“Celestial Pearl danio” is a miniature Danio

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“Celestial Pearl danio” is a miniature Danio
Zootaxa 1686: 1–28 (2008)
www.mapress.com / zootaxa/
ISSN 1175-5326 (print edition)
Copyright © 2008 · Magnolia Press
ISSN 1175-5334 (online edition)
ZOOTAXA
The “Celestial Pearl danio” is a miniature Danio (s.s) (Ostariophysi: Cyprinidae): evidence from morphology and molecules
KEVIN W. CONWAY, WEI-JEN CHEN & RICHARD L. MAYDEN
Department 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, is
described in detail and briefly compared with that of other members of the Rasborinae (notably Danio (s.s), Danionella,
Devario, Esomus, Microrasbora, Paedocypris and Sundadanio). Celestichthys margaritatus possesses an “A” stripe on
the anal fin and two pigment stripes on the caudal fin (apomorphic features of Danio 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 species of Danio (including D. erythromicron) amongst the Cyprinidae, and a lateral projection on the lateral face of the
dentary (present only in Danio, Sundadanio and Paedocypris). Phylogenetic analysis of 1,494 bp of the RAG1 nuclear
gene for 31 rasborine taxa, including 5 species of Danio, places C. margaritatus as the sister group to D. erythromicron,
and part of a larger monophyletic group including all other species of Danio included for analysis. Based on characters of
morphology and its position in a molecular phylogeny of the Rasborinae it is proposed that Celestichthys be placed in the
synonymy of Danio, its only member referred to as Danio 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 either Microrasbora or Rasbora 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. Despite
the choice of common name, however, Roberts did not place this new species within Danio but instead created 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” erythromicron) might also be a congener.
Little is known about the ecology of the “Celestial Pearl danio” other than that it inhabits small and shallow 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 previously
known Asian Cyprinidae by its distinctive head and body shape, small upturned mouth with shortened jaws,
Accepted by A. Gill: 28 Nov. 2007; published: 21 Jan. 2008
1
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 pigmentation 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, something that Roberts (2007) did not attempt in its original description. We also redescribe its colour pattern using
appropriate Danio 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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CONWAY ET AL.
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 protocol outlined in Weitzman (1974) (hyoid arches removed with the branchial arches) under a Leica MZ95 stereomicroscope. 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 photographs. 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 Sørensen (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 during
this 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 2004.4.26.18—21 (3); B. maculata — BMNH
1995.5.17.112–126 (6); B. merah — BMNH 2004.4.26.10–17 (4); B. micros — BMNH 2004.4.29.1–3 (2); B.
urophthalmoides — BMNH 2004.4.26.2–9 (2); Campostoma anomalum — AMNH 40260 (1); Celestichthys
margaritatus — BMNH 2007.10.9.15–16 (2); Chela oxygasteroides — AMNH 36368 (1); Chelaethiops bibie
— BMNH 2006.3.9.46–93 (4). — UMMZ 166632 (1); Chondrostoma nasus — UMMZ 185029 (2); Clinostomus elongatus — AMNH 45955 (5); Couesius plumbeus — AMNH 41266 (5); Culter alburnus — UMMZ
66525 (2); Cyclocheilichthys apogon — BMNH 2001.1.15.699–718 (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 2001.3.12.76–92 (3),
BMNH 1983.7.11.15–29 (2); Danionella mirifica — USNM 372848 (36); Devario cf. aequipinnatus —
BMNH 2005.7.5.502–539 (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 2000.6.10.8031–8258 (3); Exoglossum maxillingua
— KU 18925 (11); Garra dembeensis — BMNH 1984.9.7.50–60 (2); Hampala macrolepidota — BMNH
2000.6.10.7891–7900 (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 2006.3.9.108–162 (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); Microrasbora kubotai — BMNH 2004.6.25.6–10 (3); M. nana — BMNH 2004.6.25.1–5 (3); M. rubescens —
BMNH 2004.6.25.11–13 (2), UAIC 14297.01 (1); Nocomis effusus — KU 18932 (11); Notemigonus crysoleucas — KU 1357 (1); Notropis altipinnis — UAIC 7960.03 (10); N. buccatus — KU 17764 (4); N. buccula —
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KU 14286 (6); N. chiliticus — KU uncat. (12); N. dorsalis — 11166.02 (2); N. harperi — KU 21251 (5); N.
hudsonius — 1685.07 (2); N. nubilus — KU 17189 (8); N. ortenburgeri — KU 12462 (5); Opsariichthys
bidens — UMMZ 64240 (1); Oxygaster hypophthalmus — AMNH 48924 (2); Paedocypris micromegethes
BMNH 2004.11.16.41–60 (20); Paralaubuca riveroi — UMMZ 181128 (1); Phenacobius mirabilis — KU
7918 (4); P. teretulus — KU 18929 (17); P. uranops — KU 19619 (2); Phoxinus erythrogaster — UAIC
14176. (7); P. neogaeus — KU 18882 (12); P. phoxinus — AMNH 36873 (4); Pteronotropis welaka — KU
18895 (9); Ptychocheilus grandis — KU 18920 (12); Romanogobio albipinnatus — UMMZ 185111 (1);
Rasbora argyrotaenia — UMMZ 157150 (5); R. cephalotaenia — BMNH 2000.10.18.34–40 (2); R. spilocerca — UAIC 14185 (4); R. sumatrana — BMNH 2001.1.15.6780–6801 (2); R. tornieri — BMNH 2000.
7.9.63–81 (2); Rasboroides vaterifloris — BMNH 2004.6.25.26–30 (3); Rhinichthys evermanni — KU 18910
(7); Rhodeus sericeus — AMNH 39117 (2), — UMMZ 18511 (2); Rutilus rutilus — AMNH 36897 (4);
Salmophasia bacaila — UMMZ 187849 (1); Semotilus corporalis — KU 18854 (9); Squalidus japonicus —
UMMZ 142961 (1); Squalius borysthenicus — UMMZ 185112 (2); Sundadanio axelrodi — BMNH
1982.3.29.50–54 (2), BMNH 2005.11.10.1–8 (3), BMNH 2005.11.10.9–16 (5), CMK 8424 (3), CMK 9633
(3), CMK 10876 (3), CMK 16731(2), ZRC 46313 (2), ZRC 34255 (2); Telestes souffia — UMMZ 185042 (1);
Tribolodon hakonensis — UMMZ 187536 (2), UMMZ 188890 (4); Trigonostigma heteromorpha — BMNH
2004.6.25.26–30 (2); Yuriria alta — KU 21247 (8).
Colour pattern description: The colour pattern description of Celestichthys margaritatus is based on
preserved specimens. Colour pattern terminology follows that of Fang (1998).
Laboratory molecular work: Tissue extraction was performed using Qiagen DNAeasy extraction kit
(Qiagen, Valencia, CA) according to the manufacturer's instructions. DNA amplification was conducted by
PCR (Mullis & Faloona, 1987; Saiki et al., 1988) for fragments of exon 3 of RAG1 (recombination-activating
gene 1). Primers used in this study have been published by López et al. (2004) and Chen et al. (2007). Conditions for amplification were as follows: Takara Ex Taq (0.5 units) (Tekara Bio. Inc.), 1x reaction buffer, 2 mM
of MgCl2, 200 um of each dNTP, 0.2 mM of each primer, and 25–50 ng of genomic DNA in a 25-μl final reaction volume. Thermocycler conditions for PCR were: initial denaturing step at 95°C for 4 min followed by 35
cycles of 95°C (for 40 s), 53°C (for 40 s), and 72°C (for 1.5 min.), and then a final extension step of 72°C (for
7 min). PCR cleanup procedure followed the AMPure magnetic bead cleanup protocol (Agencourt Bioscience
Corporation) and resuspended in 30 µL of sterile water. Sequences were then determined by Macrogen Inc.
(Seoul, South Korea) using ABI 3730xl analyzer (Applied Biosystems).
Molecular data analysis: The DNA sequences were edited and managed using Se-Al v2.0a11 (Rambaut,
1996). The data were composed of sequences from 31 rasborin samples (including Celestichthys margaritatus) plus eight representative taxa from different cyprinid subfamilies and three outgroup taxa from the cypriniform families Gyrinocheilidae, Balitoridae and Cobitidae (Table 1). The methods utilized for phylogenetic
analyses included: partitioned Bayesian inference (BI), as implemented in MrBayes parallel version v3.1.1
(Huelsenbeck & Ronquist, 2001); Maximum Parsimony (MP), as implemented in PAUP* -version 4.0b10
(Swofford, 2002), and maximum likelihood (ML), as implemented in RAxML-VI-HPC (v2.2.3) (Stamatakis,
2006). Optimal trees for MP were obtained by heuristic searches with random stepwise addition sequences
followed by TBR swapping, for 100 replications each. We used mix model, which allows an individual model
of nucleotide substitution to be estimated independently from each gene partition, for maximum likelihood
based methods. Partitions were assigned with respect to the codon positions of RAG1. Likelihood ratio tests
(Goldman, 1993), as implemented in MrModeltest 2.2 (Nylander, 2004), were used to choose models for each
gene coding position in Partitioned BI. The GTR+G+I model (Yang, 1994) for the first and second codon
position and GTR+G model for third codon position were suggested. The parameters for running MrBayes
were set as follows: “lset nst=6” (GTR), “rates=invgamma” (G+I), or “rates=gamma” (G), “unlink” (unlinking of model parameters across data partitions), and “prset ratepr = variable” (rate multiplier variable across
data partitions). Four independent MCMC chains were performed with 1,000,000 replicates, sampling one
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CONWAY ET AL.
TABLE 1. Taxa used for molecular analyses in this study. Subfamily nomenclature follows Nelson (2006).
Family/subfamily
Taxon
GenBank accession no.
Gyrinocheilus aymonieri
EU292682
Lefua echigonia
EF458305
Leptobotia pellegrini
EU292683
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
EU292697
Rasborinae
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
EU292707
Rasborinae
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
EU292712
Rasborinae
Zacco sieboldii
EU292713
Gyrinocheilidae
Balitoridae
Nemacheilinae
Cobitidae
Botiinae
Cyprinidae
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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 program
from 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 posteriori probabilities from partitioned BI.
Results
Preserved colour pattern of Celestichthys margaritatus: In males, 5–6 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 6th or 7th branched ray. Submarginal 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 3rd branched ray, and basal stripe, extending along middle of pelvic fin, ending at distal tip of 5th branched ray. Females with similar distribution of longitudinal rows of spots and fin stripes. Fin
stripes 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 anterior margin of the orbit. It projects outwards from the ethmoid region in an anteroventral direction and terminates 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 in D. 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 ossification that forms the floor of the ethmoid region.
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CONWAY ET AL.
FIGURE 2. Neurocranium of Celestichthys margaritatus BMNH 2007.10.9.15–16, 15.2 mm SL. A. dorsal view; B. lateral view, left side; C. ventral view. Cartilage grey. Abbreviations: AV–VII, anterior opening of trigeminal-facial chamber; Apto, autopterotic; Asph, autosphenotic; BL, Baudelot’s ligament; Boc, basioccipital; Exoc, exoccipital; Epoc,
epiotic; Fr, frontal; LE, lateral ethmoid; MP, masticatory plate of the basioccipital; ME, mesethmoid; Osph, orbitosphenoid; Psph, parasphenoid; PV–VII, posterior opening of trigeminal-facial chamber; PE, prethmoid; Pro, prootic; Pt, parietal; Ptsph, pterosphenoid; SO, supraorbital; Soc, supraoccipital; STF, subtemporal fossa; Vo, vomer; FIX, foramen for
glossopharyngeal 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 endochondral 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 hyomandibular 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 laterally, 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 contributes 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 by
the 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 parietal, 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 magnum, 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 basioccipital
forms 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 surface, 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 processes 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 2007.10.9.15–16, 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, dentary; Ecpt, ectopterygoid; Enpt, endopterygoid; Hy, hyomandibular; Iop, interopercle; Mx, maxilla; MC, Meckel’s cartilage; 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 cartilage. 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 posteriormost 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 posteroventral
process (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 palatoquadrate 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 posterior
edge. 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 Meckel’s 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 anguloarticular 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 triangular retroarticular articulating on the posteroventralmost tip of the anguloarticular. It exhibits a small needle-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 Meckel’s 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 attachment 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 ventralmost 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 2007.10.9.15–16, 15.2 mm SL. Cartilage
grey. Abbreviations: Apal, autopalatine; Fr, frontal; IO1, infraorbital 1; LE, lateral ethmoid; KE, kinethmoid; Mx, maxilla; 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 posteriorly. It extends far forward in front of the anteriormost tips of the ventral hypophyals.
The anterior copula contains three basibranchial (Bb) ossifications, Bb1–3. All are thin, rod shaped elements, only slightly narrower in width than the basihyal. Bb1 is the shortest of the three. Bb2 and Bb3 are similar 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, Hb2–3. 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 cartilage
situated at the anterior tip of Cb1. Ceratobranchials 1–4 (Cb1–4) are rod-shaped elements. All are tipped in
cartilage anteriorly and posteriorly and exhibit 2–7 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, Eb1–4, and 2 pharyngobranchials, Pb2–3. Eb1–
3 lack uncinate processes and exhibit a small flange of membrane bone along their posterior edges. Eb4 exhibits 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.
Pb2–3 are small perichondral ossifications surrounding Pb2–3c. 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 2007.10.9.15–16, 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 1–3, basibranchial 1–3; Bh, basihyal; Br, branchiostegal rays; Cb 1–5, ceratobranchial 1–5; DHh, dorsal hypohyal; Eb1–4, epibranchial 1–4; Eb5C, epibranchial 5 cartilage; Hb1C, hypobranchial 1
cartilage; Hb2–3, hypobranchial 2, 3; IhC, interhyal cartilage; Lev, levator process; Pb2–3, pharyngobranchial 2–3; PCh,
posterior ceratohyal; PC; posterior copula cartilage; Uh, urohyal; VHh, ventral hypohyal.
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Hyoid arch and urohyal (Figure 5): The interhyal cartilage is small, its upper arm articulating with the
hyopalatine arch at the junction between the hyomandibular and sympletic and its lower tip with the posterior
end of the posterior ceratohyal. The posterior ceratohyal is relatively short and exhibits a rounded posterior
tip. It is transversed by the hyoidean artery, which enters posterodorsally, via a small foramen, and exits
anterodorsally, via a larger foramen, to continue along a shallow groove on the dorsal surface of the anterior
ceratohyal. The posterior ceratohyal is separated from the anterior ceratohyal by a thin cartilaginous strip,
which extends anteriorly along the posteroventral surface of the anterior ceratohyal. At its posteriormost point
the anterior ceratohyal is similar in depth to the posterior ceratohyal, but continues to decrease in depth anteriorly, where it is bifurcated, serving as a point of articulation for the hypohyals. The ventral hypohyal is triangular in shape and much larger than the dorsal hypohyal, its posteroventral face serving as a point of
attachment for the ligament attached to the anterior process of the urohyal.
There are three branchiostegal rays of similar length and thickness. The anteriormost articulates on the
medial face of the anterior ceratohyal close to its midsection, the posteriormost articulates on the lateral face
of its posteroventralmost tip. The middle branchiostegal also articulates on the lateral face of the anterior ceratohyal, between the other two.
The urohyal is ventrally flattened with a dorsally directed blade-like process extending along the midline,
giving the bone a ‘T’-shape appearance in cross-section. Two long processes extend from its anterior edge,
which attach to the ventral hypohyals by short ligaments.
Infraorbitals (Figure 6A): There are four infraorbitals (IO1, 3–5) rimming the anterior, ventral, and posterior margin of the orbit and a large supraorbital rimming its dorsal margin (Fig. 2). IO2 is absent and there is
no enclosed infraorbital sensory canal. IO1 is a small, weakly ossified dermal ossification with a large concave depression at its center. It sits at the anterior edge of the orbit, lateral to the point of articulation between
the lateral ethmoid and the autopalatine. Its anteriormost edge overlaps the posterior edge of the maxilla. IO3
is the largest of the infraorbital bones, rimming the ventral margin of the orbit. It is most heavily ossified
along its dorsalmost edge and only weakly ossified anteriorly and ventrally. IO4 is a fan-shaped bone, bordering the posterior margin of the orbit. Its posteriormost edge extends close to the anteriormost edge of the opercle. IO4 is most heavily ossified along its anteriormost edge, as is IO5. IO5 is a roughly triangular bone,
similar in width to IO4. Its anterodorsalmost point is closely associated with the posteriormost tip of the
supraorbital.
The supraorbital is a weakly ossified, plate-like bone, which sits at the lateral edge of the frontal, rimming
the anterior margin of the orbit (Fig. 2).
Weberian apparatus (Figure 7A): The Weberian apparatus incorporates the 4 anterior abdominal vertebral centra and associated elements. The first centrum (V1) is much shorter than the three succeeding centra,
which are similar in shape. All Weberian centra are much shorter than non-Weberian centra, which are almost
twice as long, hourglass shaped bones. The second centrum bears a large lateral process close to its anteroventral edge, which extends laterally into the body musculature. All Weberian ossicles are well formed. The
scaphium is a large element that sits dorsal to the first vertebral centrum, to which it is attached by a small
nodular foot. It exhibits a large ascending process on its posterodorsal edge, which extends dorsally lateral to
the cartilage rimming the anterior face of the neural complex. The claustrum is a thin lamina of bone that sits
medial to the scaphium, anterior to the anterior edge of supraneural 2. The intercalarium is a small ‘v’-shaped
ossification, which is attached by a small nodular base to V2. The tripus is a large sickle-shaped element that
attaches to V3 via a broad, twisted, medial process. It exhibits a narrow transformator process on its posteriormost tip, which curves medially, before attaching to the anterior face of the anterior swimbladder chamber.
The inner arm of the os suspensorium is a long thin element that extends ventrally, surpassing the ventralmost
part of the outer arm of the os suspenorium, and terminating at the level of the dorsal most point of the post
cleithrum. The inner arms of both sides are tightly bound to each other over their entire length. The outer arm
of the os suspenorium is much shorter than the inner arm. It is autogenous with V4, to which it is attached via
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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. Supraneural 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 margaritatus BMNH 2007.10.9.15–16, 15.2 mm SL, right side, lateral
view, (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 UAIC
14297.01, 23.7 mm SL, right side, lateral view, (image reversed). Abbreviations: IO1–5, infraorbital 1–5.
Vertebral column (Figures 7, 8): Both specimens exhibit 32 vertebrae, consisting of 15 abdominal + 17
caudal. Pleural ribs start on V5 and continue to V14–V15. 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 without contact to its associated centrum (not illustrated). The head of the 5th pleural 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 10th on V14), articulate at the midpoint of the base of the neural arch. The last parapophysis is fused to its centrum. The neural arches of all vertebrae, excluding those on PU2–3, are narrow and situated on the anterior half of the centra. Neural arches on
V6–V13 exhibit long prezygopophyses on their anterior edge. V5–V29 exhibit small dorsally oriented postzygopophyses. Hemal spines are borne on the anterior half of V16–31. Small ventral postzygopophyses are
borne on the posterior edge of V16–28.
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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 2007.10.9.15–16, 15.2
mm SL; B. Danio erythromicron UAIC 14166.23, 19.4 mm SL; C. Esomus metallicus BMNH 2000.6.10.8031–8258,
27.5 mm SL; D. Devario devario UMMZ 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 the
os 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 5th or 6th vertebral centrum.
There are five small plate-like supraneurals, situated between the neural spines of V4–V9. 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 posterior. They are attached to the base (V16–21) or midregion (V211–29) of the hemal spines of their associated
centra 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 V11–17. 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 supernumerary 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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FIGURE 8. Celestichthys margaritatus BMNH 2007.10.9.15–16, 15.2 mm SL. A. mid-region of vertebral column, dorsal and anal fin skeleton, lateral view, left side; B. pelvic skeleton, ventral view; C. caudal skeleton, lateral view, left side.
Cartilage grey. Abbreviations: AFS, anal fin style; Bp, basipterygium; CC, compound centrum; DFS, dorsal fin style; Dr,
distal radial; DrC, distal radial cartilage; Epl, epipleural; Epn, epineural; Ep, epural; Ha, hemal arch; Hs, hemal spine;
H1–5, hypural 1–5; Na, neural arch; Ns, neural spine; P, parapophyses; Ph, parhypural; Pls, pleurostyle; PU2, 3, Preural
centra 2, 3; Pr, pelvic radial; PrC, pelvic radial cartilage; Ps, pelvic splint; P-Mr, proximal-middle radial; R, fin ray; S,
supernumerary ray; V13, 24, 28, 13th, 24th or 28th vertebral centrum; 13–14r, rib of 13th or 14th vertebral centrum.
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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 perichondral 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 inserting between the hemal spines of V16–22. The first four anal 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 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 radials are ossified endochondrally. Distal radial cartilages posterior to the third distal radial may exhibit small
perichondral ossifications around their points of articulation with the base of the associated fin ray.
Pelvic girdle (Figure 8B): The pelvic girdle consists 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. Caudal fin rays are supported by the neural and hemal spines of the 2nd and 3rd preural caudal centra, the pleu rostyle, a single epural, 5 hypural elements and the parhypural. The 2nd and 3rd preural 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 2nd preural centra is autogenous from the centrum. Hemal
spines of the 2nd and 3rd preural centra bear expanded tips that provide support for ventral procurrent rays. The
posteriormost tip of the neural spine of the 3rd preural centrum is not expanded and does not support dorsal
procurrent rays, unlike the tip of the neural spine of the 2nd preural centrum which is expanded and provides
support for the three anteriormost dorsal procurrent rays. There are no cartilaginous radial elements in the caudal 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 each
other 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 3rd hypural, which is
comparable in length and width to the 2nd, abuts with the compound centrum in the ‘v’ formed between the
pleurostyle and 2nd hypural. 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.6–7.iii–iv) 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. Baudelot’s 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 2007.10.9.15–16, 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; Pr1–4, pectoral radial 1–4; 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 postcleithrum, medially with the mesocoracoid, and posteroventrally with the scapula and coracoid. The postcleithrum 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 endochondral 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 elements. The second is the largest of the three and exhibits a large flange of membrane bone on its medial surface, 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 5th branched
pectoral-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’, including Danio, Devario, Danionella, Esomus, Microrasbora and Sundadanio.
Danio (= Devario) malabaricus by Howes (1979: 192), is present in several genera of South East Asian cyprinids, including Danio, Devario, Danionella, Esomus, and is also reported as present in Parabarillius (Roberts, 1986). It is not present in Sundadanio axelrodi or species of Microrasbora (Fang, 2003) but is present in
specimens of D. 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 in Devario 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 in Danionella; and once along the branch
grouping all Devario, but reversed in D. devario) suggesting that this character is not synapomorphic for danionins (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 Danionella and Devario (Fig.
10D). In Danio and Esomus the notch is positioned slightly anterior to the anteriormost tip of Meckel’s cartilage. 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. In Danionella sp. the notch is long and deep, and adjacent to the anterior portion of
Meckel’s 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 between Danio and Esomus and Danionella may be
due to the developmentally truncated form of the latter (Britz, 2003). In Devario devario the notch is short and
shallow (Fig. 10D), and does not cut into the dentary as far as it does in species of Danio or Esomus.
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FIGURE 10. Lower jaw, left side, ventrolateral view (image reversed) of: A. Celestichthys margaritatus BMNH
2007.10.9.15–16, 15.2 mm SL; B. Danio erythromicron UAIC 14166.23, 19.4 mm SL; C. Esomus metallicus BMNH
2000.6.10.8031–8258, 27.5 mm SL; D. Devario devario UMMZ 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; 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 of Danio is the presence 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 passage 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 compared to that of most Danio species, excluding D. erythromicron, where the projection exhibits a sharp, backwards pointed tip (Fig. 10E). In D. 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 in Danionella, Devario (Fig. 10D),
Esomus (Fig. 10C) or Microrasbora 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; Rüber, 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 Sundadanio is much larger than
that of Danio 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. margaritatus is also reduced (IO2 absent; Fig. 6A). A similar condition is also present in our material of D. nigrofasciatus (Fig. 6C). Reduction in the size of IO2 appears to be characteristic for Danio (excluding D.
erythromicron) and some species of Esomus (Fang, 2003). In D. erythromicron IO2 is similar in width to IO1,
a condition also exhibited by M. 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 of Danio (excluding D. 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 and Microrasbora (Fig. 6D). In D. 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 in D. nigrofasciatus (Fig. 6C).
Weberian apparatus: Celestichthys margaritatus exhibits 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 swimbladder chamber. This feature was observed in all other species of Danio examined (Fig. 7B, E) but not in Danionella, Devario (Fig. 7D), Esomus (Fig. 7C), Microrasbora (Fig. 7F), Paedocypris or Sundadanio. In C.
margaritatus and D. erythromicron the median projection is slightly anteriorly oriented (Fig. 7A,B). In other
species of Danio 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) and Paedocypris (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 internal indels were found among the aligned sequences. Of these, 834 sites were constant and 544 sites were parsimony-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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CONWAY ET AL.
group containing species of Rasbora, Boraras and their relatives. Robust nodes with resulting Bayesian posterior 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 gene
sequences (1,494 bp), depicting relationships among the taxa from the Rasborinae and its cyprinid allies. Tree based on
Maximum-Likelihood (ML) analysis is shown on the left. The branch length is proportional to inferred character substitutions under GTR+G model. Strict consensus tree from 20 equally parsimonious trees (tree length of 2,546) resulting
from the Maximum Parsimony (MP) analysis is presented on the right. Numbers on the branches of topology present ML
bootstraps (left) and MP bootstraps (right) respectively. Values below 50% are not shown. Bold branches at left topology
indicate that the resulting a posteriori probabilities 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 body
size (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 Rasborinae2 was not recovered as monophyletic in the MP strict consensus tree (Fig. 11, right side). Rasborinae-1
(Zacco, Opsariichthys and Aphocypris) and Ischikauia steenackeri (Cultrinae) form a well supported monophyletic group (node support 91%, 85% and 1.00 for ML and MP bootstrap and Bayesian posterior probabilities, respectively). This indicates that members of Rasborinae-1 are more closely related group are more
closely 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;
Rüber, 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 including Luciosoma, Opsaridium and Barilius; a clade including Esomus and
Sundadanio; a clade including all species of Boraras and Rasbora used in this study, Horadandia atukorali
and Trigonostigma heteromorpha; a clade including all species of Microrasbora and Chela sampled, Devario
regina and Inlecypris auropurpureus; a clade including both species of Danionella sampled; and finally, a
clade containing all Danio species in this study and our target taxon, C. margaritatus. In all analyses, C. margaritatus is the sister group to D. erythromicron. Monophyly of the clade containing Danio plus 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 to species of Danio and the closest relative of D. erythromicron.
Discussion
Comments on Roberts (2007): Though Roberts (2007) did not attempt to describe the osteology of C. margaritatus 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 clarification. Firstly, Roberts (2007: 136) noted that the expanded ventral tip of the maxilla of C. margaritatus and
Microrasbora rubescens was “embedded in a mobile cartilaginous element…. connected to the coronoid process of the lower jaw.” This strange jaw configuration, described, but not illustrated by Roberts, is not present
in our material of D. margaritatus, nor is it present in our material of M. rubescens, in which the only cartilaginous element present in the lower jaw is Meckel’s cartilage, medial to the dentary. An additional cartilaginous
element, the ‘maxillo-mandibular cartilage’, is present between the upper and lower jaws of species of Danionella (Roberts, 1986; Britz, 2003) but is not present in D. margaritatus, M. rubescens or any other species of
cyprinid that we examined.
Roberts (2007: 134) listed the modal number of total vertebrae for C. margaritatus as 31 (N=40) with frequencies 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 exhibiting a full hemal spine (following Hubbs & Lagler, 1958) whereas Roberts refered to caudal (his “postabdominal”) 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 radiographs. Using Roberts method of counting vertebrae our total vertebrae counts would not change but the number of abdominal and caudal would both be 16 (vs. 15 and 17, respectively).
Sister-group relationship between C. margaritatus and D. erythromicron: Amongst South East Asian
cyprinids Roberts (2007) believed that C. margaritatus appeared to be most closely related to two small “danioins” endemic to Lake Inle, Microrasbora rubescens and D. erythromicron (his “Microrasbora” erythromicron). Of the two species, Roberts appeared to believe that C. margaritatus was more closely related to D.
erythromicron than to M. 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” erythromicron Annandale, 1918, endemic to Inle
Lake.” (p. 132); “In most respects, “Microrasbora” erythromicron is again like Celestichthys [in reference to
anal fin and caudal peduncle shape].” (p.132); “Body deep and strongly compressed, much more so than in M.
rubescens but similar to “M.” erythromicron.” (p. 134).
In addition to the similarities identified by Roberts (2007), both C. margaritatus and D. 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 also
shares 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 with D. 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 and D. erythromicron as the result of a single miniaturization event from their most recent common ancestor.
Celestichthys as a synonym of Danio: Fang (2003) restricted the genus Danio to those species assigned
previously to the “Danio dangila species group” (Fang, 2000), based on the shared presence of two apomorphic states: (1) an “A stripe” on the anal-fin rays (a dark stripe extending along the middle of the anal-fin rays
and 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 of Danio, 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 original taxonomic placement by Roberts (2007).
Celestichthys margaritatus also 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)
in Danio erythromicron, a species originally placed with in the genus Microrasbora by Annandale (1918) but
later moved to Danio (Kottelat and Witte, 1999). Sanger and McCune (2002) later reported the presence of
this trait in their “slender bodied” Danio clade as did Fang (2003: character 34(1)) in all members of her D.
dangila species group and in D. erythromicron. However, according to the Fang’s 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 to Microrasbora rubescens. Though Fang (2003) did not recover D. 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 phylogenetic 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 for Danio.
In addition to the morphological features mentioned above, C. margaritatus exhibits one further feature in
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common with all other species of Danio, a projection on the lateral face of the dentary, a feature that is also
present in two other miniature cyprinids, Sundadanio (Roberts, 1989; Kottelat and Witte, 1999; Fang, 2003;
Fig. 10G) and Paedocypris (Kottelat et al. 2006). Sundadanio axelrodi was resolved with strong BI but mediocre support in bootstraps (70% and 89% for ML and MP respectively) as sister-group to Esomus metallicus
(Fig. 11), a taxon lacking this particular feature (Fig. 10C). Rüber et al. (2007) recovered Sundadanio as the
sister group to Paedocypris (a species which we did not include in our analysis) with strong support. A projection on the lateral face of the dentary appears to have evolved at least twice in cyprinds, once each in the lineage of Danio and the lineage of Sundadanio plus Paedocypris (Rüber et al., 2007).
Roberts (2007) distinguished Celestichthys from all other genera of South East Asian cyprinids by a distinctive head and body shape, small upturned mouth with shortened jaws, unique colouration, and 9/8 principal caudal fin rays. As we have stated previously the colour pattern of Celestichthys, composed of a series of
stripes, is not unique, and is simply a variation of the striped pattern common to all species of Danio (Fang,
1998). In addition, C. margaritatus shares two derived traits with all species of Danio (excluding D. erythromicron): (1) the presence of an “A” stripe on the anal-fin rays (Fang, 2003) and (2) the presence of two pigment stripes on the caudal-fin rays (Fang, 2003). It also exhibits one unique and unreversed trait with all
species of Danio: the presence of a median projection on the outer arm of the os suspensorium, and exhibits a
projection on the lateral face of the dentary, common to all species of Danio but not unique to that genus.
Though there are reductive characters that serve to unite C. margaritatus and D. erythromicron together
(see above) as a monophyletic grouping, we have discovered no additional apomorphic features that would
serve to unit this clade with any other species of Danio, or clade within Danio. Though the ML analysis places
C. margaritatus and D. erythromicron within Danio as the sister group to a clade containing D. nigrofasciatus,
D. rerio and D. albolineatus, there is only weak support for this grouping (bootstrap <50%) and such a grouping is not recovered in the MP analysis (Fig. 11). In addition, we have discovered no single apomorphic feature that would serve to unite all other species of Danio, exclusive of these two miniature species, together as
monophyletic (sensu Fang, 2003). As the characters proposed herein to unit C. margaritatus and Danio
together as a monophyletic group include the two characters proposed by Fang (2003) to define Danio we feel
that the most appropriate action is to recognise a broader concept of Danio, inclusive of Celestichthys.
Though accepting a broader concept of Celestichthys (including D. erythromicron) as the sister group to
Danio could be considered a feasible option, we feel that this approach would only unnecessarily inflate the
number of generic names within Cyprinidae. We therefore place Celestichthys in the synonymy of Danio.
Despite this nomenclatural change, the characters proposed by Roberts as diagnostic for Celestichthys still
serve to identify D. margaritatus from all other species of Danio. We reinterpreted these characters as diagnostic at the species level.
Acknowledgments
We are grateful to B. Brown, R. Arrindell (AMNH), D. Nelson (UMMZ), D. Catania (CAS), J. Maclaine, O.
Crimmen, P. Campbell (BMNH), A. Bentley (KU), B. Kuhajda (UAIC), M. Kottelat (CMK) and K. Lim
(ZRC) for the loan of specimens, Masaki Miya and Kenji Saitoh for providing specimens for molecular analyses, P. Mabee (USD), K. Tang and C. Dillman (SLU) for discussion and comments on an earlier draft of this
manuscript, and R. Britz (BMNH) and A. Gill (ASU) for critical review. Finally, we wish to thank T. Moritz
for providing us with excellent colour photographs of live individuals of D. margaritatus. This research was
supported by Saint Louis University and the National Science Foundation’s Cypriniformes Tree of Life Initiative as part of the NSF Assembling the Tree of Life Initiative (EF 0431326).
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