yolk sac occupies most of the gut region, (b) 280 mmLSpreflexion-stage larva, 1 DAH, AMS I.38139-035. No separate yolk is visible, (c) 365 mmLSpreflexion-stage larva, 6 DAH, AMS I.38139-023, (d) 457 mmLSflexion- stage larva, 7 DAH, AMS I.38139-026, (e) 598 mmLS postflexion-stage larva, 11 DAH, AMS I.38139-028, (f) 730 mmLSpostflexion-stage larva, 13 DAH, AMS I.38139-030. Some scales are present, but are not illustrated and (g) 880 mmLSpostflexion-stage larva, 15 DAH, AMS I.38139- 031. Scales are present over most of the body, but are not illustrated.
FIG. 2. 1062 mm standard length reared larva ofPomacentrus amboinensisapproaching settlement stage, 21 days after hatch. AMS I.38139-033.
external olfactory development was observed from scanning electron micrographs (SEM) of a developmental series (methods are described in Kavanagh & Alford, 2003).
Adult P. amboinensis captured on the central Great Barrier Reef constituted the broodstock that provided the laboratory-reared developmental series used. Larvae from the Australian Museum, Sydney (AMS I.38139) were reared at the research aquaria facilities at James Cook University, Townsville (JCU), in November to December 1994 at 28–29° C using the methods described in Kavanagh & Alford, 2003. Sixteen larvae from this series were examined in detail and measured, and another 10 larvae were examined in less detail to ascertain particular developmental events. A single settlement-stage larva reared with methods similar to those used for I.38319 at JCU (S. Job, pers. comm.) was examined in detail. Two wild settlement-stage larvae were cap- tured using light traps at a depth of 1–3 m at Lizard Island, Great Barrier Reef in 2001.
Seven cleared and stained larvae were prepared from specimens from the AMS I.38139 series. Specimens examined had a LS range of 25–130 mm and ages of reared larvae ranged from 0 to 27 days after hatching (DAH). Larvae were fixed in 5–10% formalin and preserved in 70% ethanol. All descriptions are of preserved specimens.
Material examined is deposited at the AMS. Registration numbers are AMS I.39844- 010, I.38139-022 to -035 (all specimens examined in detail), -036 to -040 (some specimens examined in less detail), I.43594-001 to -007 (cleared and stained examined specimens), I.41401-142 and -143 (wild examined specimens).
As reared larvae were spawned by known adults, their identity is assured. Field-caught settlement-stage larvae were identified as P. amboinensis using the characters listed by Allen (1991), particularly the fin meristics: dorsal (D) XIII, 14–16; anal (A) II, 14–16;
and pectoral (P1) 17–18 (Allen, 1991, reports only 17 P1 rays, but some settlement-stage P. amboinensisfrom Lizard Island on the Great Barrier Reef have 18 P1 rays); and the bright yellow live colour, absence of blue lines on the head, combined with the presence of an ocellus on the dorsal fin spanning the posterior-most spines and anterior-most soft rays (Masuda & Kobayashi, 1994; Randall, 2005). No other pomacentrid found at Lizard Island (Great Barrier Reef) has this colour pattern at settlement (pers. obs.).
RESULTS
G R O W T H
At 28–29°C, the rearedP. amboinensis hatched in 4 days, and settled at 19–21 DAH at 112–125 mm LS. Growth (Fig. 3) was best described by separate linear relationships for: 1) larvae before notochord flexion was complete
FIG. 3. Growth in standard length (LS) ofPomacentrus amboinensislarvae reared in the laboratory in November to December 1994. Notochord flexion was complete by 10 days after hatch (DAH) and settlement took place by 24 DAH.
(DAH 0–6) (LS ¼ 0282t þ 2760, where t ¼ DAH) and 2) larvae after noto- chord flexion was complete (DAH 7–23) (LS ¼ 0492t þ 1200, where t ¼ DAH). The r2 values for the two relationships were 075 and 092, respec- tively, with P < 0001 for both. The 95% CI of the two slopes (i.e. growth rates) did not overlap, confirming that the growth rates of the two portions of the larval phase were different. Therefore, preflexion and flexion-stage lar- vae grew at a mean 95% CI 0282 0057 mm day1, whereas postflexion larvae grew nearly 75% faster, 0492 0021 mm day1. In addition, growth rate varied considerably among individuals. By 9 DAH, the range of LS was 44–56 mm, and at 18 DAH, a range of 84–116 mm was attained, constitut- ing a maximum range of daily for the series. Growth of AM was allometric:
AM¼00181L29092S ðr2 ¼099Þ:
G E N E R A L M O R P H O L O G Y ( T A B L E I I A N D F I G S 1 A N D 2 ) At hatching, neither the mouth nor the eye are complete, and there is a large yolk sac. Within 24 h, the mouth is open, the eye heavily pigmented and the
TABLEII. Morphometrics of Pomacentrus amboinensis larvae. Measurements were not recorded for cleared and stained specimens
Age LS PAL PDL HL ED SnL BDP1 VAFL CPL P2L
(DAH) (mm)
Preflexion
0 250 085 060 045 020 013 045
1 280 100 070 055 028 010 045
2 282 092 070 064 032 016 052
6 365 140 120 100 044 018 076
Flexion
5 436 168 148 112 044 036 100
7 440 176 156 132 044 042 090
7 457 184 164 136 052 032 096 006
Postflexion
8 465 216 212 148 060 040 126 040 016
11 598 315 241 216 080 068 184 032 092 068
12 697 349 265 249 092 068 220 042 104 112
13 730 382 282 274 092 064 248 042 108 132
15 880 465 299 324 096 088 308 040 104 188
17 963 498 349 365 116 088 380 044 112 236
21 1062 614 398 416 152 100 433 032 108 289
F 1079 598 365 412 133 100 415 032 120 260
F 1129 631 398 415 149 108 432 034 140 280
27 1295 697 432 415 176 115 581 048 160 320
DAH, days after hatch (of reared larvae); F, the larva is field-caught;LS, standard length; PAL, preanal length; PDL, predorsal length; HL, head length; ED, eye diameter; SnL, snout length;
BDP1, body depth at pectoral-fin base; VAFL, vent to anal-fin length; CPL, caudal peduncle length;
P2L, length of pelvic fin.
yolk largely exhausted. Larvae are laterally compressed and initially elongate, but gradually become deep and relatively more compressed. Body depth is 16% at hatching, reaches 23% by the start of flexion, 27% when flexion is complete and 45% by settlement. There are 26 myomeres, initially 6 þ 20 (preanal þ postanal), becoming 11þ 15 in postflexion larvae due to posterior extension of the gut. The preanal length is 33–38% in preflexion larvae and 47–
58% in postflexion larvae as the gut extends posteriorly. The gut is coiled and compact, with a gap between anus and anal fin that becomes smaller but does not completely disappear before settlement: 86% at the end of flexion, decreas- ing to 32% at settlement. The head is initially small (18–27%), but becomes moderate in size by flexion (26–30%) and is large (32–39%) in postflexion lar- vae. The snout is short (less than eye diameter) throughout development. It is blunt at hatching, but quickly becomes concave, and remains so until the end of flexion, after which it is more rounded and convex, but it becomes shorter relative to head length. The eye is round and large, constituting 8–14%LS. The mouth is moderately oblique in preflexion larvae, becoming much less oblique in postflexion larvae. Once the mouth is open, the posterior tip of the maxilla extends past the anterior edge of the eye, but not to the pupil. By settlement, the tip of the maxilla does not reach the eye. Small conical teeth are present on both the upper and lower jaw by 96 mm, but are not readily visible due to the lips. The nasal pit begins to roof over atc. 60 mm, and two nostrils are present at 70 mm. Two nostrils are still present in the largest settlement-stage larvae (130 mm), although only a single nostril is present in adults. Scales begin to form atc. 7 mm, ventral to the base of the P1fin and in one or two rows along and above the lateral midline. A full complement of scales is present at 9–10 mm.
Head spination is weak. No head spines are present until after 3 mm. Two small spines form on the outer border of the preopercle by 37 mm just before notochord flexion commences. The single spine on the lower, outer preopercle border persists, and from 60 mm a second may be present. The spine on the upper, outer preopercle border is joined by a second byc. 6 mm, and five are present by 88 mm. The upper, outer preopercle border becomes serrate at c.
93 mm. A small spine develops on the upper, inner preopercle border by the commencement of flexion and a small spine forms on the lower, inner preop- ercle border at 44 mm. The position of these spines along the inner preop- ercle border is variable, and one or two spines may be found at the preopercular angle in some postflexion larvae between 6 and 9 mm. There- fore, a total of between two and four small spines may be present on the inner preopercle border from 6 mm. Inner preopercular spines become reduced at 106 mm and are lost by 130 mm. One subopercular spine and one interopercular spine form just prior to notochord flexion (37 mm): both are small. These remain as single spines, except in one 100 mm specimen that has two subopercular spines. The subopercular and interopercular spines become reduced in late postflexion larvae (103 mm) and were visible only as two bony plates on the edge of the bones in a 108 mm larva. One supra- cleithral spine appears at 37 mm, a second develops in early postflexion lar- vae by 60 mm, and three or four supracleithral spines are present in a 118 mm specimen. A single opercular spine forms following flexion (60 mm) and becomes less prominent as the larva develops.
F I N D E V E L O P M E N T ( T A B L E I I I )
At hatch, only the rayless medial finfold and pectoral fin are present. Noto- chord flexion begins at c. 4 mm and is complete at c. 5 mm. A very weak caudal-fin anlage is first present at c. 28 mm. The first rays are present at the start of flexion and the principal rays are formed by the completion of flexion.
Cleared and stained specimens confirm there are 9 þ 7 principal caudal rays, with six ventral principal rays attached to the hypural and one attached to the parhypural. Anlagen of the dorsal and anal fins form during notochord flexion;
incipient rays form early in the postflexion stage, and the full complement of TABLEIII. Fin development in Pomacentrus amboinensis.Values in parentheses indicate countable incipient elements; square brackets indicate a soft ray that will transform (arabic numeral) or is transforming (roman numerals) into a spine. For cleared and stained specimens, the vertebrae count is given instead of the myomere count. Larvae are
reared unless otherwise indicated
LS(mm) Dorsal Anal Pectoral Pelvic Caudal
Myomeres (vertebrae) Preflexion stage
250 6þ20¼26
280 Anlage 5þ21¼26
282 Anlage 6þ20¼26
365 Weak anlage Weak anlage Anlage 5þ21¼26
Flexion stage
436 Weak anlage Weak anlage 7þ6 7þ19¼26
440 Anlage Anlage 7þ7 6þ20¼26
457 10 bases 10 bases Buds 8þ7 7þ19¼26
Postflexion stage
465 (10), 17 bases (I, 9), 15 bases (5) Buds 9þ7 8þ18¼26 598 X(II)[1], 12(2) II, 13(2) 15þ I, 4þ 9þ7 10þ16¼26 631cþs VIII(IV)[1],
10 (4)
II, 10 (5) 8þ Buds 9þ7 (11þ15¼26)
697 XII[I], 14 II, 15 10(3)þ I, 5 9þ7 8þ18¼26
730 XII[I], 14 II, 15 16þ I, 5 9þ7 10þ16¼26
755cþs XII[I], 14 II, 14 D I, 5 9þ7 (11þ15¼26)
805cþs XII[I], 14 II, 14 15(2) I, 5 9þ7 (11þ15¼26)
830cþs XIII, 15 II, 15 17 I, 5 9þ7 (11þ15¼26)
880 XIII, 14 II, 14 17 I, 5 9þ7 11þ15¼26
930cþs XIII, 14 II, 15 17 I, 5 b 9þ7 (11þ15¼26)
963 XIII, 14 II, 14 17 I, 5 9þ7 11þ15¼26
996cþs XIII, 14 II, 14 17 I, 5 b 9þ7 (11þ15¼26)
1062 XIII, 14 II, 14 17 I, 5 b 9þ7 11þ15¼26
1079f XIII, 15 II, 15 18 I, 5 b 9þ7 S
1129f XIII, 15 II, 16 18 I, 5 b 9þ7 S
1179cþs XIII, 14 II, 14 16 I, 5 b 9þ7 (11þ15¼26)
1295 XIII, 15 II, 15 17 I, 5 b 9þ7 S
þ, additional incipient elements that were not countable; b, the rays are branched; D, damaged;
S, scales obscured myomeres.
fThe larva is field-caught.
cþsA cleared and stained, reared larva.
elements is present by 63 mm. In the dorsal fin, soft rays develop from anterior to posterior and begin to form prior to the spines, although full complements of both spines and rays are present at about the same time. The last spine of the dorsal fin forms as a soft ray by 6 mm, but does not fully transform into a spine until 83 mm. Formation of the anal fin parallels that of the dorsal fin, but both spines of the anal fin form directly. Incipient rays form in the pectoral fin at c.
47 mm, the first rays form early in the postflexion stage, and a full complement of rays is present at c. 8 mm. Pelvic-fin buds form during flexion, the spine is present at 60 mm and a full complement of I, 5 is present at c. 7 mm.
O L F A C T O R Y O R G A N S
On the day of hatching (0 DAH, meanLS ¼28 mm), a round patch of cilia is present on the snout where the olfactory nares will form. At 4–6 DAH (mean LS ¼35–44 mm), the nasal pit forms and deepens. The pit elongates, and by 12 DAH (mean LS ¼ 74 mm), divides by ‘pinching in’ of tissue along the middle. At 23 DAH (mean LS ¼ 123 mm), the nares are cleanly divided.
The dorsal-most nostril of the pair then enlarges. Only following settlement, does the adult condition of one nostril on each side of the snout develop.
E Y E
The eye placode differentiates from the cranial tissue 2 days before hatching.
The inner nuclear layer of the retina differentiates by hatching. The pigment layer, cone layer and all other retinal layers begin to differentiate over the next week and by 12 DAH (mean LS ¼ 74 mm), rods appear in the retina. By 17 DAH (mean LS ¼ 99 mm), the pigment layer can move, allowing light adap- tation of the retina. Following settlement, at 23 DAH (mean LS ¼ 123 mm), rod and cone layer thicknesses are roughly equal. The eye is not pigmented at hatching, but becomes so by 1 DAH.
P I G M E N T
Larvae are lightly to moderately pigmented and much of the pigment is vari- able. At hatching, pigment is confined to a series of c. 13–14 melanophores along the ventral midline of the tail, 1–2 melanophores on the ventral surface of the yolk sac, a shield of melanophores over the gut, a bilateral pair of mel- anophores on the forebrain, a single, medial melanophore dorsally on the mid- brain and two melanophores on the nape at the level of the pectoral-fin base.
Pigment on the head increases during development. One to two melanophores on the tip of the lower jaw and several melanophores on the tip of the upper jaw develop during the early postflexion stage (70 mm), but those on the lower jaw may be absent in settlement-stage larvae. One to a few melanophores on the angle of the lower jaw may be present from 60 mm. Melanophores along the isthmus are present in some larvae (c. 30%) from 37 mm, but are present in most specimens from c. 6 mm. On the upper opercle, an external, expanded melanophore appears by 37 mm. The number of melanophores on the opercle increases, particularly from c. 70 mm, to as many as 11, and they spread
ventrally to form a loose cluster of melanophores over the opercle at settlement.
A similar number of melanophores is present on the inner surface of the opercle from 6 mm. The paired melanophores on the forebrain in the yolk-sac larva (0 DAH, 25 mm) disappear by 1 DAH, and the forebrain then remains unpig- mented until c. 8 mm. The single dorsal melanophore on the midbrain has become three by the end of flexion (46 mm), and the number increases rapidly after that to c. 13–15 from 60 mm. By early in the postflexion stage, the mela- nophore on the nape is no longer externally apparent.
The trunk and tail also become increasingly pigmented with development.
The number of internal melanophores over the gut increases and they spread ventrally during development, but become increasingly difficult to see as set- tlement approaches due to overlying musculature and scales. The ventral pig- ment on the yolk sac disappears by 1 DAH, and the ventral surface of the gut remains unpigmented. There are three series of external melanophores on the tail: ventral, lateral and dorsal. A series of melanophores is present along the ventral midline of the tail in all specimens but the number of melanophores and their extent vary. These melanophores are initially more or less uniformly distributed along the entire ventral edge of the tail, but by 37 mm, they are divided into two groups: one of four melanophores above the anal-fin anlage, and one of three melanophores on the ventral edge of the caudal peduncle on myomeres 21–23. There are one to four anal-fin-base melanophores from 4 to 5 mm (i.e. during and just after flexion), but larger larvae have 10–15 melano- phores along the anal-fin base. The peduncle melanophores may be absent fromc. 6 mm, but cover most of the ventral edge of the peduncle in most post- flexion specimens. One to four expanded melanophores appear on the second anal-fin spine by 88 mm: otherwise, the anal fin remains unpigmented. A single melanophore at the caudal-fin anlage is present in all but one specimen, and is found on the base of a ventral principal ray of the caudal fin following flexion.
Three specimens have a second melanophore at the base of a dorsal caudal-fin ray. Another melanophore occurs dorsally on the caudal-fin base in three of 17 postflexion larvae examined. The dorsal surface of tail and trunk remains unpigmented until the first melanophore of a series appears along the middle of the base of the soft dorsal fin as early as 46 mm. Melanophores of this series extend anteriorly so that by 7 mm, 11 are present and they extend to the middle of the base of the spiny portion of the dorsal fin. Melanophores of this series spread onto the dorsal surface of the caudal peduncle from 7 mm, and they increase in number to c. 16 by 10 mm. At 9–10 mm, three of these melanophores extend dorsally onto the fin from the bases of spines XII and XIII and soft ray 5 to form the beginning of an ocellus. The ocellus is fully formed at 106 mm, extending from the posterior-most spines to the anterior- most soft rays (from about spine XI to one of the first five soft rays) and extending from the ray bases to about two thirds the height of the fin. The ocellus is surrounded by an unpigmented band, which is further surrounded by a ring of small, peppered melanophores. By 11 mm, these peppered mela- nophores cover the membrane of the dorsal fin. The external series along the lateral midline of the tail is present initially as two to four melanophores on myomeres 19–22 (just above the caudal peduncle melanophores on the ventral midline) late in the preflexion stage (37 mm). Following flexion, the series
extends anteriorly to about myomere 14 (above the anterior rays of the anal fin), and posteriorly to the urostyle, and consists of 9–11 melanophores. The most anterior melanophores in this series become at least partially internal in larger larvae. An internal series of melanophores appears along the dorsal sur- face of the notochord in the vicinity of myomeres 19–21 from about 37 mm.
The series spreads, extending approximately between myomeres 13 and 21 by 7 mm, but becomes increasingly difficult to see as the tail musculature thickens.
At the size at which the dorsal-fin ocellus appears, peppered melanophores also appear on the entire trunk and tail and along the margins of the pectoral-fin rays. The pelvic fins remain free of melanophores.
In life, the larvae are largely transparent, but with a silvery gut. They become yellow during the settlement stage.
DISCUSSION
The larval development of P. amboinensis is typical for pomacentrids: direct and with few specializations for pelagic life. Most pomacentrids, including P.
amboinensis, have a tightly coiled and compact triangular gut, inconspicuous gas bladder and weak head spination: small supracleithral, preopercular and in- teropercular spines and a small opercular spine (Kavanaghet al., 2000; Table I).
Pomacentrids also generally lack elongate fin spines and precocious development of fin elements or scales (Table I). Although development of P. amboinensis at hatching is not very advanced, within 1 day, the mouth and eyes are functional, enabling the larvae to feed, orientate visually and avoid predators. Median fins are complete by 7 mm, and the larvae are fully scaled by 10 mm, which in the reared larvae was attained at an average age of 12 and 18 DAH, respectively.
The growth data for reared larvae should be used cautiously because of the large differences between laboratory and field conditions. Nevertheless, the reared larvae had both PLD and size at settlement well within the ranges of values reported for wild P. amboinensis (Thresher et al., 1989; Wellington &
Victor, 1989; Kavanagh, 1996; Kerrigan, 1996; Kavanagh & Alford, 2003;
Bay et al., 2006), suggesting that these growth data will be at least broadly applicable to larvae in the sea. The marked increase in growth rate coincident with completion of notochord flexion and the caudal fin indicates that simply assuming growth rate in pomacentrid larvae is constant throughout the PLD may be misleading. These larvae were reared during the middle of the spawning season ofP. amboinensison the Great Barrier Reef (November) at temperatures similar to ambient water temperatures (28–29° C) on the reef at that time.
Growth and development rates would probably differ at other temperatures.
The growth rates reported here are slightly less than those reported for wild Pomacentrus coelestis Jordan & Starks (056–076 mm day1, averaged over the whole PLD: Meekan et al., 2003). Although P. coelestis settles at a larger size (c. 15 mm LS) than P. amboinensis, both have a PLD of similar duration and hatch at similar sizes (Tanaka & Nitta, 1997a), so P. coelestis must have a higher overall growth rate, regardless of whether the assumption made by Meekan et al. (2003) of constant growth rate throughout the PLD is valid.
Pomacentrus amboinensis grows relatively fast but develops relatively more slowly than the pomacentrid genera Acanthochromis, Chromis and Premnas
(Kavanagh & Alford, 2003), although data from only a few species are available for comparison. Pomacentrids exhibit an unusually broad range of life-history timings within the family (egg stages from 2 to 16 days; pelagic larval stages from 0 to 35 days). The life-history timing found in P. amboinensis is similar to that found in many pomacentrid genera and species, with a relatively short egg stage (4 days) and a relatively long pelagic larval duration (25 days).
Attempts to identify pomacentrid larvae are hampered by the dearth of knowledge on their development. In onlyc. 15% of the over 350 species is any- thing at all known of development (Table I), and the majority of published descriptions are incomplete. Although few complete descriptions of pomacen- trid larvae are available, some tentative comparisons among genera can be made, because it seems that congeneric species tend to have very similar onto- genetic patterns. Larvae of many pomacentrid genera (e.g. Abudefduf, Amphip- rion, Chromis, Dascyllus, Hypsypops, Microspathodon, Plectroglyphidodon and Stegastes) are more deep-bodied and ‘hunchbacked’ prior to and shortly after flexion than is P. amboinensis. In contrast, larvae of several other pomacentrid genera (e.g. Amblyglyphidodon, Chrysiptera, Neoglyphidodon and Paraglyphido- don) are very similar to P. amboinensis in morphology and pigmentation, but most have a second series of external, lateral melanophores on the tail dorsal to the lateral midline series, and this second series is lacking in P. amboinensis.
Development of only three other Pomacentrusspecies,P. coelestis, Pomacentrus nagasakiensis Tanaka and Pomacentrus pavo (Bloch) has been described over a range of sizes (Table I), and as might be expected, their larval morphology is essentially identical to that of P. amboinensis. Some minor differences from P. amboinensis in pigment are evident in the published descriptions of these three Pomacentrus species: in particular they seem to have less extensive pig- ment on the tail compared to larvae ofP. amboinensisdescribed here. The least pigmented of the four isP. nagasakiensiswhich lacks dorsal pigment on the tail untilc. 9 mmLS(4–5 mm in the others). The ocellus on the dorsal fin develops atc. 10–11 mmLS inP. amboinensisand P. nagasakiensis, but is more posterior in the latter, being restricted to the soft rays. The ventral pigment series on the tail is more extensive in postflexion P. amboinensis that it is in either P. pavo or P. coelestis, and the last two lack a dorsal-fin ocellus. Given the morpho- logical similarity of larvae of the four described Pomacentrus species, it is likely that larvae of species of this genus will be distinguishable only by pig- ment, if at all, as described in the present paper forP. amboinensis. It must be kept in mind, however, that pigment of reared larvae can differ from that of wild larvae (Hunter, 1984). Further, it is likely that larvae of at least some of the other 30þ species of Pomacentrus will be more similar to P. amboinensis than are the three described species. As settlement approaches, the develop- ment of the dorsal-fin ocellus of P. amboinensis at c. 10 mm helps narrow the range of choices, as only a minority of Pomacentrus species have a dor- sal-fin ocellus (pers. obs.). Differences among species in the exact location of the ocellus and in fin-ray counts will assist in narrowing the range of possible identifications. Finally, at settlement, the bright yellow colour of P. amboinensis (without blue lines on the head) combined with the ocellus is an important aid to identification, and is unique in many locations (e.g.
Great Barrier Reef).
The documentation of the ontogeny of P. amboinensis larvae provided here will facilitate correlation of behavioural development to morphological development. For example, Fisher et al. (2000) studied the development of swimming ability in larvae of P. amboinensis in the laboratory, and portrayed a marked increase in swimming speed at 7–9 mm LS. Morphologically, there was no major development event, with the unlikely exception of the completion of the pectoral-fin rays, that might be correlated with this increase in perfor- mance (the caudal fin was complete by 5 mm, and the dorsal and anal fins by 7 mm). Similarly, an even greater rate of increase in swimming endurance was found between 8 and 10 mmLS(Fisheret al., 2000), over which size range the only major development event was the completion of scalation. Neither of these morphological developments is obviously connected with increased swim- ming performance, but between 7 and 9 mm, relative body depth increased by one third, from c. 30 to 40% LS, resulting in a large increase in muscle mass.
This implies that muscle development, rather than fin development, may be a key factor in these marked increases in swimming performance. Muscle growth was allometric, increasing at a rate greater than length squared.
The authors thank A. Hay for general assistance, guidance, photography and comments on the manuscript, M. Lockett for Fig. 2, S. Bullock for inking our sketches of larvae, S.
Job for depositing reared larvae in the AMS collection, M. McGrouther for access to AMS collections, Y. Tanaka for sending copies of his valuable publications on pomacen- trid development, and C. Paris and T. Trnski for comments on the manuscript.
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