Celestial Pearl Danio is a Miniature Danio

8/11/2019 Celestial Pearl Danio is a Miniature Danio

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Accepted by A. Gill: 28 Nov. 2007; published: 21 Jan. 2008 1

ZOOTAXA

ISSN 1175-5326 (print edition)

ISSN1175-5334(online edition)Copyright 2008 Magnolia Press

Zootaxa1686: 128 (2008)www.mapress.com/zootaxa/

The Celestial Pearl danio is a miniatureDanio (s.s) (Ostariophysi: Cyprin-idae): evidence from morphology and molecules

KEVIN W. CONWAY, WEI-JEN CHEN & RICHARD L. MAYDENDepartment of Biology, Saint Louis University, 3507 Laclede Ave, St. Louis, MO 63103, USA. E-mail: [email protected]

Abstract

The osteology of the miniature cyprinid Celestichthys margaritatus Roberts, type species of Celestichthys Roberts, isdescribed in detail and briefly compared with that of other members of the Rasborinae (notablyDanio (s.s),Danionella,

Devario,Esomus,Microrasbora, Paedocyprisand Sundadanio). Celestichthys margaritatuspossesses an A stripe onthe anal fin and two pigment stripes on the caudal fin (apomorphic features ofDanio sensu Fang, 2003). In addition, C.margaritatus exhibits a median projection on the outer arm of the os suspensorium, a derived feature, present only in spe-cies of Danio (includingD. erythromicron) amongst the Cyprinidae, and a lateral projection on the lateral face of thedentary (present only in Danio, Sundadanio and Paedocypris). Phylogenetic analysis of 1,494 bp of the RAG1 nucleargene for 31 rasborine taxa, including 5 species ofDanio, places C. margaritatus as the sister group toD. erythromicron,and part of a larger monophyletic group including all other species ofDanioincluded for analysis. Based on characters ofmorphology and its position in a molecular phylogeny of the Rasborinae it is proposed that Celestichthys be placed in thesynonymy ofDanio, its only member referred to asDanio margaritatus new combination.

Key words:Cypriniformes; Celestichthys;Danio; osteology; phylogeny; RAG1; miniaturization; taxonomy

Introduction

Roberts (2007) recently described Celestichthys margaritatus as a new genus and new species of miniature

cyprinid fish from Myanmar. This description was greatly anticipated by the aquarist community, to which

this species was known under the common name of Galaxy microrasbora or Galaxy rasbora (Clarke,

2006a,b). These common names, in reference to the spectacular colour pattern of this species (Fig. 1A), are

rather confusing as they suggested that the species was a member of eitherMicrorasbora orRasbora prior to

any taxonomic assignment. In its taxonomic description Roberts (2007) inflated the situation by introducing

another common name, Celestial Pearl danio, in allusion to the small pearly spots along the flanks. Despitethe choice of common name, however, Roberts did not place this new species within Daniobut instead cre-

ated a new generic name, Celestichthys, for the sole inclusion of this miniature species, with slight reference

that one other miniature species,Danio erythromicron (Annandale)(refered to as Microrasbora erythromi-

cron) might also be a congener.

Little is known about the ecology of the Celestial Pearl danio other than that it inhabits small and shal-

low ponds with adundant aquatic vegetation (Roberts, 2007). Until recently the distribution of C. margaritatus

was believed to be resticted to the type locality (ponds at the foot of a mountain near Hopong Town, 30km

east of Taunggyi, Myanmar; Roberts, 2007). However, it is now known to have a much wider distribution

within Myanmar (Clarke, 2007) and has even been reported from Thailand (Hary, 2007).

Roberts (2007) diagnosed the genus Celestichthys and its species, C. margaritatus, from all previouslyknown Asian Cyprinidae by its distinctive head and body shape, small upturned mouth with shortened jaws,


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CONWAYET AL.2 Zootaxa1686 2008 Magnolia Press

unique coloration, and 9/8 principal caudal fin rays. During an ongoing investigation by one of us (KWC) on

the morphology of miniature cyprinid fishes, it became apparent that though striking, the underlying pigmen-

tation pattern of C. margaritatus, which consists of a series of interrupted longitudinal stripes, is not unique,

but similar to that of several species of Danio (sensu Fang, 2003). The majority of Danio species exhibit a

colour pattern consisting of a series of longitudinal stripes along the sides of the body, which may varyingly

extend to the end of the median caudal fin interradial membranes (Fang, 1997, 1998). These longitudinal

stripes may be uniformly pigmented along their entire length or interrupted to form a series of spots. Even

within a single species, individuals may exhibit either solid longitudinal stripes or a series of spots (e.g. D.

kyathit Fang) and the gene mutations that result in such colour pattern variation are well understood

(Watanabe et al., 2006).

In the present study, we provide a detailed osteological investigation of Celestichthys margaritatus, some-

thing that Roberts (2007) did not attempt in its original description. We also redescribe its colour pattern using

appropriateDanio colour pattern terminology (Fang, 1998). Nucleotide sequences of the nuclear gene RAG1

were also collected and are analyzed to evaluate the evolutionary relationships of C. margaritatus to other

rasborin species, with particular emphasis on the so-called danionine taxa.

FIGURE 1. A. Celestichthys margaritatus, adult male, photograph by Timo Moritz; B.line drawing of Celestichthys

margaritatus (redrawn from Roberts, 2007: Fig. 1), male, showing principal colour pattern components (after Fang,

1998). Abbreviations: A, anal stripe; A 1, stripe distal to anal stripe; D, submarginal stripe on dorsal fin; P + 1, stripe

dorsal to primary stripe; P 1, stripe ventral to primary stripe.


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Zootaxa1686 2008 Magnolia Press 3PHYLOGENY OF CELESTICHTHYS MARGARITATUS

Materials and methods

Descriptive Osteology:Specimens were cleared and double stained for bone and cartilage study using the

technique of Taylor and van Dyke (1985). Specimens were dissected following a modified version of the pro-

tocol outlined in Weitzman (1974) (hyoid arches removed with the branchial arches) under a Leica MZ95ste-

reomicroscope. All photographs were taken using a Leica DC300 mounted on the aforementioned

microscope. Figure illustrations were adapted from sketches obtained via a camera lucida or from photo-graphs. General osteological terminology follows that of Weitzman (1962). Weberian apparatus terminology

follows Bridge and Haddon (1893) and Chranilov (1927) except that the term os suspensorium is used in its

original sense as defined by Srensen (1890) following Conway and Britz (2007). Methods for collecting

meristic counts follow Hubbs and Lagler (1958), except that the two posteriormost rays of the dorsal and anal

fins, which articulate with the same pterygiophore, are counted as two separate elements. Material examined

is deposited in the following collections: AMNH, American Museum of Natural History, New York; BMNH,

Natural History Museum, London; CMK, personal collection of Maurice Kottelat, Cornol; KU, University of

Kansas Ichthyology Collection, Lawrence; UMMZ, University of Michigan Museum of Zoology, Michigan;

ZRC, Raffles Zoology Collection, Singapore.

Comparative material:The following represents a list of the members of the Cyprinidae utilized duringthis investigation (listed alphabetically). Only cleared and stained specimens (number in parentheses) are

listed: Abramis brama AMNH 37594 (2), UMMZ 184987 (2); Acheilognathus cyanostigma UMMZ

187566 (1);Agosia chrysogaster KU 8084 (3);Alburnus alburnus UMMZ 174614 (1);A. bipunctatus

UMMZ 184991 (1); Amblypharyngodon mola UMMZ 187844 (2);Aphyocypris chinensis UMMZ

167397 (1); Aspius aspius UMMZ 1746907 (1); Barbus barbus AMNH 54635 (3); Barbus bynni

AMNH 215380 (3); Barbus paludinosus AMNH 217300 (3); Blicca bjoerkna AMNH 37599 (2),

UMMZ 174617 (1); Boraras brigittae BMNH 2004.4.26.1821 (3); B. maculata BMNH

1995.5.17.112126 (6);B. merah BMNH 2004.4.26.1017 (4);B. micros BMNH 2004.4.29.13 (2);B.

urophthalmoides BMNH 2004.4.26.29 (2); Campostoma anomalum AMNH 40260 (1); Celestichthys

margaritatus BMNH 2007.10.9.1516 (2); Chela oxygasteroides AMNH 36368 (1); Chelaethiopsbibie

BMNH 2006.3.9.4693 (4). UMMZ 166632 (1); Chondrostoma nasus UMMZ 185029 (2); Clinosto-

mus elongatus AMNH 45955 (5); Couesius plumbeus AMNH 41266 (5); Culter alburnus UMMZ

66525 (2); Cyclocheilichthys apogon BMNH 2001.1.15.699718 (2); Cyprinella analostana UAIC

11003.01 (2); C. labrosa KU 88319 (2); C. proserpina UAIC 8354.01 (2); Cyprinus carpio AMNH

49088 (1);Danio albolineatus UMMZ 70708 (2);D. choprai UAIC 14166.09 (2);D. erythromicron

UACI 14166.23 (2); D. nigrofasciatus UAIC 14166.12 (2); D. rerio BMNH 2001.3.12.7692 (3),

BMNH 1983.7.11.1529 (2); Danionella mirifica USNM 372848 (36); Devario cf. aequipinnatus

BMNH 2005.7.5.502539 (38);D.devario UAIC 14166.18 (1), UMMZ 187873 (1);Dionda episcopa

KU 7427 (7);Engraulicypris sardella AMNH 31917 (5);Erimystax x-punctatus KU 18012 (3);Esomus

danricus UMMZ 187851 (1);E. metallicus BMNH 2000.6.10.80318258 (3);Exoglossum maxillingua

KU 18925 (11); Garra dembeensis BMNH 1984.9.7.5060 (2); Hampala macrolepidota BMNH

2000.6.10.78917900 (1);Hemitrema flammea KU 18884 (10);Hesperoleucus symmetricus KU 18917

(15); Horadandia atukorali BMNH uncatalogued (4); Hybognathus placitus KU 9766 (1); Hybopsis

boucardi KU 21256 (4); Hypopthalmichthys molitrix AMNH 10222 (1); Ischikauia steenackeri

UMMZ 187564 (1);Lavinia exilicauda 54637 (1);Leptocypris niloticus BMNH 2006.3.9.108162 (4);

Leucaspius delineatus UMMZ 160942 (1);Luxilus chrysocephalus KU 12654 (2);L. pilsbryi KU

15281 (8);Macrhybopsis gelida KU 8111 (1);Microphysogobio labeoides AMNH 10588 (4);Micro-

rasbora kubotai BMNH 2004.6.25.610 (3); M. nana BMNH 2004.6.25.15 (3); M. rubescens

BMNH 2004.6.25.1113 (2), UAIC 14297.01 (1);Nocomis effusus KU 18932 (11);Notemigonus crysole-

ucas KU 1357 (1);Notropis altipinnis UAIC 7960.03 (10);N. buccatus KU 17764 (4);N. buccula


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TABLE 1.Taxa used for molecular analyses in this study. Subfamily nomenclature follows Nelson (2006).

Family/subfamily Taxon GenBank accession no.

Gyrinocheilidae Gyrinocheilus aymonieri EU292682

Balitoridae

Nemacheilinae Lefua echigonia EF458305

Cobitidae

Botiinae Leptobotia pellegrini EU292683

Cyprinidae

Acheilognathinae Acheilognathus typus EU292688

Cultrinae Ischikauia steenackeri EU292687

Cyprininae Garra orientalis EU292684

Cyprininae Puntius titteya EU292685

Cyprininae Sawbwa resplendens EU292686

Gobioninae Gobio gobio EU292689

Leuciscinae Alburnus alburnus EU292690

Leuciscinae Notropis baileyi EU292691

Rasborinae Aphyocypris chinensis EU292692

Rasborinae Barilius bendelisis EU292693

Rasborinae Boraras merah EF452838

Rasborinae Boraras urophthalmoides EF452480

Rasborinae Chela cachius EF452845

Rasborinae Chela dadiburjori EU292694

Rasborinae Celestichthys margaritatus EU292695

Rasborinae Danio rerio U71093

Rasborinae Danio albolineatus EU292696

Rasborinae Danio dangila EU292697Rasborinae Danio erythromicron EU292698

Rasborinae Danio nigrofasciatus EU292699

Rasborinae Danionella mirifica EU292700

Rasborinae Danionella sp. EF452841

Rasborinae Devario regina EU292701

Rasborinae Esomus metallicus EU292702

Rasborinae Horadandia atukorali EU292703

Rasborinae Luciosoma setigerum EU292704

Rasborinae Microrasbora nana EU292705

Rasborinae Microrasbora kubotai EU292707Rasborinae Microrasbora rubescens EU292706

Rasborinae Inlecypris auropurpurea EU292708

Rasborinae Opsaridium sp. EF452846

Rasborinae Opsariichthys uncirostris EF452847

Rasborinae Rasbora gracilis EU292710

Rasborinae Rasbora bankanensis EU292709

Rasborinae Rasbora argyrotaenia EF452836

Rasborinae Rasbora sumatrana EF452837

Rasborinae Sundadanio axelrodi EU292711

Rasborinae Trigonostigma heteromorpha EU292712Rasborinae Zacco sieboldii EU292713


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tree per 100 replicates for each run. We repeated this procedure until stationary in log likelihoods was

observed. We discarded initial trees (first 1,537 trees in our analysis) with non-stationary log likelihood values

as part of a burn-in procedure, and used the remaining trees that resulted in convergent log likelihood scores to

construct a 50% majority rule consensus tree. For ML search with mix model of nucleotide substitution, we

used the GTR+G model (with 4 discrete rate categories) for the first, second and third codon position because

RAxML only provides GTR+G and the GTR+CAT approximation (Stamatakis, 2006) of rate heterogeneity for

nucleotide data. Optimal ML tree was obtained through 100 distinct runs by default algorithm of the programfrom 10 random starting trees (-d option). Finally, node support was assessed using the bootstrap procedure

(Felsenstein, 1985) under MP (with heuristic search described above) and ML (using only MP tree as starting

tree each run) criterion, based on 100 pseudo-replicates and the resulting a posterioriprobabilities from parti-

tioned BI.

Results

Preserved colour pattern of Celestichthysmargaritatus: In males, 56 irregular rows of spots, arranged in a

longitudinal series, extend along sides of body (Fig. 1B). P stripe not extending onto caudal fin. P + 1 and P -1 stripes extending to end of caudal fin rays. Incomplete stripes on caudal fin, dorsal to P + 1 and ventral to P

- 1, possibly continuation of P + 2 and P - 2. Anal-fin stripe (A stripe) present, extending along middle of

anal-fin rays and ending at the distal tip of the last branched ray (stripe interrupted in figured specimen; Fig.

1B). A - 1 extending along distalmost edge of anal fin, terminating at tip of 6 thor 7thbranched ray. Submar-

ginal dorsal-fin stripe (D stripe) present, extending from anterior edge of dorsal fin to tip of 5th or 6th

branched ray. Second dorsal-fin stripe, ventral to D stripe, extending along dorsal-fin base, from base of 1st

unbranched ray to tip of 8th branched ray. Pelvic fin with short marginal stripe, extending from tip of

unbranched ray to tip of 3rdbranched ray, and basal stripe, extending along middle of pelvic fin, ending at dis-

tal tip of 5

th

branched ray. Females with similar distribution of longitudinal rows of spots and fin stripes. Finstripes less prominent.

Descriptive osteology of the Celestial Pearl danio

The following osteological description is based on two specimens of Celestichthys margaritatus (BMNH

2007.10.9.15-16).

Neurocranium (Figure 2): The neurocranium is well ossified. It tapers towards the anterior from its

broader otic region, ending in a rather blunt and narrow ethmoid region.

The ethmoid region is composed of lateral ethmoid, mesethmoid, preethmoid and vomer. There is no

nasal bone. The lateral ethmoid sits at the anterolateral corner of the lamina orbitonasalis and forms the ante-

rior margin of the orbit. It projects outwards from the ethmoid region in an anteroventral direction and termi-

nates in a short spine-like process. The median mesethmoid is a cup-shaped ossification. It exhibits small and

weakly ossified wing-like membrane bone flanges on its dorsolateral edges that curve towards the midline. It

is not clear whether a dermethmoid component (usually referred to as the supraethmoid within the cyprinid

literature; see Harrington, 1955) is incorporated in to the ethmoid region of C. margaritatus as it is inD. rerio

(Cubbage & Mabee, 1996). The preethmoid is a small egg-shaped endochondral ossification that caps the

anterolateral margin of the ethmoid region. The median vomer is a roughly diamond-shaped dermal ossifica-

tion that forms the floor of the ethmoid region.


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FIGURE 2.Neurocranium of Celestichthys margaritatus BMNH 2007.10.9.1516, 15.2 mm SL. A.dorsal view; B.lat-eral view, left side; C.ventral view. Cartilage grey. Abbreviations: AVVII, anterior opening of trigeminal-facial cham-

ber; Apto, autopterotic; Asph, autosphenotic; BL, Baudelots ligament; Boc, basioccipital; Exoc, exoccipital; Epoc,epiotic; Fr, frontal; LE, lateral ethmoid; MP, masticatory plate of the basioccipital; ME, mesethmoid; Osph, orbitosphe-noid; Psph, parasphenoid; PVVII, posterior opening of trigeminal-facial chamber; PE, prethmoid; Pro, prootic; Pt, pari-

etal; Ptsph, pterosphenoid; SO, supraorbital; Soc, supraoccipital; STF, subtemporal fossa; Vo, vomer; FIX, foramen forglossopharyngeal nerve; FX, foramen for vagus nerve.


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The frontal forms the roof of the neurocranium anterodorsally. They are broad, relatively short bones, in

contact along their entire medial edges. There is no supraorbital canal. The orbitosphenoid is a large endoch-

ondral ossification. It rims the medial face of the orbit and extends ventrally but does not contact the dorsal

surface of the parasphenoid from which it is separated by a small remnant of the trabecula communis. The

pterosphenoid rims the posteromedial face of the orbit. Its posterior edge contacts the anterior edge of the

large prootic, its posterolateral edge with the anteromedial face of the autosphenotic. The anterior opening of

the trigeminal-facial chamber is situated between the pterosphenoid and the prootic.The autosphenotic forms the anterolateral margin of the otic capsule. It possesses a small ventrally

directed projection on its anterolateral face that rims the posterodorsal margin of the orbit and serves for the

attachment of the levator arcus palatini muscle. Laterally, it contributes to the anteriormost part of the hyo-

mandibular facet. The autopterotic is situated at the posterolateral corner of the neurocranium, which is its

widest point. Ventrally, the autopterotic contributes to the lateral portion of the subtemporal fossa and later-

ally, to the posteriormost part of the hyomandibular facet. There is no temporal canal.

The prootic is the dominant bone in the ventral surface of the cranium. It houses the anterior part of the

auditory bulla and forms the posterior opening of the trigeminal-facial chamber. Its posterolateral edge con-

tributes to the large subtemporal fossa. The medial edge of the prootic is occluded by the posterior wing of the

parasphenoid. The epiotic caps the posterolateral corner of the otic capsule. Its anteriormost edge is roofed bythe posterior edge of the parietal. The parietal sits dorsal to the otic capsule. It is similar in width to the frontal,

which dorsally overlaps its anteriormost edge. A small ridge of bone runs along the dorsal surface of the pari-

etal, close to its posterior edge. There is no temporal commisure. There is no intercalar.

The median supraoccipital caps the posterodorsalmost point of the occiput. Its anterior edge is overlapped

by the parietal. It exhibits a small crest of membrane bone along its midline, which serves for the attachment

of the epaxial musculature. The exoccipital is large and forms a significant portion of the posterior wall of the

occiput and the posterodorsal part of the otic bulla. It forms the roof and lateral margin of the foramen mag-

num, which is separated from the lateral occipital foramen by a bony strut. The exoccipital exhibits a large

foramen on its ventral surface, for the exit of the vagus (X) nerve. The exit of the glossopharyngeal (IX) nerve

is a tiny foramen situated anteromedial to the foramen for the exit of the vagus (X). The median basioccipitalforms the posteroventralmost point of the otic capsule, the ventral portion of the otic bulla, and the articulation

between the neurocranium and the first vertebra. There is a short and posterioly rounded pharyngeal process

of membrane bone extending posteroventrally from its posteroventralmost point. Located on its ventral sur-

face, the masticatory plate (Howes, 1981) of the basioccipital is flat and triangular in shape. The dorsal aorta

runs through a large canal, the aortic canal, in the center of the basioccipital process.

The median parasphenoid extends along the ventral surface of the neurocranium, from the ethmoid region

to the otic capsule. Narrow along its entire length, the parasphenoid possesses short wing-like ascending pro-

cesses that extend towards the anterior edge of the prootic. Its posteriormost region gently tapers, terminating

in a blunt tip between the prootics, anterior to the anterior edge of the basioccipital. The narrow anterior tip of

the parasphenoid inserts between the ventral surface of the mesethmoid and the dorsal surface of the vomer.

The vomer is accommodated in a shallow grove along the ventral surface of the parasphenoid.

Hyopalatine arch and opercular series (Figure 3):The hyopalatine arch consists of hyomandibular,

symplectic, quadrate, metapterygoid, ectopterygoid, endopterygoid, and autopalatine. All endocondral bones

are well ossified and exhibit membrane bone components to varying degrees. The two dermal ossifications,

the ectopterygoid and endopterygoid, are weakly ossified.

The hyomandibular is the largest bone of the hyopalatine arch. It is almost entirely composed of bone,

except for its ventralmost point, a remnant of the hyosymplectic cartilage. It exhibits large membrane bone

flanges along its anterior and posterior edge. There are four articular heads, two dorsal heads (the anterior and

the posterior), one posterior head, termed the opercular head, and one ventral head. The dorsal articular heads

articulate with the neurocranium via two concave facets, one situated between the autosphenotic, prootic and


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FIGURE 3.Celestichthys margaritatus BMNH 2007.10.9.1516, 15.2 mm SL. A.hyopalatine arch and opercular series,right side lateral view (image reversed); B.hyopalatine arch and opercular series, right side, medial view. Cartilage grey.* indicates danioin notch. Abbreviations: An, anguloarticular; Apal, autopalatine; CM, coronomeckelian; De, den-

tary; Ecpt, ectopterygoid; Enpt, endopterygoid; Hy, hyomandibular; Iop, interopercle; Mx, maxilla; MC, Meckels carti-lage; Mpt, metapterygoid; Op, opercle; Pmx, premaxilla; Pop, preopercle; Q, quadrate; Ra, retroarticular; Sop,subopercle; Sy, symplectic.

pterosphenoid (associated with the anterior articular head) and a more posterior one, situated between the

autopterotic and the prootic (associated with the posterior articular head). The opercular head articulates in a

concave facet on the medial face of the opercle, close to its anterior edge. A small foramen for the passage of

the hyomandibular branch of the facial (VII) nerve pierces the centre of the hyomandibular just ventral to the

opercular head (not illustrated). The ventral head forms an articulation with the dorsal tip of the interhyal car-tilage. The symplectic is an elongate endoskeletal bone, capped with cartilage anteriorly and posteriorly. The


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metapterygoid is a large, thin, irregularly shaped endoskeletal bone that exhibits a small flange of membrane

bone along its dorsal aspect. The metapterygoid possesses two small cartilage-capped heads on its posterior

edge. The ventralmost head is closely associated with the ventralmost tip of the hyomandibular and the poste-

riormost point of the symplectic. A thin strip of cartilage rims the anteriormost edge of the metapterygoid, a

remnant of pars metapterygoidea, which is confluent ventrally with the remnant of the pars quadrata, which in

turn persists as a thin strip of cartilage along the posterodorsal edge of the quadrate. The quadrate articulates

anteriorly with a small groove on the posteriormost tip of the anguloarticular. It exhibits a large posteroventralprocess (Arratia & Schultze, 1991) that extends posteriorly, running along the lateral face of the symplectic,

terminating anterior to its midpoint. The palatoquadrate cartilage persists as a thin strip of cartilage extending

between the quadrate and the autopalatine. The autopalatine is a roughly cylindrical-shaped ossification. It

exhibits a large concave facet on its medial face that articulates around the lateral face of the preethmoid. A

small dorsomedial process extends from the autopalatine, close to its anterior edge, to contact the mesethmoid

laterally. The ectopterygoid is a thin lamina of dermal bone that rims the palatoquadrate anteriorly, between

the autopalatine and quadrate. Widest ventrally, the ectopterygoid decreases in width dorsally, terminating as a

fine needle-like point close to the ventralmost tip of the autopalatine. The endopterygoid is a large dermal

ossification. It is most heavily ossified at its anteriormost tip, which forms a small concave facet around the

posteriormost point of the autopalatine, and its anterovental edge, which rims the posterior edge of the palato-quadrate cartilage.

The four bones of the opercle are thin and mostly weakly ossified. The opercle is a roughly shield-shaped

bone, ossified most heavily at its point of articulation with the opercular head of the hyomandibular. There is

no opercular canal. The subopercle is a thin strip of bone that sits medial to the opercle. It is widest anteriorly

at its point closest to the interopercle. The preopercle is thin, roughly L-shaped bone. It exhibits a short

enclosed preopercular canal along the anterior half of its horizontal arm only. The canal extends onto the base

of the vertical arm of the preopercle but remains open. The interopercle is a thin and weakly ossified lamina of

dermal bone. It lies medial to the preopercle and is similar in shape to the horizontal arm of the preopercle.

The lower jaw is composed of dentary, anguloarticular, retroarticular, and coronomeckelian. The dentary

is the largest bone of the lower jaw. It exhibits a large dorsally directed coronoid process close to its posterioredge. There is a small foramen on the dentary for the passage of the internal mandibular branches of the

trigeminal (V) nerve dorsal to the anteriormost tip of Meckels cartilage. The ventral margin of the dentary is

very weakly ossified but there is a clear indentation anteriorly on the ventromedial edge, termed the danioin

notch (Roberts, 1986). The dentary also exhibits a small lateral membrane bone process anteriorly. The angu-

loarticular inserts into a shallow groove along the medial face of the dentary. It is well ossified posteriorly,

where it exhibits a shallow groove that accommodates the articular head of the quadrate. There is a small tri-

angular retroarticular articulating on the posteroventralmost tip of the anguloarticular. It exhibits a small nee-

dle-like posterior process that serves as an attachment point for a ligament originating on the anterior tip of the

interopercle. The coronomeckelian is relatively large and situated along the dorsal edge of the posteriormost

point of Meckels cartilage. The upper jaw comprises the maxilla, premaxilla and kinethmoid (Fig. 4). The

maxilla exhibits a small rounded palatine process midway along its dorsal edge, which serves for the attach-

ment of the A1 portion of the adductor mandibulae muscle. It is bifurcated anterodorsally with the median arm

of the bifurcation inserting beneath the head of the premaxilla. There is a slight expansion of the ventral tip of

the maxilla at the point lateral to the coronoid process of the dentary. The premaxilla is long and thin. Its ven-

tralmost tip overlaps the ventralmost tip of the maxilla laterally. It extends furthest dorsally at its point closest

to the midline. The kinethmoid is a small unpaired median bone. It is cyclindrical in shape, with a bifurcated

posterior tip.


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FIGURE 4. Dorsal view of upper jaws of Celestichthys margaritatus BMNH 2007.10.9.1516, 15.2 mm SL. Cartilagegrey. Abbreviations: Apal, autopalatine; Fr, frontal; IO1, infraorbital 1; LE, lateral ethmoid; KE, kinethmoid; Mx, max-

illa; ME, mesethmoid; PE, preethmoid; Pmx, premaxilla; Vo, vomer.

Gill arches (Figure 5): The basihyal is a thin rod-like element, capped in cartilage anteriorly and posteri-

orly. It extends far forward in front of the anteriormost tips of the ventral hypophyals.

The anterior copula contains three basibranchial (Bb) ossifications, Bb13. All are thin, rod shaped ele-

ments, only slightly narrower in width than the basihyal. Bb1 is the shortest of the three. Bb2 and Bb3 are sim-

ilar in length and roughly twice as long as Bb1. The posterior copula is situated posterior to Bb3. It is

relatively short, extending between the anteromedial tips of ceratobranchial (Cb) 3 to Cb5. There are two

hypobranchial (Hb) ossifications, Hb23. Hb2 exists as a small perichondral ossification around the periphery

of the Hb2c. Hb3 is well ossified and exhibits a short ventral process. Hb1 cartilage is a small round cartilagesituated at the anterior tip of Cb1. Ceratobranchials 14 (Cb14) are rod-shaped elements. All are tipped in

cartilage anteriorly and posteriorly and exhibit 27 small triangular gill rakers along their lateral and medial

edges. The gill rakers along the lateral edge of Cb1 are much longer than other gill rakers and are ossified only

at the base. Cb3 exhibits a triangular flange of membrane bone on its posteromedial edge. Cb5 is larger and

extends much farther dorsally than other Cb elements. It exhibits three rows of pharyngeal teeth, hooked at

their tips. There are 2, 3, 5 pharyngeal teeth on each Cb5 (Roberts, 2007). The dorsal gill-arch endoskeleton is

composed of four roughly rod-shaped epibranchial elements, Eb14, and 2 pharyngobranchials, Pb23. Eb1

3 lack uncinate processes and exhibit a small flange of membrane bone along their posterior edges. Eb4 exhib-

its a small anterodorsally directed uncinate process. A small cartilaginous nodule, termed Eb5 cartilage,

extends between the tip of the levator process, on the posterior edge of Eb4, and the cartilaginous head of Cb4.Pb23 are small perichondral ossifications surrounding Pb23c. Pb2 is completely separated from Pb3. This


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differs somewhat from the typical Pb condition in cyprinids where Pb2 is usually dorsally overlapped by Pb3

(Siebert, 1987; Cavender & Coburn, 1992). There is no separate Pb4c and Pb3c extends posteriorly to abut

with the cartilaginous head of Eb4.

FIGURE 5. Celestichthys margaritatus BMNH 2007.10.9.1516, 15.2 mm SL. A. hyoid arch right side, lateral view(image reversed); B. urohyal, left side, lateral view (above) and ventral view (below); C.dorsal gill-arch endoskeleton,right side, dorsal view; D. ventral gill-arch endoskeleton, dorsal view, gill rakers shown on left side only. Cartilage grey.Abbreviations: ACh, anterior ceratohyal; Bb 13, basibranchial 13; Bh, basihyal; Br, branchiostegal rays; Cb 15, cera-tobranchial 15; DHh, dorsal hypohyal; Eb14, epibranchial 14; Eb5C, epibranchial 5 cartilage; Hb1C, hypobranchial 1cartilage; Hb23, hypobranchial 2, 3; IhC, interhyal cartilage; Lev, levator process; Pb23, pharyngobranchial 23; PCh,posterior ceratohyal; PC; posterior copula cartilage; Uh, urohyal; VHh, ventral hypohyal.


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a broad fenestrated head. It exhibits a large medial process on its medial edge, close it its terminal most tip.

The base of the third neural arch is broad and articulates with almost the entire dorsal surface of V3. The base

of the fourth neural arch, much narrower than the third, articulates with V4. The fourth neural arch bears a

short neural spine, which exhibits an expanded dorsal tip. Supraneural 3, exhibits a large crest of membrane

bone, which extends dorsally closely associated with the neural spine associated with neural arch 4. Supraneu-

ral 2 is similar in size to the endochondral portion of supraneural 3. It exhibits a large semicircular indentation

on its anterior edge, which rims the posteriormost remnant of the tectum synoticum.

FIGURE 6.Infraorbital series: A.Celestichthys margaritatusBMNH 2007.10.9.1516, 15.2 mm SL, right side, lateralview, (image reversed); B.Danio erythromicron UAIC 14166.23, 19.4 mm SL, right side, lateral view, (image reversed);C.Danio nigrofasciatus UAIC 14166.12, 22.2 mm SL, left side, lateral view; D.Microrasbora rubescens UAIC14297.01, 23.7 mm SL, right side, lateral view, (image reversed). Abbreviations: IO15, infraorbital 15.

Vertebral column (Figures 7, 8):Both specimens exhibit 32 vertebrae, consisting of 15 abdominal + 17caudal. Pleural ribs start on V5 and continue to V14V15. All pleural ribs are similar in length and thickness,

except for the last rib (on V15) of the larger specimen examined, which is greatly reduced in length and with-

out contact to its associated centrum (not illustrated). The head of the 5 thpleural rib is much smaller than that

of succeeding ribs (excluding the last), which exhibit a small membrane bone flange close to their point of

articulation with the parapophyses. The first parapophysis articulates on V5 at the base of the neural arch,

close to its anterior edge. Remaining parapophyses, excluding the last (the 10thon V14), articulate at the mid-

point of the base of the neural arch. The last parapophysis is fused to its centrum. The neural arches of all ver-

tebrae, excluding those on PU23, are narrow and situated on the anterior half of the centra. Neural arches on

V6V13 exhibit long prezygopophyses on their anterior edge. V5V29 exhibit small dorsally oriented postzy-

gopophyses. Hemal spines are borne on the anterior half of V1631. Small ventral postzygopophyses areborne on the posterior edge of V1628.


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Figure 7 to be continued...


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FIGURE 7.Weberian apparatus, left side, lateral view, of: A.Celestichthys margaritatus BMNH 2007.10.9.1516, 15.2mm SL; B.Danio erythromicronUAIC 14166.23, 19.4 mm SL; C.Esomus metallicusBMNH 2000.6.10.80318258,27.5 mm SL; D.Devario devarioUMMZ 187873, 56.6 mm SL; E.Danio nigrofasciatus UAIC 14166.12, 22.2 mm SL;

F.Microrasbora rubescens UAIC 14297.01, 23.7 mm SL.* indicates median flange on outer arm of os suspenorium.Cartilage grey. Abbreviations: Boc, basioccipital; C, claustrum; Exoc, exoccipital; I, intercalarium; Ios, inner arm of theos suspenorium; L1, lateral process of the first vertebral centrum; L2, lateral process of the second vertebral centrum; Na,neural arch; Ns, neural spine; Oos, outer arm of the os suspenorium; S, scaphium; Sn, supraneural; Sn2, 3, supraneural 2,

3; Soc, supraoccipital; T, tripus; 5r, 6r, rib of 5thor 6thvertebral centrum.

There are five small plate-like supraneurals, situated between the neural spines of V4V9. There are small

epineural and epipleural intermuscular bones. Epineural bones extend from V15 to V30 and increase in length

gradually towards the posterior. They are attached to the base of the neural spines of their associated centra via

a short tendon. Epipleural bones extend from V16 to V29 and similarly increase in length towards the poste-

rior. They are attached to the base (V1621) or midregion (V21129) of the hemal spines of their associatedcentra via a short tendon.

Dorsal fin (Figure 8A):There are 10 dorsal-fin rays (ii,7,i) supported by well ossified pterygiophores

inserted between the neural spines of V1117. The first two pterygiophores comprise a large proximal-middle

radial and a small, spherical distal radial. The remaining pterygiophores are composed of proximal, middle

and distal radials. The first proximal-middle radial supports a large unbranched ray plus a smaller unbranched

supernumerary ray. The last pterygiophore supports a small branched ray plus a smaller unbranched supernu-

merary ray. Remaining pterygiophores support each a single branched ray, which embrace the distal radials

distally and rest on the anterodorsal corner of the succeeding proximal-middle (the last unbranched ray only)

or middle radials. Each proximal-middle or proximal radial exhibits a flange of membrane bone on its anterior

and posterior edge.Membrane bone flanges are best developed on the first proximal-middle radial.All middle


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radials are well ossified endochondrally. The last middle radial exhibits a small flange of bone, termed the end

piece or dorsal-fin stay (Weitzman, 1962), on its posteroventralmost edge. The two anteriormost distal radials

are ossified endochondrally. Distal radial cartilages posterior to the second distal radial may exhibit small per-

ichondral ossifications around their points of articulation with the base of the associated fin ray.

Anal fin (Figure 8A):There are 13 anal-fin rays (iii,9,i) supported by well ossified pterygiophores insert-

ing between the hemal spines of V1622. The first four anal pterygiophores comprise a large proximal-middle

radial and a small, spherical distal radial. The remaining pterygiophores are composed of proximal, middleand distal radials. The first proximal-middle radial is the largest of the series in the two specimens examined.

The first proximal-middle radial supports a large unbranched ray plus two smaller unbranched supernumerary

rays. The last anal pterygiophore supports a small branched ray plus a smaller unbranched supernumerary ray.

Remaining pterygiophores support a single branched ray, which embrace the distal radials distally and rest on

the anteroventral corner of the succeeding proximal-middle (the last unbranched ray + first three branched

rays) or middle radials. Each proximal-middle or proximal radial exhibits a flange of membrane bone on its

anterior and posterior edge. Membrane bone flanges are best developed on the first proximal-middle radial.

All middle radials are well ossified endochondrally. The last middle radial exhibits a small flange of bone,

termed the end piece or anal-fin stay (Weitzman, 1962), on its dorsal edge. The three anteriormost distal radi-

als are ossified endochondrally. Distal radial cartilages posterior to the third distal radial may exhibit smallperichondral ossifications around their points of articulation with the base of the associated fin ray.

Pelvic girdle (Figure 8B):The pelvic girdleconsists of a pair of anteriorly bifurcated basipterygia. Each

basipterygium is well ossified, except for the posterolateralmost tip, which remains cartilaginous. Posteriorly,

each basipterygium supports three pelvic radials, a large pelvic splint and 7 fin rays, and exhibits a long

ischiac process on the posteromedial edge. The two lateralmost pelvic radials are small and round and exhibit

small perichondral ossifications on their ventral surface. The medialmost radial is roughly boomerang

shaped and sits lateral to the ischiac process.

Caudal skeleton (Figure 8C):There are 9+8 principal rays and 6 dorsal and ventral procurrent rays. Cau-

dal fin rays are supported by the neural and hemal spines of the 2ndand 3rdpreural caudal centra, the pleu ros-

tyle, a single epural, 5 hypural elements and the parhypural. The 2 ndand 3rdpreural centra bear large neural

and hemal spines that varyingly exhibit laminar flanges of membrane bone on the anterodorsal (neural) or

anteroventral (hemal) edges. The hemal arch of the 2ndpreural centra is autogenous from the centrum. Hemal

spines of the 2ndand 3rdpreural centra bear expanded tips that provide support for ventral procurrent rays. The

posteriormost tip of the neural spine of the 3rdpreural centrum is not expanded and does not support dorsal

procurrent rays, unlike the tip of the neural spine of the 2 ndpreural centrum which is expanded and provides

support for the three anteriormost dorsal procurrent rays. There are no cartilaginous radial elements in the cau-

dal skeleton.

The compound centrum (sensu Fink & Fink, 1981) of C. margaritatus bears a large neural process, which is

firmly ankylosed to the centrum. The anteriormost tips of the parhypural and 1st

hypural are fused to eachother and are firmly attached to the compound centrum but remain autogenous from this element. The 2nd

hypural is firmly ankylosed to the posteroventral edge of the compound centrum. The 3 rdhypural, which is

comparable in length and width to the 2nd, abuts with the compound centrum in the v formed between the

pleurostyle and 2ndhypural. The remaining hypurals, which decrease in size dorsally, are loosely bound to the

pleurostyle. There is no autogenous uroneural.

Pectoral girdle (Figure 9):The pectoral girdle consists of a posttemporal, a supracleithrum, a cleithrum,

a postcleithrum, a coracoid, a mesocoracoid, a scapula, four pectoral radials, and 11 (i.67.iiiiv) fin rays. The

posttemporal is a small irregularly shaped dermal ossification, which articulates with the medial face of the

supracleithrum ventrally and the posterolateral corner of the neurocranium (autopterotic and epiotic) dorsally.

The supracleithrum is a small, blade-like, dermal ossification that articulates dorsally with the medial face of


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the posttemporal and ventrally with the lateral face of the cleithrum. Baudelots ligament appears to originate

on the dorsalmost tip of the cleithrum, rather than exhibit the usual supracleithral origin, and inserts on the

exoccipital, ventral to the foramen for the exit of the vagus (X) nerve. The cleithrum is the largest element of

FIGURE 9.Pectoral girdle of Celestichthys margaritatus BMNH 2007.10.9.1516, 15.2 mm SL. A. left side, lateral

view; B. left side, medial view. Cartilage grey. Third to fifth unbranched pectoral fin rays damaged. Abbreviations: Cl,

cleithrum; Co, coracoid; DrC, distal radial cartilage; Ms, mesocoracoid; Pr14, pectoral radial 14; Pcl, postcleithrum;

Pt, posttemporal; R, fin ray; Sc, scapula; Scl, supracleithrum.


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the pectoral girdle. It articulates dorsally with the medial face of the supracleithrum, posteriorly with the post-

cleithrum, medially with the mesocoracoid, and posteroventrally with the scapula and coracoid. The postclei-

thrum is a long, thin dermal ossification that attaches to the posteromedial face of the cleithrum. The

mesocoracoid is a small strut-like endochondral ossification which articulates dorsally on the medial face of

the cleithrum and ventrally in the suture between the scapula and coracoid. The scapula is an endochondral

ossification that articulates on the medial face of the cleithrum close to its posteroventral edge. It exhibits a

large foramen centrally for the passage of a branch of the pterygial nerve. The coracoid is a large endochon-dral ossification that articulates dorsally with the ventral surface of the cleithrum and posteriorly with the

scapula. It exhibits a pronounced ridge along its lateral face, close to its suture with the cleithrum. There is no

cleithro-coracoid fenestra.

All four pectoral radials are ossified endochondrally, except for the distal tips of second to fourth, which

remain cartilaginous. The first radial is round in shape and exhibits a shallow groove on its medial face, which

articulates tightly with the posteroventral face of the scapula. The three outermost radials are elongate ele-

ments. The second is the largest of the three and exhibits a large flange of membrane bone on its medial sur-

face, which extends farther dorsally than the first. The third is slightly smaller than the second but larger than

the fourth, which is closely associated with its ventral edge. There are six small distal pectoral radial cartilages

associated with the distal tips of all radials. The largest (associated with the base of the 4th and 5 thbranchedpectoral-fin rays) exhibits a small endochondral ossification at its center.

Comparative osteology

In the following section we compare certain aspects of the osteology of C. margaritatus with that of certain

taxa referred to as danionin, includingDanio,Devario, Danionella,Esomus, Microrasbora and Sundadanio.

Danio (=Devario) malabaricus by Howes (1979: 192), is present in several genera of South East Asian cyp-

rinids, includingDanio,Devario,Danionella,Esomus, and is also reported as present in Parabarillius (Rob-

erts, 1986). It is not present in Sundadanio axelrodi or species ofMicrorasbora(Fang, 2003) but is present inspecimens ofD. erythromicron that we have examined (Fig. 10B) as suggested by Kottelat and Witte (1999),

Jaws:The jaws of C. margaritatus are similar in many respects to other danionin taxa.

Firstly, there is a large semicircular indentation on the anteroventral part of the dentary (Fig. 10A). This

structure, referred to as the danioin notch by Roberts (1986: 236), and first reported in Danio dangila and

but not recorded as such for this taxon by Fang (2003: 719). Fang (2003: 719) also reported this character as

absent inDevario devario but it is present in our material of that species (Fig. 10D). Fang (2003) recovered

three independent origins of the danioin notch (her character 9) in her phylogenetic treatment of danionin

taxa (once on the branch leading to the Esomus+Danio clade; once inDanionella; and once along the branch

grouping allDevario, but reversed inD. devario) suggesting that this character is not synapomorphic for dan-

ionins (s.l).The danioin notch of C. margaritatus (Fig. 10A)is more similar in terms of shape and position to that of

other species of Danio (Fig. 10B, E)and Esomus (Fig. 10C) than to that of Danionellaand Devario (Fig.

10D). InDanio andEsomus the notch is positioned slightly anterior to the anteriormost tip of Meckels carti-

lage. It is short and deep, so that when viewed in lateral view the concavity formed by the notch causes the

width of the dentary at the deepest point of the notch to be approximately half the width of the widest point

posterior to the notch. InDanionella sp. the notch is long and deep, and adjacent to the anterior portion of

Meckels cartilage, which extends almost to the anteriormost tip of the lower jaw (see Robert, 1986; fig 6).

The differences in shape and position of the danioin notch betweenDanioandEsomus andDanionella may be

due to the developmentally truncated form of the latter (Britz, 2003). InDevario devario the notch is short and

shallow (Fig. 10D), and does not cut into the dentary as far as it does in species of DanioorEsomus.


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FIGURE 10. Lower jaw, left side, ventrolateral view (image reversed) of: A. Celestichthys margaritatus BMNH2007.10.9.1516, 15.2 mm SL; B.Danio erythromicronUAIC 14166.23, 19.4 mm SL; C.Esomus metallicusBMNH2000.6.10.80318258, 27.5 mm SL; D.Devario devarioUMMZ 187873, 56.6 mm SL; E.Danio nigrofasciatus UAIC14166.12, 22.2 mm SL; F.Microrasbora rubescens UAIC 14297.01, 23.7 mm SL; G.Sundadanio axelrodi ZRC 46313,18.6 mm SL. * indicates lateral flange on dentary. Cartilage grey. Abbreviations: An, anguloarticular; De, dentary; Ra,retroarticular.

Another feature of the lower jaw of C. margaritatus that it shares with other species ofDanio is the pres-

ence of a projection on the lateral face of the dentary (termed the danioin mandibular knob by Roberts, 2007:

136), situated slightly posterior to the danioin notch and closely associated with a small foramen for the pas-

sage of the internal mandibular branches of the trigeminal (V) nerve. Fang (2003) suggested that this structure

provided support for a fleshy flap on the lower jaw, a structure that Roberts (2007: 135) referred to as the

manidular pad. In C. margaritatus this projection is blunt-ended, and only weakly developed when com-

pared to that of mostDaniospecies, excludingD. erythromicron, where the projection exhibits a sharp, back-

wards pointed tip (Fig. 10E). InD. erythromicron the projection is short and rounded and similar in size and

shape to that of C. margaritatus (Fig. 10B). This projection is not present inDanionella,Devario (Fig. 10D),

Esomus (Fig. 10C)orMicrorasbora rubescens (Fig. 10F)but a similar projection is present in Sundadanio


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(Roberts, 1989; Kottelat & Witte, 1999; Fang, 2003; Fig. 10F) and Paedocypris (Kottelat et al. 2006). In these

latter miniature species (which are sister groups; Rber, et al. 2007) the projection on the dentary provides

support for a cluster of large conical tubercles and appears to be more highly developed in males (Roberts,

1989; Kottelat et al. 2006). The projection on the lateral face of the dentary in Sundadaniois much larger than

that ofDanio species and is shaped somewhat differently (two prominent rounded heads vs. one rounded or

sharp head) and does not appear to be a homologous structure (Fang, 2003).

Infraorbital series: Members of the Cyprinidae usually exhibit 5 infraorbital bones (IO1-5). Nelson(1969) suggested that the reduced number of bones in the infraorbital series of cyprinids may have resulted

from the fusion of two middle bones of the series. Within the Cyprinidae, reduction in the number of bones of

the infraorbital series is common, particularly in species with small adult body sizes (e.g. only IO1 present in

Barboides (Conway & Moritz, 2006) and Danionella mirifica (Britz, 2003); IO series completely absent in

Paedocypris (Kottelat et al. 2006). Like other species of miniature cyprinids, the infraorbital series of C. mar-

garitatus is also reduced (IO2 absent; Fig. 6A). A similar condition is also present in our material ofD. nigro-

fasciatus (Fig. 6C). Reduction in the size of IO2 appears to be characteristic for Danio (excluding D.

erythromicron)and some species ofEsomus(Fang, 2003). InD. erythromicron IO2 is similar in width to IO1,

a condition also exhibited byM. rubescens (Fig. 6D) and considered plesiomorphic (Fang, 2003). Strangely,

C. margaritatus retains the contact between the anterior tip of IO5 and the posterior tip of the supraorbital.The loss of this contact was considered a derived condition within the Cyprinidae by Cavender and Coburn

(1992). Other species ofDanio (excludingD. erythromicron) exhibit the derived condition, likely due to the

reduced nature of IO5 (Fang, 2003).

Celestichthys margaritatus also lacks an ossified infraorbital canal (Fig. 6A). A similar condition is also

exhibited by Sundadanio andMicrorasbora (Fig. 6D). InD. erythromicron the infraorbital sensory canal is

only enclosed posteriorly, on IO4-5 (Fig. 4B). In other species of Danio examined the infraorbital canal may

be completely enclosed (e.g.D. albolineatus) or only partially enclosed, as inD. nigrofasciatus (Fig. 6C).

Weberian apparatus:Celestichthys margaritatusexhibits a median projection on the outer arm of the os

suspenorium (Fig. 7A) close to its ventral tip, which attaches to the tunic surrounding the anterior swimblad-

der chamber. This feature was observed in all other species of Danioexamined (Fig. 7B, E) but not inDan-

ionella, Devario (Fig. 7D), Esomus (Fig. 7C),Microrasbora (Fig. 7F), Paedocypris or Sundadanio. In C.

margaritatus andD. erythromicron the median projection is slightly anteriorly oriented (Fig. 7A,B). In other

species ofDanio examined the projection is oriented posteriorly (Fig. 7E).

Unlike several other miniature cyprinid species, C. margaritatus does not appear to exhibit any sexual

dimorphism of the Weberian apparatus, as reported in Danionella (Britz, 2004), Sundadanio (Conway &

Britz, 2007) andPaedocypris(Britz & Conway, in prep).

Molecular Phylogeny of the Rasborinae and Phylogenetic Position of Celestichthys margaritatus

A total of 1,494 bp were aligned for the exon3 of RAG1 sequences for 42 taxa sampled in this study. No inter-

nal indels were found among the aligned sequences. Of these, 834 sites were constant and 544 sites were par-

simony-informative. MP analysis yielded 20 equally parsimonious trees (Tree length = 2,546, CI = 0.41, RI =

0.56). A strict consensus tree is presented in Figure 11 (topology on the right). ML analysis (ML of -

13623.5258) provided similar results (Fig. 11, topology on the left) to that of the MP analysis. Although the

ML tree is more resolved than the strict consensus resulting from the MP analysis, its internal branches,

depicting higher-level relationships of cyprinids, appear relatively short when compared with its long terminal

branches, possibly reflecting a rapid radiation of cyprinid lineages early in their evolutionary history. 50%

majority rule consensus tree of all post burn-in trees from partitioned BI (not shown) generated an almost

identical result to the ML tree, with only slight differences in relationships, particularly among taxa in the


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group containing species ofRasbora,Borarasand their relatives. Robust nodes with resulting Bayesian poste-

rior probabilities equal to or higher than 0.95 are highlighted in bold branches on the ML topology (Fig. 11).

FIGURE 11. Phylogenetic trees obtained from different analytical methods used in this study based on RAG1 genesequences (1,494 bp), depicting relationships among the taxa from the Rasborinae and its cyprinid allies. Tree based onMaximum-Likelihood (ML) analysis is shown on the left. The branch length is proportional to inferred character substi-tutions under GTR+G model. Strict consensus tree from 20 equally parsimonious trees (tree length of 2,546) resultingfrom the Maximum Parsimony (MP) analysis is presented on the right. Numbers on the branches of topology present MLbootstraps (left) and MP bootstraps (right) respectively. Values below 50% are not shown. Bold branches at left topologyindicate that the resulting a posterioriprobabilities from partitioned Bayesian analysis are equal to or higher than 0.95.

The targeted taxon in this study, Celestichthys margaritatus, is marked in bold. * indicates taxa with miniature adult bodysize (26 mm SL or below; sensu Weitzman & Vari, 1988).


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In all resulting phylogenies (all analyses), the cyprinid subfamily Rasborinae was not found to represent a

monophyletic grouping (Fig. 11). As shown in ML analysis, species currently placed within the Rasborinae

appear to have at least two distinct origins among cyprinids (Rasborinae-1 and 2; Fig. 11), though Rasborinae-

2 was not recovered as monophyletic in the MP strict consensus tree (Fig. 11, right side). Rasborinae-1

(Zacco, Opsariichthys andAphocypris) andIschikauia steenackeri (Cultrinae) form a well supported mono-

phyletic group (node support 91%, 85% and 1.00 for ML and MP bootstrap and Bayesian posterior probabili-

ties, respectively). This indicates that members of Rasborinae-1 are more closely related group are moreclosely related to members of the Cultrinae and other cyprinid subfamilies (including the Acheilognathinae,

Gobioninae and Leucisinae) than they are to other species currently placed within the Rasborinae (a frequent

result in molecular phylogenetic investigations of the Cyprinidae: Saitoh et al., 2006; Mayden et al. 2007;

Rber, et al. 2007; Wang et al., 2007). Rasborinae-2 includes the majority of the rasborin specieses that we

sampled in this study (Fig. 11). Within the rasborin-2, six major groups were consistently recovered (with

strong nodal support) in all analyses, although the interrelationships among these groups were not well

resolved. They include: a clade includingLuciosoma, OpsaridiumandBarilius; a clade includingEsomusand

Sundadanio; a clade including all species of BorarasandRasboraused in this study,Horadandia atukorali

and Trigonostigma heteromorpha; a clade including all species ofMicrorasboraand Chela sampled,Devario

reginaand Inlecypris auropurpureus; a clade including both species of Danionellasampled; and finally, aclade containing allDaniospecies in this study and our target taxon, C. margaritatus.In all analyses, C. mar-

garitatusis the sister group toD. erythromicron. Monophyly of the clade containingDanioplus Celestichthys

was highly supported by MP and ML bootstraps (100%) and Bayesian posterior probabilities (1.00).

Our results, based on comparative osteology and molecular phylogeny, provide strong evidence that C.

margaritatus is closely related tospecies ofDanioand the closest relative ofD. erythromicron.

Discussion

Comments on Roberts (2007):Though Roberts (2007) did not attempt to describe the osteology of C. mar-garitatus in detail, due to problems encountered with clearing and double staining (Roberts, 2007:132),he did

make comments on several aspects of its osteology. Based on our examination of cleared and double stained

specimens of C. margaritatus we have found several of these comments to be inaccurate and in need of clari-

fication. Firstly, Roberts (2007: 136) noted that the expanded ventral tip of the maxilla of C. margaritatus and

Microrasbora rubescenswas embedded in a mobile cartilaginous element. connected to the coronoid pro-

cess of the lower jaw. This strange jaw configuration, described, but not illustrated by Roberts, is not present

in our material ofD. margaritatus, nor is it present in our material ofM.rubescens, in which the only cartilag-

inous element present in the lower jaw is Meckels cartilage, medial to the dentary. An additional cartilaginous

element, the maxillo-mandibular cartilage, is present between the upper and lower jaws of species of Dan-

ionella(Roberts, 1986; Britz, 2003) but is not present inD. margaritatus, M. rubescensor any other species ofcyprinid that we examined.

Roberts (2007: 134) listed the modal number of total vertebrae for C. margaritatus as 31 (N=40) with fre-

quencies 30(7), 31(26), 32(7), composed of 13-16 abdominal +15-17 caudal vertebrae. Both specimens of C.

margaritatus that we examined possess 32 total vertebrae, composed of 15+17. However, our method of

counting vertebral centra differs somewhat from Roberts. We refer to caudal vertebrae as all vertebrae exhib-

iting a full hemal spine (following Hubbs & Lagler, 1958) whereas Roberts refered to caudal (his postabdom-

inal) vertebrae as all vertebrae posterior to the first elongate anal-fin pterygiophore. Roberts (2007: 132)

stated that the first elongate pterygiophore of C. margaritatus was actually the second anal fin pterygiophore

(the first pterygiophore is very short). It is clear from our description of C. margaritatus and Figure 8A that

the first elongate pterygiophore is actually the first and not the second pterygiophore, as suggested by Roberts,


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as is the case in all other species of the Cyprinidae that we examined. Roberts may have mistaken the flange of

membrane bone on the anterior edge of the first proximal-middle radial for the first pterygiophore in radio-

graphs. Using Roberts method of counting vertebrae our total vertebrae counts would not change but the num-

ber of abdominal and caudal would both be 16 (vs. 15 and 17, respectively).

Sister-group relationship between C. margaritatus andD. erythromicron: Amongst South East Asian

cyprinids Roberts (2007) believed that C. margaritatus appeared to be most closely related to two small dan-

ioins endemic to Lake Inle,Microrasbora rubescens andD. erythromicron(his Microrasbora erythromi-cron). Of the two species, Roberts appeared to believe that C. margaritatus was more closely related to D.

erythromicron than toM. rubescens, a point which he returned to frequently throughout the description of C.

margaritatus: In size and shape of head, jaws, body and fins it [C. margaritatus] is most similar to another

diminutive and highly colourful cyprinid, Microrasbora erythromicronAnnandale, 1918, endemic to Inle

Lake. (p. 132); In most respects, Microrasbora erythromicronis again like Celestichthys[in reference to

anal fin and caudal peduncle shape]. (p.132); Body deep and strongly compressed, much more so than inM.

rubescens but similar to M. erythromicron. (p. 134).

In addition to the similarities identified by Roberts (2007), both C. margaritatus andD. erythromicron

exhibit a miniature adult body size (sensu Weitzman & Vari, 1988), lack barbels, the mandibular sensory canal

(Fig. 3C, 8A,B), and the autogenous uroneural of the caudal skeleton (Fig. 6C). Celestichtys margaritatus alsoshares one reductive feature in common with D. nigrofasciatus, absence of IO2 (Fig. 6A,C). However, D.

nigrofasciatus exhibits a mandibular sensory canal (Fig. 10E), barbels and the autogenous uroneural of the

caudal skeleton and thus C. margaritatus shares more reductions in common withD. erythromicron then it

does with D. nigrofasciatus or any other species of Danio. We conclude here that C. margaritatus and D.

erythromicon are sister-group. Our molecular analyses support this sister-group relationship (Fig. 11) and we

interpret the shared reductive features of C. margaritatus andD. erythromicron as the result of a single minia-

turization event from their most recent common ancestor.

Celestichthys as a synonym ofDanio: Fang (2003) restricted the genusDanio to those species assigned

previously to the Danio dangilaspecies group (Fang, 2000), based on the shared presence of two apomor-

phic states: (1) an A stripe on the anal-fin rays (a dark stripe extending along the middle of the anal-fin raysand ending at the distal tip of the last branched anal-fin rays; character 15(state1)), and (2) the presence of two

or more pigment stripes on the caudal-fin rays (character 16(1). No other genus of the Cyprinidae from South

or South East Asia possesses this combination of shared derived characters and only one species ofDanio,D.

erythromicron, is known to lack these two traits. Celestichthys margaritatus also possesses an A stripe and

exhibits two pigment stripes on the caudal fin (Fig. 1B), traits which first aroused our suspicion about its orig-

inal taxonomic placement by Roberts (2007).

Celestichthys margaritatusalso exhibits a median projection on the outer arm of the os suspenorium (= 4th

pleural rib of other authors) (Fig. 7A). This same derived trait was first observed by Kottelat and Witte (1999)

inDanio erythromicron,a species originally placed with in the genusMicrorasbora by Annandale (1918) but

later moved toDanio(Kottelat and Witte, 1999). Sanger and McCune (2002) later reported the presence ofthis trait in their slender bodiedDanioclade as did Fang (2003: character 34(1)) in all members of herD.

dangila species group and in D. erythromicron. However, according to the Fangs phylogenetic hypothesis

based on morphology, this character was shown to arise twice, once on the branch leading to members of the

D. dangila species group (= Danio s.s.), and once in D. erythromicron, which was recovered as the sister

group toMicrorasbora rubescens. Though Fang (2003) did not recoverD. eryrthromicron as a member of her

Danio (s.s), this species was hypothesised to belong to Danio by Kottelat and Witte (1999) and a recent

molecular phylogenetic analysis supports such a grouping (Mayden et al. 2007), as do the results of the phylo-

genetic analysis presented herein (Fig. 11). No other member of the Cyprinidae examined by us was found to

exhibit a similar projection on the outer arm of the os suspensorium. The presence of a median projection on

the outer arm of the os suspensorium should be considered synapomorphic forDanio.In addition to the morphological features mentioned above,C. margaritatus exhibits one further feature in


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Celestial Pearl Danio is a Miniature Danio