# A new species of Indo-Pacific fish, Canthigaster criobe, with comments on other Canthigaster (Tetraodontiformes: Tetraodontidae) at the Gambier Archipelago

Jeffrey T Williams, Erwan Delrieu-Trottin, S Planes

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Jeffrey T Williams, Erwan Delrieu-Trottin, S Planes. A new species of Indo-Pacific fish, Canthigaster criobe, with comments on other Canthigaster (Tetraodontiformes: Tetraodontidae) at the Gambier Archipelago. Zootaxa, 2012, 3523 (1), pp.80-88. 10.11646/zootaxa.3523.1.9. hal-04905624

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# A new species of Indo-Pacific fish, *Canthigaster criobe*, with comments on other *Canthigaster* (Tetraodontiformes: Tetraodontidae) at the Gambier Archipelago

Jeffrey T. Williams<fnref n="1" />, Erwan Delrieu-Trottin<fnref n="2" />, Serge Planes<fnref n="2" />

**Abstract.** A new species of the tetraodontid fish genus *Canthigaster* was discovered during a recent expedition to the Gambier Archipelago, French Polynesia. The new species, named *Canthigaster criobe* herein, is the only known species of *Canthigaster* having 12-14 brown stripes along the body (stripes beginning in front of the eye, extending along the body, and abruptly ending at the base of the caudal fin). It also has 17 pectoral rays, the origin of the anal fin inserts posterior to a vertical from the rear base of the dorsal fin and lacks spots on the caudal fin. *Canthigaster criobe* is currently known from a single specimen collected at the Gambier Archipelago. It appears to be most similar to the white-spotted *C. janthinoptera*, a wide-ranging, Indo-Pacific species, which also inhabits the Gambier Archipelago and to the Hawaiian endemic *C. jactator* forming a species complex that exhibits incomplete lineage sorting. Specimens of *C. axiologus*, or an undescribed but very similar sibling species, were also collected at the Gambier Archipelago. Molecular analysis of these samples reveals an affiliation with *C. axiologus* specimens collected from disjunct localities in the western Central Pacific. *C. axiologus* was not previously known to occur east of the Tonga Islands. Geographic range expansions are also reported for *C. rapaensis* and *C. amboinensis*.

**Key words:** taxonomy, Tetraodontidae, *Canthigaster*, new species, French Polynesia, Gambier Archipelago

## Introduction

The pufferfish family Tetraodontidae is divisible into two subfamilies, the Tetraodontinae and the Canthigasterinae. The latter subfamily, popularly known as tobies or sharpnose puffers, consists of a single genus, *Canthigaster* Swainson. These fishes are differentiated from the Tetraodontinae by a laterally compressed body, elongate pointed snout, erectile ridge of skin middorsally and midventrally, small gill opening, inconspicuous lateral line, 17 vertebrae, and small size. Their bold color patterns probably serve to advertise their repelling skin toxin (Eger & Starkus 1973).

Randall et al. (2008) summarized previous work on the genus and discussed the historical problems faced by taxonomists in distinguishing the species of *Canthigaster*.

<footnote n="1">¹ Division of Fishes, Department of Vertebrate Zoology, National Museum of Natural History, 4210 Silver Hill Road, Suitland, MD 20746, USA, e-mail williamsjt@si.edu</footnote>

<footnote n="2">² Labex “CORAIL”, USR 3278 CNRS – EPHE, Centre de Recherche Insulaire et Observatoire de l'Environnement (CRIOBE), Université de Perpignan, 52 Av. Paul Alduy - 66860 Perpignan cedex, France</footnote>
In addition to the uniformity of morphological characters among *Canthigaster* species,
Randall et al. (2008) found a high degree of genetic uniformity within the genus. As
noted by Randall et al. (2008), color pattern reigns as the primary character used to
distinguish the known species of *Canthigaster*. Randall et al (2008) described a new
species and resurrected a species from synonymy, resulting in the recognition of 35 valid
species of *Canthigaster*.

In September and October 2010, the authors collected fish specimens during an
expedition to the Gambier Archipelago, French Polynesia, using a variety of collecting
techniques and took tissue samples and photographs of voucher specimens of each fish
species collected. Among the specimens collected was a distinctively colored new species
of *Canthigaster*. The new species described herein increases the number of recognized
*Canthigaster* species to 36.

Geographic range expansions are also reported for *Canthigaster axiologus*, *C. rapaensis*, and *C. amboinensis*. These three species are recorded for the first time from the Gambier Archipelago.

Using the tissue samples obtained during the expedition, we attempted to genetically
differentiate the new species from its congeners using mitochondrial DNA sequences.

**Materials and Methods**

All specimens examined are deposited in the National Museum of Natural History,
Washington, D.C. (USNM).

The length of specimens is given as standard length (SL), measured from the median
anterior end of the upper dental plate to the base of the caudal fin (posterior end of the
hypural plate); body depth is the slightly oblique measurement from the origin of the
dorsal fin to the origin of the anal fin (the inflatable abdomen makes the maximum body
depth highly variable in species of *Canthigaster*); body width is measured at the base of
the pectoral fins; head length (HL) is taken from the front of the upper dental plate to the
dorsal end of the gill opening; orbit diameter is the greatest diameter of the unpigmented
skin over the eye, and interorbital width the least bony width; snout length is measured
from the upper dental plate to the nearest unpigmented cutaneous edge of the eye; length
of the gill opening is taken by inserting divider tips and spreading for the maximum firm
edges; caudal-peduncle depth is the least depth, and caudal-peduncle length the
horizontal distance between verticals at the rear base of the anal fin and the caudal-fin
base; lengths of fins are the length of the longest rays.

Morphometric data for the new species are presented in the descriptions as
proportional measurements of the SL or HL, rounded to the nearest 0.5. Counts of
pectoral rays include the short uppermost ray.

To conduct our genetic analysis, whole genomic DNA was extracted from fin clips
preserved in 96% EtOH. DNA extraction was performed using QIAxtractor (QIAGEN,
Crawley) according to manufacturer's protocols. A fragment of the mitochondrial DNA
control region of *Canthigaster* was amplified with the primers CANT-CR-F and CANT-
CR-R (Randall et al. 2008). PCR amplifications were performed on a Mastercycler® pro
(Eppendorf) thermocycler, in a final 25 µl reaction containing: 2.5 µl of the buffer
provided by the manufacturer (5 PRIME), 1.25 µl of MgCl2 (25 mM), 3 µl of dNTP (2
mM), 0.4 µl of each primer (10 mM), 5 U Taq DNA polymerase (5 PRIME), 2.0 mM, and
1-3 µl of total genomic DNA. The thermocycle procedure (35 cycles) was a modified hot-start touchdown PCR, with an initial soak at 94 °C for 3 min, followed by 35 cycles at 94°C for 25s, 52°C for 25s and 72°C for 1 min. The final phase of the procedure was a 3-min elongation period at 72 °C.

The result was visualized on ethidium-bromide stained agarose gels. Sequencing was
performed using an ABI3730 Automated Sequencer using the same primers. All samples
were sequenced in forward and reverse directions to ensure accuracy of nucleotide
designations. Sequences were analyzed using Geneious v5.4 (Drummond et al. 2009) and
aligned by eye with no ambiguities. All sequences are deposited in Genbank (Accession
numbers: JQ241772-JQ241781).

A 678 base-pair fragment of the mitochondrial control region was sequenced for comparative purposes among the three species (*C. axiologus*, *C. cyanospilota*, *C. coronata*) of the *coronata* complex (Randall et al. 2008), four other species found during the campaign and five specimens obtained from Genbank (Table 1) with a representative of the Tetraodontinae used as the outgroup.

Two different tree-building methods were used to construct branching diagrams.
Neighbor-joining (NJ) analysis was conducted using the software package MEGA 5
(Tamura et al. 2011). For the maximum-likelihood (ML) approach, the appropriate model
of nucleotide substitution was determined using jModeltest version 3.07 (Posada 2008).
The ML analysis was run on Phylogeny.fr server (Dereeper et al, 2008) with parameters
fixed according to jModeltest specifications. Confidence in topologies was evaluated
based on 1000 bootstrap replicates or posterior probability. The two analyses resulted in
identical tree topologies. The NJ tree is presented herein.

*Canthigaster criobe* new species
**Common Name: Striped toby**
(Figure 1, 2)

*Holotype:* USNM 400521, 38.7 mm SL when fresh (37.6 mm SL after preservation), Gambier Archipelago, Kouaku Island, on SE side of archipelago, small surge channel in outer reef; 23.22 degrees S, 154.88 degrees W; 15-20 m depth; field number GAM-2010-01; tissue sample number GAM-001; collected by S. Planes, J.T. Williams, E. Delrieu-Trottin, M. Kulbicki, P. Sasal. 29 September 2010.

**Diagnosis:** Dorsal rays 9; anal rays 9; pectoral rays 17; body depth between origins of dorsal and anal fins 2.6 in SL; head length 2.3 in SL; fleshy pigmented skin interorbital width 3.1 in HL; gill opening 8.2 in HL; origin of anal fin posterior to rear base of dorsal fin 3.1 in SL, the preanal length 1.2 in SL; longest dorsal ray 2.3 in HL; color in life: body pale tan with orangish brown stripes; head with uniformly orangish brown snout anteriorly, at middle of snout about 4 narrow, orangish brown stripes radiate posteriorly below eye, three stripes extend posteriorly from rear margin of eye, one stripe runs along ridge of erectile tissue on top of head, at deepest part of body there are about 14 narrow orangish brown stripes, middle five of those stripes continue to base of caudal fin where they abruptly end.
**Description:** Dorsal rays 9; anal rays 9; dorsal and anal rays branched, except the first; principal caudal rays 9, all branched but lowermost; a single small segmented upper and lower recurrent caudal ray; pectoral rays 17 (on right side; 16 on left, but left side appears to have been damaged earlier in life and healed, possibly losing a ray in the middle); vertebrae 17; mouth small, oval, and terminal, exposing about terminal half of upper dental plate; lips fleshy and papillose.

Body depth between origins of dorsal and anal fins 2.6 in SL; body moderately compressed, the width 1.6 in depth; head length 2.3 in SL; snout length 1.7 in HL; dorsal profile of snout slightly concave; eye diameter (unpigmented skin over eye) 3.9 in HL; bony orbit diameter 3.0 in HL; interorbital space slightly concave, the least bony interorbital width 3.7 in HL; gill-opening length 8.2 in HL; caudal-peduncle depth 2.3 in HL; caudal-peduncle length 2.7 in HL; predorsal length 1.3 in SL; dorsal-fin base 4.9 in HL; third dorsal ray usually longest, but adjacent rays nearly as long, 2.3 in HL; origin of anal fin below rear base of dorsal fin, the preanal length 1.2 in SL; anal-fin base 6.2 in HL; fourth anal ray longest, but adjacent rays nearly as long, 2.7 in HL; caudal fin truncate, 3.4 in SL; upper and lower portions of pectoral fins rounded, fin deeply incised in the middle; third pectoral ray longest, 2.5 in HL.

Skin with numerous small spinules directed posteriorly, each fitting into a longitudinal groove; skin smooth when stroked posteriorly, but spinules apparent when stroked anteriorly; spinules densely cover head, nape and belly, but are absent from the body immediately behind the pectoral-fin base and from body posterior to an imaginary line between origin of dorsal fin and origin of anal fin.

Color of holotype when fresh shown in Fig 1. Body pale tan with orangish brown stripes; head with uniformly orangish brown snout anteriorly, at middle of snout about 4 narrow, orangish brown stripes radiate posteriorly below eye, three stripes extend posteriorly from rear margin of eye, one stripe runs along ridge of erectile tissue on top of head, at deepest part of body there are about 14 narrow orangish brown stripes, middle five of those stripes continue to and end abruptly at base of caudal fin; lips brown; orangish brown coloration on snout becomes paler, but continues posteriorly across chest and abdomen as a diffuse orangish tint that reaches anus; fins unpigmented and translucent; base of dorsal fin with a poorly developed dark blotch.

Color of holotype in alcohol similar to life colors except stripes are dark brown on tan background.

**Etymology:** We name this species *criobe* after the Centre de Recherche Insulaire et Observatoire de l'Environnement (CRIOBE), Moorea, French Polynesia, in recognition of the laboratory's continuing support of marine research in French Polynesia. The species name is treated as a noun in apposition.

**Remarks:** Although it is known from only a single specimen, the longitudinally striped color pattern of *Canthigaster criobe* is unique within the genus *Canthigaster* and easily distinguishes *C. criobe* from all other described *Canthigaster* species. Although *C. janthinoptera* has its body covered with blue centered white spots, the orangish brown reticulations are similar in color to the stripes of *C. criobe*. In both of these species the base of the caudal fin has narrow brown lines extending onto the bases of the fin rays (Figs. 1, 3). Some specimens of *C. janthinoptera* have a few lines on the upper part of the
body (Fig. 3), but never a striped pattern like that of *C. criobe*. *Canthigaster criobe* and *C. janthinoptera*, along with the Hawaiian endemic *C. jactator* form a species complex within *Canthigaster*.

**Comments on other Canthigaster at the Gambier Archipelago.**

During the 2010 expedition to the Gambier Archipelago, we collected six additional species of *Canthigaster* including: *C. janthinoptera* (Bleeker, 1855), *C. amboinensis* (Bleeker, 1864), *C. axiologus* Whitley, 1931, *C. solandri* (Richardson, 1844), and *C. rapaensis* Allen and Randall, 1977. The sixth species, *C. bennetti* (Bleeker, 1854), was observed during the expedition and a single specimen was collected on the last day, but was not tissue sampled or retained as a voucher.

The discovery and collection of several specimens of *Canthigaster axiologus* (USNM 400503, 400522, 400533, 400534) at the Gambier Archipelago was surprising as the geographically closest known population of *C. axiologus* is at the Tonga Islands over 4,000 km to the west. There is a slight difference in coloration in that the Gambier specimens have 0-1 yellow spots (versus numerous yellow and blue spots) on the caudal peduncle and no spots (versus numerous spots at least basally) on the caudal fin (Fig. 4). The Gambier specimens of *C. axiologus* are currently under study to determine if this isolated population has evolutionarily diverged from the western Pacific form and warrants recognition as a distinct species (see below).

The collection of a specimen (Fig. 5) of *Canthigaster rapaensis* (USNM 400531) at the Gambier Archipelago represents a geographic range extension from Rapa, Austral Islands. This is the first record of *C. rapaensis* occurring at a locality other than Rapa.

The single specimen (Fig. 6) of *Canthigaster amboinensis* (USNM 399907), taken on the eastern side of the Gambier Archipelago on the outer reef slope at a depth of 10-25 m, represents the first report of the species from the Gambier Archipelago. There is one record of this species from the Galapagos (presumably a waif), but previously in French Polynesia the easternmost reports of this species were from the Society Islands and the Marquesas.

**Molecular analysis**

The topology of our NJ tree (Fig. 7) is similar to that of Alfaro et al. (2007) based on 12S and 16S sequences and that of Randall et al. (2008) based on the control region. In our Fig. 7, the two *C. cyanospilota* do not group together. The Randall et al. (2008) tree was supported by a low bootstrap probability (53) in the Randall et al. (2008) study and our study includes additional species.

*Canthigaster criobe* appears to belong to a group of closely related species including *C. criobe*, *C. janthinoptera* and *C. jactator* (Jenkins, 1901). Alfaro et al. (2007) reported that the Hawaiian endemic *C. jactator* and the Indo-Pacific *C. janthinoptera* were genetically very similar. Incomplete lineage sorting or mitochondrial introgression might explain this finding as these processes have been reported to produce similar phylogenetic patterns (Avise and Ball 1990; Morando et al. 2004). Mitochondrial introgression, as discussed by Allendorf et al. (2001) and McGuire et al (2007),
describes two divergent haplotypes of geographically separated taxa becoming very
similar in intermediate areas where the taxa overlap in distribution. The mitochondrial
haplotypes of the *C. janthinoptera* sampled at the Gambier Archipelago are similar to
those of *C. criobe*, currently known only from the Gambier Archipelago, but that of *C.
jactator* also is similar, even though it is a Hawaiian endemic found over 5,500 km to the
northwest. This similarity with a geographically separated taxon suggests incomplete
lineage sorting instead of mitochondrial introgression. The speciation events may have
occurred so recently that the neutral genes have not had time to accumulate independent
mutations (McCartney et al. 2003, Shultz et al. 2007, McGuire et al. 2007, Rocha et al.
2007). The absence of lineage partitioning coinciding with distinctive species-specific
color pattern characters has been reported in other genera of Osteichthyes in addition to
that reported for *Canthigaster* (Randall et al. 2008), such as: *Centropyge* (Pomacanthidae,
Bowen et al. 2006), *Halichoeres* (Labridae, Rocha 2004) and *Scaphirhynchus*
(Acipenseridae, Campton et al., 2000). *Canthigaster criobe* appears to be another
example of incomplete lineage sorting in the subfamily Canthigasterinae. Our analysis
provides additional support to the Randall et al. (2008) hypothesis that "this group
undergoes fast colour/morphological differentiation combined with slow mtDNA
evolution."

Molecular analysis groups the four Gambier specimens of *C. axiologus* with the western Pacific *C. axiologus* lineage (Fig. 7). Yet, a well-supported differentiation can be observed for two of the specimens. Considering the slight difference in the coloration and the geographic isolation of the Gambier population, we consider this population to be in an early stage of evolutionary differentiation. Other populations of this species may occur between Tonga and Gambier, but the species has not been reported from the intervening geographic regions.

**Acknowledgements**

We thank Diane Pitassy and Jerry Finan, National Museum of Natural History, for
specimen processing. Particular thanks to the crew of the M/V Claymore II for their
invaluable assistance during the Gambier Expedition. We thank Tea Frogier and Pierre
Mery for their support of the Coralspot project at the Gambier Archipelago. The
Coralspot expedition was funded by the "Contrat de projet Etat-Polynésie", by the ANR
"IMODEL" and the French Ministry for Environment, Sustainable Development and
Transport (MEDDTL). The first author's travel to Moorea to participate in the expedition
was funded by a grant from the Leonard P. Schultz Fund (Division of Fishes, National
Museum of Natural History).

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<figure><img src="image_2.png" /><figcaption>**FIGURE 1.** Holotype of *Canthigaster criobe*, USNM 400521, 38.7 mm SL, Gambier Archipelago (J.T. Williams).</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**FIGURE 2.** Radiograph of holotype of *Canthigaster criobe*, USNM 400521, 38.7 mm SL, Gambier Archipelago, showing skin spinule pattern (J.T. Williams).</figcaption></figure>
<figure><img src="image_4.png" /></figure>

**FIGURE 3.** *Canthigaster janthinoptera*, USNM 400502, 50.6 mm SL, Gambier Archipelago (J.T. Williams).

<figure><img src="image_5.png" /></figure>

**FIGURE 4.** *Canthigaster axiologus*, USNM 400503, 73.9 mm SL, Gambier Archipelago (J.T. Williams).
<figure><img src="image_6.png" /></figure>

**FIGURE 5.** *Canthigaster rapaensis*, USNM 400531, 71.4 mm SL, Gambier Archipelago (J.T. Williams).

<figure><img src="image_7.png" /></figure>

**FIGURE 6.** *Canthigaster amboinensis*, USNM 399907, 81.6 mm SL, Gambier Archipelago (J.T. Williams).
<figure><img src="image_8.png" /><figcaption>**FIGURE 7.** Neighbor-Joining tree based on K2P model of sequence evolution (with 1,000 bootstrap replicates) for the subfamily Canthigasterinae, with *Arothron meleagris* as the outgroup.</figcaption></figure>
<table><caption>**TABLE 1.** Specimens included in the molecular analysis for this study. Asterisk (*) denotes specimens collected and sequenced by us.</caption><thead><tr><th>Species</th><th>Reference for the sequences</th><th>GeneBank Accession Number</th><th>Sampling location</th></tr></thead><tbody><tr><td>Canthigaster axiologus</td><td></td><td>AP006743</td><td>Japan</td></tr><tr><td>Canthigaster axiologus</td><td>Randall et al., 2008</td><td></td><td>Australia</td></tr><tr><td>Canthigaster coronata</td><td>Randall et al., 2008</td><td></td><td>Hawaii</td></tr><tr><td>Canthigaster coronata</td><td>Randall et al., 2008</td><td></td><td>Hawaii</td></tr><tr><td>Canthigaster cyanospilota</td><td>Randall et al., 2008</td><td></td><td>La Réunion</td></tr><tr><td>Canthigaster cyanospilota</td><td>Randall et al., 2008</td><td></td><td>La Réunion</td></tr><tr><td>Canthigaster axiologus*</td><td></td><td>JQ241775</td><td>Gambier archipelago</td></tr><tr><td>Canthigaster axiologus*</td><td></td><td>JQ241776</td><td>Gambier archipelago</td></tr><tr><td>Canthigaster axiologus*</td><td></td><td>JQ241777</td><td>Gambier archipelago</td></tr><tr><td>Canthigaster axiologus*</td><td></td><td>JQ241778</td><td>Gambier archipelago</td></tr><tr><td>Canthigaster solandri*</td><td></td><td>JQ241779</td><td>Gambier archipelago</td></tr><tr><td>Canthigaster jactator</td><td></td><td>AP011911</td><td>Hawaii</td></tr><tr><td>Canthigaster janthinoptera*</td><td></td><td>JQ241773</td><td>Gambier archipelago</td></tr><tr><td>Canthigaster janthinoptera*</td><td></td><td>JQ241774</td><td>Gambier archipelago</td></tr><tr><td>Canthigaster criobe*</td><td></td><td>JQ241772</td><td>Gambier archipelago</td></tr><tr><td>Canthigaster amboinensis*</td><td></td><td>JQ241780</td><td>Gambier archipelago</td></tr><tr><td>Canthigaster rapaensis*</td><td></td><td>JQ241781</td><td>Gambier archipelago</td></tr><tr><td>Canthigaster rivulata</td><td></td><td>AP006744</td><td>Japan</td></tr><tr><td>Canthigaster valentini</td><td></td><td>AP011912</td><td>Tonga</td></tr><tr><td>Arothron meleagris</td><td></td><td>AP011931</td><td>NA</td></tr></tbody></table>