On the validity of the Cirrhitid fish genus Itycirrhitus.
The 33 species of fishes of the perciform family Cirrhitidae, popularly known as hawkfishes, are found in the tropical and subtropical Indo-Pacific region, except for three in the Atlantic and three in the eastern Pacific (two of which, Cirrhitichthys oxycephalus and Oxycirrhites typus, are range extensions from the Indo-Pacific). Cirrhitid fishes all have X dorsal spines, III anal spines, 14 pectoral rays (the lower five to seven rays unbranched and thickened), two flat opercular spines, a serrate preopercle, cycloid scales, one to several cirri at the tip of each membrane of the dorsal spines, and no swim bladder. Most species occur in shallow water on coral reefs or rocky substrata, often in areas exposed to wave action. When in a surge zone, they use their thickened lower pectoral rays to wedge themselves in cracks in the reef or within branches of coral. Hawkfishes feed mainly on benthic crustaceans and occasionally on small fishes. Exceptions are Cyprinocirrhites polyactis that feeds well above the substratum on zooplankton, and O. typus that often makes short forays from the bottom to prey on the larger animals of the demersal plankton. At least some of the species of the family are protogynous hermaphrodites (Sadovy & Donaldson 1995).
The generic classification of the Cirrhitidae has a long and confused history, Gunther (1860) recognized eight genera in the family. A century and 11 cirrhitid publications later, Schultz in Schultz & collaborators (1960) listed 13 genera, including Isobuna and Serranocirrhitus, now known to be serranids. Randall (1963) included 10 genera and 34 species in his revision of the family. Randall (2001) again revised the genera of cirrhitids, adding three new monotypic genera from species formerly classified in Cirrhitus. One of these, Itycirrhitus, was described for a small species, Cirrhitus wilhelmi, from Easter Island. This species was first described by Lavenberg & Yanez (1972) and later reclassified in Amblycirrhitus by Pequeno (1989). Randall (1999) extended the range to the Pitcairn Islands. The key to genera of Randall (2001) is reproduced here.
After the separation of Oxycirrhites in the key, mainly by its snout length, and Cyprinocirrhites by its lunate caudal fin, the characters leading to the other genera are less incisive, in particular those leading to Itycirrhitus. This study was initiated to determine if an analysis of DNA sequence data from species of cirrhitid fishes will support the generic classification. Specifically we collected mitochondrial sequence data to compare Itycirrhitus wilhelmi (Fig. 1) with the similarly colored species of Cirrhitops, C. fasciatus (Fig. 2) and C. mascarenensis. Additionally, we investigated the level of genetic divergence between the two monotypic genera Notocirrhitus and Neocirrhites which are closely grouped in the key.
MATERIALS AND METHODS
For genetic analysis a 1cm fin clip was obtained from three specimens of Itycirrhitus wilhelmi from Easter Island. DNA was isolated using the modified HotSHOT method (Meeker et al. 2007; Truett et al. 2000). We amplified approximately 660 bp the mitochondrial cytochrome c oxidase subunit I (COI) gene and approximately 750 base pairs of cytochrome b (cyt b) gene using the primers employed in Randall and Schultz (2009). Polymerase chain reactions (PCR) were carried out in a 20 [micro]l volume containing 5-20 ng of template DNA, 0.4 [micro]M of each primer, 10 [micro]l of the premixed PCR solution BioMix Red (Bioline Inc., Springfield, NJ, USA), and deionized water to volume. After an initial 7 min denaturation at 95[degrees]C, each of 35 cycles consisted of denaturation for 30 s at 94[degrees]C, annealing at 56[degrees]C for COI and 58[degrees]C for cyt b for 30 s, and extension at 72[degrees]C for 45 s with a final 10 min extension at 72[degrees]C. Amplification products were purified using 0.75 units of Exonuclease I: 0.5 units of Shrimp Alkaline Phosphatase (ExoSAP, USB, Cleveland, OH, USA) per 7.5 [micro]l PCR products at 37[degrees]C for 60 minutes, followed by deactivation at 80[degrees]C for 15 minutes. DNA sequencing was performed with fluorescentlylabeled dideoxy terminators on an ABI 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) at the University of Hawai'i Advanced Studies of Genomics, Proteomics and Bioinformatics sequencing facility. Sequences for each locus were aligned, edited, and trimmed to a common length using the DNA sequence assembly and analysis software Geneious Pro 5.0 (Biomatters, LTD, Auckland, NZ). In all cases, alignment was unambiguous with no indels or frameshift mutations. Unique sequences were deposited in Gen-Bank (http://www.ncbi.nlm.nih.gov/genbank/; see Table I for accession numbers).
Table I. Mitochondrial cytochrome oxidase I (COI) and cytochrome b (cyt b) sequences used in this study. Species name, Barcode of Life Data systems (http://www.boldsystems.org/views/login.php, Ratnasingham & Hebert 2007) sequence identification numbers and GenBank accession numbers (in italics; http://www.ncbi.nlm.nih.gov/genbank/), and museum voucher IDs (in parentheses) are listed. Sequences generated in this study are in bold. Number of individuals (if > 1) in which haplotype was detected is listed in parentheses. Species COI cyt b Amblycirrhitus tzaib036-06 (hlc-12147) pinos A. bimacula JX645648 eu684136, JX645657 Cirr/iitops EU684132, EU684133 (bpbm EU684137, EU684138 (bpbm fasciatus 40485, 40888) 40485, 40888) tzaib307-06 (hlc-12324) gbgc10204-09, gbgc10205-09 C. mascarenensis EU684134, EU684135 (bpbm EU684139, EU684140 (bpbm 40889-90) 40889-90) Cirrhitus JX645649, JX645650 (2) JX645661, JX645662, pinnulatus JX645663 Itycirrhitus JX645651,JX645652(2) JX645658, JX645659, wilhelmi JX645660 Neocirrhites tzaic178-05 (hlc-10878) JX645664, JX645665, armatus tzaib505-06 (hlc-13128) JX645666 tzaic695-06 (hlc-11757) JX645653, JX645654, JX645655 Notocirrhitus JX645656(2) (ma655017, splendens ma655018) Paracirrhites tzaib143-06 (hlc-12066) arcatus tzaib144-06 (hLC-12067) tzaib145-06 (hLC-12068) tzaib147-06 (hLC-12070) tzaib867-07 (hLC-15197) P. forsteri dsfsg451-11(adc11_214.7 dsfsg566-11(adc11_214.7 #2) #4) Epinephelus HQ174825, HQ174826 HQ174838, HQ174839 lanceolatus Plectropomus gbgc1814-06, gbgc1208-06, AY963555, AY963556 leopardus fscs615-07
Maximum Likelihood (ML) trees were constructed using the default settings implemented in the program MEGA 5.05 (Tamura et al. 2007). COI and cyt b sequences of Cirrhitops fasciatus (N = 5, Hawai'i) and Cirrhitops mascarenensis (N = 2, Mauritius), were obtained from GenBank (Randall & Schultz 2009, Table I). For inclusion in the phylogenetic
tree COI sequences from representatives of three genera in the family Cirrhitidae were downloaded from the Barcode of Life Data (BOLD) Systems 2.5 website (www.barcodinglife.org) (Table I). Corresponding cyt b sequences were available from GenBank for only Amblycirrhitus bimacula. Additionally, tissue samples of Amblycirrhitus bimacula (N = 1, Hawai'i), Cirrhitus pinnulatus (N = 3, Guam), Neocirrhites armatus (N = 3, Guam), and Notocirrhitus splendens (N = 2, Kermadec Islands, New Zealand) were obtained and sequenced at both loci and deposited in GenBank (Table I). Available voucher specimens and tissues (Table I) are deposited in the: Biodiversity Institute of Ontario (HLC), Guelph, Canada; Bernice P. Bishop Museum (BPBM), Honolulu, USA; South African Institute of Aquatic Biodiversity (ADC), Grahamstown, South Africa; and the Auckland Museum (MA), Auckland, New Zealand.
Phylogenetic trees were rooted with two species from the family Serranidae (Table I, Epinephelus lanceolatus and Plectropomus leopardus). For analysis, all COI and cyt b sequences were trimmed to 557 bp and 671 bp, respectively. Bootstrap support values were calculated using default settings with 1000 replicates. For comparison we ran a Bayesian Markov Chain Monte Carlo (MCMC) analysis as implemented in the program MRBAYES 3.1.1 (Huelsenbeck & Ronquist 2001). We employed default settings and ran simulations for 1,000,000 generations until the standard deviation of split frequencies were below 0.01 as recommended in the MRBAYES manual. Average percent divergence (d) between genera was calculated in ARLEQUIN 3.5 (Excoffier & Lischer 2010).
Our comparisons of Itycirrhitus wilhelmi and the two species of the genus Cirrhitops (C. fasciatus and C. mascarenensis) at two mitochondrial genes confirmed the distinction of these two genera. Across all COI sequences analyzed we detected 197 variable nucleotide sites. There were two haplotypes in three I. wilhelmi specimens. The average pairwise difference between C. fasciatus and C. mascarenensis was 39 bp (d = 7%), while I. wilhelmi differs from the two species of Cirrhitops by an average of 85 bp (d = 15%). All generic level comparisons resulted in average base pair differences that range between 82 bp (d = 15%; Cirrhitops vs. Neocirrhites) and 113 (d = 20%; Neocirrhites vs. Amblycirrhitus). The exception is I. wilhelmi and Neocirrhites armatus which differ by an average of only 47 bp (d = 8%).
Analyses of cyt b sequences were similarly robust. We detected 256 variable nucleotide sites. There were three haplotypes in three I. wilhelmi specimens. The average pairwise difference between C. fasciatus and C. mascarenensis was 75 bp (d = 11%). In contrast, the average pairwise differences between I. wilhelmi and the two species of Cirrhitops were 140 and 132 respectively (d = 21% and 20%, respectively). Generic level comparisons resulted in average base pair differences that ranged between 84 bp (d = 13%; Neocirrhites vs. Itycirrhitus) and 138 bp (d = 21%; Amblycirrhitus vs. Cirrhitops).
After repeated attempts, we were able to amplify the Notocirrhitus splendens specimens at COI but not cyt b. When comparing N. splendens with Neocirrhites armatus, which are closely grouped in the key, we found an average pairwise difference between these monotypic genera of 84 bp (d = 15%). A similar level of divergence was detected between N. splendens and I. wilhelmi (87 bp, d = 16%), which are also closely grouped in the key.
Phylogenetic analyses revealed a single tree topology for both mitochondrial markers with only the relative positions of the outgroups varying (Fig. 3). Both Maximum Likelihood and Bayesian analyses indicated that N. armatus is more closed related to I. wilhelmi than I. wilhelmi is to the members of the genus Cirrhitops.
Phylogenetic analyses indicate strong concordance between our molecular data and generic level classifications within the family Cirrhitidae (see key above). Species within the same genus always grouped together with intragenetic divergences ranging from 7-14% at COI. The genus Itycirrhitus was described by Randall (2001) based on seemingly small morphological differences. Here we report sequence data that support the generic level designation for Itycirrhitus wilhelmi. This species is 15% divergent at COI from either of the species of the genus Cirrhitops; a level of divergence common among the other genera of the family (8 to 20%). Our finding of a similar level of divergence between Neocirrhites armatus and Notocirrhitus splendens supports the designation of these monotypic genera.
Interestingly, I. wilhelmi did not cluster in the phylogenetic tree with the morphologically similar Cirrhitops (Figs 1-2). Instead, despite significant morphological differences, Neocirrhites armatus was found to be only 8% divergent from I. wilhelmi, a similar level of divergence between the two species of Cirrhitops (7%). In this case we found surprising incongruence between the morphological characters used to delineate these species and the level of molecular divergence detected.
We thank the following for providing tissue samples for this study: Dr. Gerald R. Allen, Dr. Alfredo Cea, Dr. Mark V. Erdmann, Daniel Pelicier, and Dr. Gordon W. Tribble. Support for the first author was provided by a grant/cooperative agreement from the National Oceanic and Atmospheric Administration, Project R/HE-1, which is sponsored by the University of Hawaii Sea Grant College Program, SOEST, under Institutional Grant No. NA09OAR4170060. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its subagencies. UNIHI-SEAGRANT-JC-09-48. This is contribution #1516 from the Hawai'i Institute of Marine Biology and #8739 from the School of Ocean and Earth Science and Technology.
Received: 6 June 2012 - Accepted: 17 September 2012
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Key to the Genera of Cirrhitidae la. Snout not elongate, its length about 2 2.8-4.1 in head length; body not slender, the depth 2.0-3.4 in SL; canine teeth in jaws markedly longer than inner villiform teeth, those at front of upper jaw and side of lower jaw enlarged lb. Snout elongate, its length 1.85-2.0 Oxycirrhites in head length; body slender, the depth 4.4-4.6 in SL; canine teeth in jaws only slightly longer than inner villiform teeth and nearly uniform in size 2a. Caudal fin rounded, truncate, or 3 slightly emarginate; dorsal soft rays 11-15; snout not short, its length 2.7-3.SL 2b. Caudal fin lunate; dorsal soft rays Cyprinocirrhites 16 or 17; snout short, its length 3.6-4.1 in head length 3a. Small scales on cheek in more than 4 12 rows 3a. Rows of large scales on cheek 4-6 8 (small scales also usually present) 4a. Lower 7 pectoral rays unbranched and 5 thickened; first 2 supraneural bones in space before second neural spine; more than 40 cirri in 2 series on posterior flap of anterior nostril 4b. Lower 6 pectoral rays unbranched and 6 thickened; first 3 supraneural bones in space before second neuralspine; fewer than 15 cirri on posterior flap of anterior nostril 5a. Supraorbital ridge high, continuing Cristacirrhitus more than half eye diameter posterior to orbit; lower opercular spine acute, forming an angle of 45[degrees] or less; no scales in interorbital space; pectoral fins reaching slightly beyond a vertical at tips of pelvic fins; body depth 3.1-3.35 in SL 5b. Supraorbital ridge low and not Cirrhitus continuing posterior to eye; lower opercular spine forming an angle of 90[degrees]; a V-shaped band of scales in posterior half of interorbital space; pectoral fins short, not reaching a vertical at tips of pelvic fins; body depth 2.6-3.1 in SL 6a. Dorsal soft rays 13; three-fourths Neocirrhites or more of preopercular margin coarsely serrate; palatine teeth absent; body very deep, the depth 2.0- 2.4 in SL, and very compressed, the width 2.9-3.1 in depth; longest pectoral rays not extending beyond a vertical at pelvic-fin tips. 6b. Dorsal soft rays 11-12; about upper 7 half of preopercular margin finely or coarsely serrate; palatine teeth present; body not very deep, the depth 2.6-3.3 in SL, and not very compressed, the width 1.9-2.3 in depth; longest pectoral rays extending beyond a vertical at pelvic-fin tips 7a. Upper margin of preopercle finely Notocirrhitus serrate (25 or more serrae); exposed end of posttemporal finely serrate; first (most medial) branchiostegal ray strongly curved and nearly parallel with second ray; gill membrane across throat naked; scales on cheek separated from serrate edge of preopercle by a broad naked zone crossed by irregular sensory channels (may show as ridges); no scales on snout; scales ventrally on chest extremely small; 3 rows of large scales above lateral line in middle of body; dorsal soft rays 12 7b. Upper margin of preopercle coarsely Itycirrhitus serrate (fewer than 13 serrae); exposed end of post-temporal with 3-5 serrae; first (most medial) branchiostegal ray nearly straight and not parallel to second ray; gill membrane across throat scaled; scales on cheek extending to base of preopercular serrae; scales on snout extending to below anterior nostrils; scales ventrally on chest one-half or more size of scales on side of body; 4 rows of large scales above lateral line in middle of body; dorsal soft rays 12-14 (rarely 12 or 14) 8a. Rows of large scales above lateral Paracirrhites line to base of spinous portion of dorsal fin 5; a single cirrus from membrane near tip of each spine of dorsal fin; membranes between longest dorsal spines incised at most one-fifth spine length; palatine teeth absent 8b. Rows of large scales above lateral line to base of spinous portion of dorsal fin 3 or 4; a tuft of cirri from membrane near tip of each spine of dorsal fin; membranes between longest dorsal spines incised one-third or more of spine length; palatine teeth present or absent 9 9a. Palatine teeth absent; maxilla Isocirrhitus reaching to or beyond a vertical through middle of eye; dorsal spines short, the longest 2.9-3.2 in head length; dorsal profile of head convex; lower 5 pectoral rays unbranched; interorbital fully scaled; snout not pointed; preorbital without a free posterior margin 9b. Palatine teeth present; maxilla not 10 reaching a vertical through middle of eye; dorsal spines 1.7-3.0 in head length; dorsal profile of head straight to slightly convex; lower 5 to 7 pectoral rays unbranched; interorbital scaled or naked; snout pointed or not pointed; preor-bital with or without a free posterior margin. 10a. Dorsal soft rays 14-15 (rarely 15); Cirrhitops first 2 pectoral rays unbranched; snout not pointed, the dorsal profile from interorbital to upper lip convex; interorbital naked 10b. Dorsal rays 11-13 (rarely 13); first 11 pectoral ray unbranched, second branched; snout pointed, the dorsal profile straight; interorbital scaled or naked 11a. Preopercular margin finely serrate; Ambfycirrhitus first 2 supraneural bones in space before second neural spine; preorbital without a free hind margin; interorbital scaled; first dorsal soft ray not produced into a filament; lower 5 (rarely 6) pectoral rays unbranched 11b. Preopercular margin coarsely Cirrhitichthys serrate; all 3 supraneural bones in space before second neural spine; preorbital with a free hind margin for one-fourth to one-half distance from lower edge to eye; interorbital not scaled; first dorsal soft ray usually produced into a filament; lower 6 or 7 pectoral rays unbranched
Michelle R. Gaither (1-2) and John E. Randall (3)
1) California Academy of Sciences, 55 Music Concourse Dr., San Francisco, CA 94118, USA. E-mail: email@example.com 2) Hawai'i Institute of Marine Biology, P.O. Box 1346, Kaneohe, HI 96744, USA. 3) Bishop Museum, 1525 Bernice St., Honolulu, HI 96817-2704, USA. E-mail: firstname.lastname@example.org.
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|Author:||Gaither, Michelle R.; Randall, John E.|
|Publication:||aqua: International Journal of Ichthyology|
|Date:||Oct 15, 2012|
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