Ocean Sci. J. (2012) 47(2):147-153 http://dx.doi.org/10.

1007/s12601-012-0014-6

Available online at www.springerlink.com

Note

Morphometric and Molecular Data on Two Mitochondrial Genes of a Newly Discovered Chimaeran Fish (Hydrolagus melanophasma, Chondrichthyes)
Jos De La Cruz-Agero1*, Francisco Javier Garca-Rodrguez1, Vctor Manuel Cota-Gmez1, Felipe Neri Melo-Barrera2, and Rogelio Gonzlez-Armas3
1

Coleccin Ictiolgica, Centro Interdisciplinario de Ciencias Marinas, Instituto Politcnico Nacional, Baja California Sur 23096, Mexico 2 Lab de Dinmica Poblacional, Centro Interdisciplinario de Ciencias Marinas, Instituto Politcnico Nacional, Baja California Sur 23096, Mexico 3 Lab de Ictioplancton, Centro Interdisciplinario de Ciencias Marinas, Instituto Politcnico Nacional, Baja California Sur 23096, Mexico
Received 21 August 2011; Revised 27 March 2012; Accepted 29 April 2012 KSO, KIOST and Springer 2012

Abstract Fresh and preserved (type material) specimens of the black ghost chimaera Hydrolagus melanophasma were compared for morphometric characteristics. A molecular comparison was also performed on two mitochondrial gene sequences (12S rRNA and 16S rRNA gene sequences). While significant differences in measurements were found, the differences were not attributable to sexual dimorphism or the quality of the specimens, but to the sample size and the type of statistical tests. The result of the genetic characterization showed that 12S rRNA and 16S rRNA genes represented robust molecular markers that characterized the species. Key words Holocephali, genetics, DNA, dimorphism, Baja California Sur

1. Introduction
The family Chimaeridae (Chondrichthyes: Holocephali) is the most diverse of the three families in the Chimaeriformes order. This family includes two genera: the Chimaera with eleven described species, and the Hydrolagus with twentytwo described species (Kemper et al. 2010), sixteen of which have been found in the Pacific Ocean (Andrade and Pequeo 2006; James et al. 2009). Of these, only six live in the Eastern Pacific: two are considered endemic to the Galapagos Islands (the white-spot ghost shark Hydrolagus
*Corresponding author. E-mail: jcruz@ipn.mx

alphus Quaranta, Didier, Long and Ebert, 2006; and the Galpagos ghost shark Hydrolagus mccoskeri Quaranta, Didier, Long and Ebert, 2006), the other two are found only in the coasts of Chile and Peru (bigeye chimaera Hydrolagus macrophthalmus de Buen, 1959; and the pallid chimaera Hydrolagus pallidus Hardy and Stehmann, 1990), and the remaining, spotted ratfish Hydrolagus colliei (Lay and Bennett 1839) and black ghost chimaera Hydrolagus melanophasma James, Ebert, Long and Didier, 2009, exclusively reside in the northern portion of the Eastern Pacific. The latter species, H. melanophasma, whose distribution is known only around the coasts of California, USA and Baja California, Mexico, including the Gulf of California, was described less than three years ago based on adult male specimens that had been preserved for more than thirty years in two museums in the United States of America. Hydrolagus melanophasma can be distinguished from its congeners by its long curved dorsal spine whose length exceeds the height of the dorsal fin. The second dorsal fin is long and of uniform height along its length; the large pectoral fins extend beyond the insertion of the pelvic fins. The males have an intromittent organ which is trifurcated, and the trifurcated part is at least a quarter of the organs total length, and the body is uniformly black or dark brown (James et al. 2009).

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Chimaeran fish is rarely collected and deposited in museums, particularly from the species H. melanophasma (its description is limited to the six known male specimens). There is no information available from fresh specimens of this species, particularly for females, to our knowledge. Therefore, the objective of this brief report is to provide new morphometric and molecular data for this rare and poorly known chimaeroid species. We compared the morphometric characters of fresh specimens of both sexes with respect to those used in the description and diagnosis of H. melanophasma (James et al. 2009). We also collected molecular data for this new kind of chimaera, from two mitochondrial gene sequences (12S rRNA and 16S rRNA). We chose these genes since mitochondrial DNA (mtDNA) can be used as a DNA-based taxonomic system (Tautz et al. 2002). These genes sequences have been proven to be a powerful phylogenetic tool for studies of vertebrates (Douady et al. 2003), and can be used to obtain the best match with the sequence data (except COI sequences) used in chimaeran studies (e.g. Inoue et al. 2010; Licht et al. 2012).

2. Materials and Methods

Morphological measurements were taken to the nearest millimeter according to the criteria of James et al. (2009). A total of 37 body measurements were obtained, corresponding to those used for the description of a new species. Morphometric measurements were presented as a proportion to the body length (% BDL). The data relating to measurements and their abbreviations are shown in Table 1. We used Friedmans test (FT, a non-parametric test used to compare observations repeated on the same subjects belonging to three or more paired groups) to compare the fresh and preserved specimens (type material) in terms of morphometric measurements. Also called non-parametric randomized block analysis of variance, FT makes no assumptions about the distribution of the data (e.g. normality or equality of variance) (Zar, 2009) as opposed to parametric repeated measures ANOVA or a paired t-test. Total genomic DNA extraction, from pectoral muscle tissue samples of each specimen, was performed using a commercial extraction kit (Qiagen) following the protocol recommended by the manufacturer. We amplified a fragment of 12S rRNA gene and one 16S rRNA gene of mitochondrial DNA (mtDNA) by polymerase chain reaction (PCR). Two sets of primers, L1091 and H1478 (Kocher et al. 1989), and 16Sar-L and 16Sbr-H (Palumbi

1996), were used for 12S rRNA and 16S rRNA respectively. In both cases, the volume of each reaction was 35 l containing: 1X PCR Buffer (Invitrogene), 0.2 mM dNTP mix, 0.48 mM of each primer, 4.0 mM MgCl2, and 2.5 U of Taq DNA polymerase (Invitrogene). The thermocycler conditions were similar to both genes: 2 minutes at 94 C, followed by 30 cycles, each of which consisted of 1 minute at 94 C, 1 minute at 60 C, and 2 minutes at 72 C. A final extension was performed for 4 minutes at 72 C. PCR products were purified and sequenced using forward and reverse primers for each gene (Macrogen, Seoul, Korea). The obtained sequences were arranged and edited using Sequencher ver. 4.5. Subsequently, the alignment and genetic characteristics of the sequences were obtained with MEGA ver. 4 (Tamura et al. 2007). The sequences were deposited in the GenBank (accession numbers: HQ645967HQ645968 for 16S rRNA, and HQ645969 for 12S rRNA sequence). We deposited only one 12S rRNA sequence because we found only one haplotype. 12S rRNA and 16S rRNA sequences of other species of the genera Hydrolagus and Chimaera (outgroup) were obtained from Genbank to assess the levels of divergence between specimens of H.

Fig. 1. Distribution map of the eastern north Pacific region for the Hydrolagus melanophasma specimens analyzed. Filled rectangles are the locations of the type material of James et al. (2009) and filled circle is the location of the present specimens (CICIMAR-CI 7102, CICIMAR-CI 7103). Map modified from James et al. (2009)

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Fig. 2. Specimens of the fish chimaera black ghost chimaera Hydrolagus melanophasma photographed fresh and deposited in the Ichthyological Collection of CICIMAR-IPN. A) Female, 662 mm BDL (CICIMAR-CI 7102). B) Male, 623 mm BDL (CICIMAR-CI 7103)

melanophasma and the other chimaeran species. The inter and intra species divergence was determined from genetic distances based on the Kimura-2 parameter model. A tree for each gene was generated by using the Neighbour Joining procedure (Saitou and Nei 1987) from 1000 replications. All analysis was performed using MEGA Ver 4 (Tamura et al. 2007). Specimens of H. melanophasma cataloged in the Ichthyological Collection of CICIMAR-IPN (http://coleccion. cicimar.ipn.mx) were collected in the area of Cabo San Lucas, Baja California Sur, Mexico (Fig. 1), by fishermen of a sport fishing fleet, who found the floating dead specimens. The specimens were labeled with the numbers 7102 for female (Fig. 2A) and 7103 for male (Fig. 2B), and preserved using standard curatorial procedures. Additional samples of muscle tissue were deposited in the CIs tissue collection, which are available upon request.

3. Results and Discussion

Friedmans test (FT) was used to compare data for each specimen (n=4) for all measurements, except for BDL and clasper measurements: CLT, CLM, CLL and FTL (see Table 1 for abbreviations of body measurements). The FTs Q statistics were 7.81 (critical) and 8.24 (observed) (P<0.04, =0.05; d.f.=3). The P-value suggested that the probability of making an error in rejecting the null hypothesis was lower than 4.12%. The null hypothesis that there was no

difference among specimens was then rejected, i.e., the samples were significantly different. This conclusion indicates the probable influence of preservative (formaldehyde) on measurements of museum specimens. Multiple comparison procedures allow identification of specimens that are different from each other. The post-hoc test used was the Nemenyis procedure, which is analogous to a Tukey test, but using ranked sums instead of means. Considering the fact that there were multiple comparisons among the four specimens, the Bonferroni correction was used (e.g. significance level divided by the number of tests or comparisons). Although significant differences were found between specimens by FT, the Nemenyis procedure did not show differences between individuals in multiple pair wise comparisons (Table 2). This may have been due to the fact that Nemenyis test is extremely conservative and is not powerful enough to detect any significant differences. Therefore, it is necessary to increase the sample size for a more robust comparison among fresh and preserved specimens. In the present case, the female was the larger specimen analyzed, yet their body proportions did not show significant statistical differences, with respect to the other specimens analyzed. However, it is noteworthy that the female body is more voluminous, hence their higher relative proportions of the trunk and the snout (e.g. TRL, D1P1, D1P, D2P1, D2P2 and PRN, POB, respectively). Sexual dimorphism in corporal dimensions for chimaeroid species has been reported for several species (Garrick and

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Table 1. List and description of the measurements used. Morphometric measurements as a proportion of body length (%BDL) of holotype and paratype of Hydrolagus melanophasma and comparative materials. Institutional abbreviations: Los Angeles County Museum of Natural History (LACM), the Scripps Institute of Oceanography (SIO) and Centro Interdisciplinario de Ciencias Marinas, Instituto Politcnico Nacional (CICIMAR-CI) Holotype Paratype Female Male Abbrevia- Male SIO Male CICIMAR-CI CICIMAR-CI Measurement tion 77-211 LACM 39805-1 7102 7103 % BDL % BDL % BDL % BDL Body length (mm) BDL 577 631 662 623 Total length TL 160.5 155.6 151.4 150.1 Precaudal length PCL 126.9 121.9 126.9 124.3 Length of snout to anus SVL 60.1 56.9 63.4 62.1 Trunk length TRL 37.3 35.5 39.3 34.3 Head length HDL 29.5 25.0 24.8 27.6 Preoral length POR 10.6 11.5 10.6 10.8 Prenarial length PRN 7.1 8.2 9.8 6.0 Preorbital length POB 13.7 13.2 14.5 13.0 Eye length EYL 6.5 6.5 6.2 5.5 Height of eye EYH 4.1 4.4 4.1 3.7 Pre-second dorsal length PD2 47.8 47.4 48.6 45.7 Pre-first dorsal length PD1 27.4 27.7 22.7 11.7 Length of first dorsal fin base D1B 15.3 13.9 14.0 12.4 Dorsal spine length along anterior margin DSA 25.9 25.9 16.6* 28.9 Maximum height of first dorsal fin D1H 19.5 19.0 20.4 21.0 Length of the base of the second dorsal fin D2B 81.1 77.3 78.9 77.7 Maximum height of the second dorsal fin D2H 4.0 4.1 4.5 4.0 Interdorsal space IDS 6.4 9.0 3.9 3.2 Length of dorsal margin of caudal fin CDM 23.1 20.5 15.6 16.5 Maximum height of dorsal lobe of caudal fin CDH 2.0 3.3 2.9 1.9 Total caudal fin length CTL 33.3 34.5 23.9 24.9 Length of ventral margin of caudal fin CVM 28.8 26.0 18.6 23.0 Maximum height of ventral lobe of caudal fin CVH 2.4 2.9 2.6 2.6 Caudal peduncle height CPH 2.4 2.4 2.6 2.2 Pectoral-fin anterior margin P1A 40.9 38.5 40.3 37.6 Pelvic-fin anterior margin P2A 21.0 19.5 19.5 18.0 Length of the rear base of the pectoral fin to anterior P2P 29.8 32.2 28.1 31.6 base of pelvic fin Length of space between the pelvic fins and caudal fin PCA 57.9 60.1 56.3 58.7 Origin of first dorsal fin to origin of pectoral fin D1P1 19.6 16.4 22.4 20.4 Origin of first dorsal fin to origin of pelvic fin D1P2 39.5 42.5 46.1 41.1 Origin of second dorsal fin to origin of pectoral fin D2P1 25.6 26.6 29.0 25.7 Origin of second dorsal fin to origin of pelvic fin D2P2 20.3 24.6 27.9 25.7 Total clasper length (intromittent organ) CLT 15.0 13.7 18.8 Length of medial clasper branch from fork to tip CLM 4.0 3.8 8.7 Length of lateral clasper branch from fork to tip CLL 3.9 4.2 8.5 Frontal tentacle length FTL 4.4 3.6 7.9 Weight ? ? 5,300 g 3,800 g
*Broken spine

Inada 1975; Gonzlez et al. 2007). In H. colliei, found off the U.S. westcoast, maximum body size and size at median

maturity were greater for females than males (Barnett et al. 2009). In this sense, it has been reported for some fish

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Table 2. Nemenyis procedure, a post-hoc test of the Friedmans test, between fresh (*) and preserved specimens (type material) and body measurements of the black ghost chimaera, Hydrolagus melanophasma. Type material described in James et al. (2009). H=female (CICIMAR-CI 7102). M=male (CICIMAR-CI 7103) BDL (body length) in mm Pair wise comparisons BDL Holotype 577 Paratypes 631 CICIMAR H* 662 CICIMAR M* 623 Holotype 0 No No No Paratypes -0.031 0 No No CICIMAR H -0.109 -0.078 0 No CICIMAR M -0.797 -0.766 -0.688 0
Bonferroni corrected significance level: 0.0083,=0.05. Critical difference: 0.851

species that larger females may provide more nutrients to their young and potentially increase offspring fitness (Berkeley et al. 2004). Unfortunately, data on Hydrolagus species is scarce and catches are typically not reported, so it is not possible to test the latter hypothesis (Barnett et al. 2009). The edited size of the 12S rRNA fragment was composed of 419 base pairs (bp). We found no variable site between the two individuals studied. The nucleotide composition was 22.7% of Thymine, 25.3 of Cytosine, 32% of Adenine and 20% of Guanine. Thus, the pyrimidines (T + C = 48%) and purines (A + G = 52%) contents were relatively similar (Table 3). The edited size of the 16S rRNA fragment was composed of 583 pb. Only one variable site, characterized by a transition between a Thymine and Cytosine, was found. The general nucleotide compositions in both specimens were also relatively similar (26% or less of Thymine, 21.5 or more of Cytosine, 31.7% of Adenine and 20.8% of Guanine). Thus, pyrimidines (T + C = 47.5%) and purines (A + G = 52.5%) contents were relatively similar in both individuals (Table 3). The two individuals of H. melanophasma were differentiated from other chimaeran species by both 12S rRNA and 16S rRNA fragments (Fig. 3A and 3B, respectively). The two 12S rRNA sequences were identical, as also found in other species of the family. The two sequences of 16S rRNA for H. melanophasma showed small differences with variation in only one nucleotide site, resulting in a relatively small genetic distance between them (0.17%). None of the other species of Hydrolagus showed variation in 16S rRNA. The estimated average genetic distance of 12S rRNA and 16S rRNA between Hydrolagus species were

6.15% and 3.45%, respectively. This latter figure is 15 times higher than found between two 16S rRNA sequences of H. melanophasma (0.17%). The result of the genetic characterization showed that 12S rRNA and 16S rRNA represented robust molecular markers that characterized the species. Similar genetic approaches using these two genes in the molecular identification of species have been employed previously by other authors (Douady et al. 2003; Ishizaki et al. 2006; Ward et al. 2008; Cui et al. 2010; Licht et al. 2012). Both showed high similarity characterized by the absence of variation in 12S rRNA and one change (p = 1 / 583 = 0.0017) in 16S rRNA in the two sequences. This one change may have been due to higher genetic diversity, since no change was found in the five specimens of H. colliei (Fig. 3B). The phylogenetic relationships found did not support the existence of clades associated with two genera of the family (Chimaera and Hydrolagus) (Fig. 3). This observation was supported by the low levels of average divergence between both genus (5.31% estimated from 12S rRNA and 4.15% estimated from 16S rRNA). Both gene sequences did not allow for the resolution of the issue of paraphyly with regard to the Chimaera and Hydrolagus. They have been properly used in molecular phylogenetic analysis (Douady et al. 2003), and similar conclusions have been derived using COI sequences (Ward et al. 2008) and cytochrome b gene (Licht et al. 2012). Our results supported the contention that neither of these two genera integrate a monophyletic clade, indicating the need for additional molecular markers and the need to revise the taxonomy of

Table 3. Nucleotide composition of fragments of 12S rRNA and 16S rRNA of the black ghost chimaera, Hydrolagus melanophasma (pb: pairs base) Gen Thymine Cytosine Adenine Guanine Size (pb) 12S rRNA 22.7 25.3 32.0 20.0 419 16S rRNA 1 26.1 21.4 31.7 20.8 583 16S rRNA 2 25.9 21.6 31.7 20.8 583

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Fig. 3. Neighbour-joining trees obtained using partial 12S (A) sequences and 16S (B) of the black ghost chimaera, Hydrolagus melanophasma and related species. Bootstrap value and scale are indicated. Code added to each scientific name is the Genbank access number. For H. melanophasma, only one 12s sequence was deposited in Genbank because we found only one haplotype (accession number: HQ645969)

the genera of Chimaeridae (e.g. Chimaera and Hydrolagus). In this sense, Licht et al. (2012) provided molecular phylogenetic analysis of holocephalan fishes using three mitochondrial genes (cytochrome b, 12S rRNA, and 16S

rRNA) and suggested that the cause of the paraphyly is by use of the anal fin as a taxonomic characteristic in order to distinguish between the genera Chimaera and Hydrolagus, arguing that the possible multiple losses of the anal fin

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within the Chimaeridae could have ocurred. Discussion of chimaeroid evolution is far beyond the scope of this short report. Nevertheless, our findings of morphometric and molecular data for this newly discovered species provide more information on its variation within the group, which may be useful for future research. A taxonomic and phylogenetic revision using a combination of molecular (nuclear and mitocondrial DNA genes), morphological and morphometric data, using a greater number of species, and detailed analysis (e.g. geometric morphometrics, appropiated models of DNA evolution and phylogenetic methods: Bayes, maximum likelihood, and maximum parsimony) should be conducted to build a systematic knowledgebase regarding the Chimaeridae family.

Acknowledgements
We thank T. Ehrenberg (Pisces Sportfishing, Cabo San Lucas, B.C.S., Mexico) for the donation of the chimaeroid specimens. J.D.A., F.J.G.R, V.M.C.G., F.N.M.B., and R.G.A. would like to thank Comisin de Operacion y Fomento de Actividades Acadmicas (COFAA) from Instituto Politcnico Nacional (IPN) and Estmulo al Desempeo de los Investigadores (EDI) from IPN for their grants. J.D.A., F.J.G.R and R.G.A. also thank the Sistema Nacional de Investigadores (SNI) for Consejo Nacional de Ciencia y Tecnologa (CONACyT) grant. Finally, we also thank D. Tung for his assistance in preparing the manuscript in English language. We thank the anonymous reviewers for their constructive comments.

References
Andrade I, Pequeo G (2006) Primer registro de Hydrolagus pallidus Hardy and Stehmann, 1990 (Chondrichthyes: Chimaeridae) en el Ocano Pacfico, con comentarios sobre los holocfalos de Chile. Rev Biol Mar Oceanogr 41(1):111-115 Barnett LAK, Earley RL, Ebert DA, Cailliet GM (2009) Maturity, fecundity, and reproductive cycle of the spotted ratfish, Hydrolagus colliei. Mar Biol 156(3):301-316 Berkeley SA, Chapman C, Sogard SM (2004) Maternal age as a determinant of larval growth and survival in a marine fish, Sebastes melanops. Ecology 85(5):1258-1264 Cui Z, Liu Y, Liu J, Luan W (2010) Molecular identification of Pampus fishes (Perciformes, Stromateidae). Ichthyol Res 57(1):32-39 Douady CJ, Dosay M, Shivji MS, Stanhope MJ (2003) Molecular

phylogenetic evidence refuting the hypothesis of Batoidea (rays and skates) as derived sharks. Mol Phylogenet Evol 26(2):215-221 Garrick JAF; Inada T (1975) Dimensions of long-nosed chimaera Harriotta raleighana from New Zealand. New Zeal J Mar Fresh 9(2):159-167 Gonzlez C, Teruel J, Lpez E, Paz X (2007) Feeding habits and biological features of deep-sea species of the Northwest Atlantic:large-eyed Rabbitfish (Hydrolagus mirabilis), Narrownose Chimaera (Harriotta raleighana) and Black Dogfish (Centroscyllium fabricii). Northwest Atlantic Fisheries Organization. Scientific Council Meeting 5423:1-9 Ishizaki S, Yokoyama Y, Oshiro N, Teruya N, Nagashima Y, Shiomi K, Watabe S (2006) Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene. Comp Biochem Phys D 1(1):139-144 James KC, Ebert DA, Long DJ, Didier DA (2009) A new species of chimaera, Hydrolagus melanophasma sp. nov. (Chondrichthyes: Chimaeriformes: Chimaeridae), from the eastern North Pacific. Zootaxa 2218:59-68 Kemper JM, Ebert DA, Didier DA, Compagno LJV (2010) Description of a new species of chimaerid, Chimaera bahamaensis from the Bahamas (Holocephali: Chimaeridae). B Mar Sci 86(3):649-659 Kocher TD, Thomas WK, Meyer A, Edwards SV, Paabo S, Villablanca FX, Wilson AC (1989). Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers. P Natl Acad Sci USA 86(16):6196-6200 Licht M, Schmuecker K, Huelsken T, Hanel R, Bartsch P, Paeckert M (2012) Contribution to the molecular phylogenetic analysis of extant holocephalan fishes (Holocephali, Chimaeriformes). Org Divers Evol 12 Palumbi SR (1996) Nucleic acids II: the polymerase chain reaction. In: Millis DM, Mortiz C, Mable BK (eds) Molecular Systematics, Sunderland, 1996, pp 205-247 Saitou N, Nei M (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406-425 Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596-1599 Tautz D, Arctander P, Minelli A, Thomas RH, Vogler AP (2002) DNA points the way ahead in taxonomy. Nature 418:479 Ward RD, Holmes BH, White AWT, Last PR (2008) DNA barcoding Australasian chondrichthyans: results and potential uses in conservation. Mar Freshwater Res 59(1):57-71 Zar JH (2009) Biostatistical Analysis. 5th ed. Prentice Hall, Upper Saddle River, 960 p