sm81n4-4601

Integrative taxonomy supports the presence of two species of Kyphosus (Perciformes: Kyphosidae) in Atlantic European waters

Rafael Bañón 1,2, David Barros-García 3,4, Alejandro de Carlos 5

1 Instituto de Investigaciones Marinas, CSIC, c/ Eduardo Cabello 6, 36208 Vigo, Pontevedra, Spain.
2 Grupo de Estudos do Medio Mariño (GEMM), Puerto deportivo s/n 15960 Ribeira, A Coruña, Spain.
(RB) (Corresponding autor) E-mail: anoplogaster@yahoo.es. ORCID-iD: http://orcid.org/0000-0001-6038-9335
3 Centro de Apoyo Científico y Tecnológico a la Investigación, Universidad de Vigo, c/ Fonte das Abelleiras s/n,
36310 Vigo, Spain.
4 Programa de Doctorado en Metodología y Aplicaciones en Ciencias de la Vida, Facultad de Biología,
Universidad de Vigo, c/ Fonte das Abelleiras s/n, 36310 Vigo, Spain.
(DB-G) E-mail: davbarros@uvigo.es. ORCID-iD: http://orcid.org/0000-0002-5283-2605
5 Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo,
c/ Fonte das Abelleiras s/n, 36310 Vigo, Spain.
(AC) E-mail: adcarlos@uvigo.es. ORCID-iD: http://orcid.org/0000-0003-0138-4918

Summary: The taxonomic identification of one Kyphosus sectatrix and two Kyphosus vaigiensis specimens caught in the European Atlantic waters of Galicia, northwestern Spain, was carried out by means of integrative taxonomy, combining the examination of morphological characters and DNA barcodes. Taxonomical assignation based on morphological characters of these specimens was tested by comparing their COI sequences with available data of Kyphosus deposited in public repositories. The resulting neighbour-joining tree defined four clades corresponding to Barcode Index Number (BIN) and indicated that some nucleotide sequences from Kyphosus, previously deposited, probably originate from misidentified specimens, as would be expected from cryptic and sympatric species. The specimens of Kyphosus vaigiensis represent a new record for the waters of Galicia and the northernmost record in the eastern Atlantic. This kind of herbivorous tropical fishes could play an important role in the tropho-dynamic context of this temperate coastal ecosystem.

Keywords: Kyphosus vaigiensis; Kyphosus sectatrix; molecular systematic; northern limit; Galician waters; ichthyogeography.

Identificación de dos especies de Kyphosus (Perciformes: Kyphosidae) en aguas del Atlántico europeo mediante taxonomía integrativa

Resumen: Un ejemplar de Kyphosus sectatrix y dos de Kyphosus vaigiensis fueron capturados en aguas del Atlántico europeo, en Galicia, al noroeste de España. La identificación se llevó a cabo mediante taxonomía integradora, combinando el examen de caracteres morfológicos y códigos de barras de ADN. La asignación taxonómica basada en los caracteres morfológicos de estos especímenes fue testada comparando sus secuencias de COI con los datos de Kyphosus depositados en los repositorios públicos. El árbol filogenético resultante definió cuatro clados correspondientes al número de índice de códigos de barras (BIN), e indicó que algunas secuencias de nucleótidos de Kyphosus, previamente depositadas, provienen probablemente de especímenes identificados erróneamente, como cabría esperar de especies crípticas y simpátricas. Los especímenes de Kyphosus vaigiensis representan un registro nuevo para las aguas de Galicia y el más septentrional en el Atlántico oriental. La proliferación de peces tropicales herbívoros podría desempeñar un papel importante en el contexto trofo-dinámico del ecosistema costero templado.

Palabras clave: Kyphosus vaigiensis; Kyphosus sectatrix; identificación molecular; límite norte; aguas de Galicia; ictiogeografía.

Citation/Como citar este artículo: Bañón R., Barros-García D., de Carlos A. 2017. Integrative taxonomy supports the presence of two species of Kyphosus (Perciformes: Kyphosidae) in Atlantic European waters. Sci. Mar. 81(4): 467-475. doi: http://dx.doi.org/10.3989/scimar.04601.08A

Editor: G. Pequeño.

Received: December 21, 2016. Accepted: May 19, 2017. Published: September 6, 2017.

Copyright: © 2017 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License.

Contents

Summary
Resumen
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References

INTRODUCTIONTop

The number of species in the perciform family Kyphosidae is controversial, mainly depending on the number of genera included. According to Nelson (2016)Nelson J.S., Grande T.C., Wilson M.V.H. 2016. Fishes of the world. John Wiley and Sons, Hoboken, New Jersey., this family contains 53 species divided into 14 genera, and Froese and Pauly (2017)Froese R., Pauly D. 2017. FishBase. World Wide Web electronic publication, version (02/2017). recognize the same number of genera but 54 species. On the other hand, Knudsen and Clements (2013) reduce this number to 12 species in 2 genera. The sea chubs Kyphosus species typically inhabits shallow waters over sandy, rocky or grassy bottoms around coral reefs, mainly in the Atlantic, Indian and Pacific Oceans, and juveniles are commonly found among floating algae or below flotsam (Tortonese 1986Tortonese E. 1986. Kyphosidae. In Whitehead P.J.P. Bauchot M.L., et al. (eds), Fishes of the northeastern Atlantic and the Mediterranean. Vol. 2, UNESCO, Paris. pp. 883-907.).

Only two species of Kyphosus have been reported so far in the northeastern Atlantic and the Mediterranean: the Bermuda sea chub Kyphosus sectatrix (L., 1758) and the yellow sea chub Kyphosus incisor (Cuvier, 1831) (Tortonese 1986Tortonese E. 1986. Kyphosidae. In Whitehead P.J.P. Bauchot M.L., et al. (eds), Fishes of the northeastern Atlantic and the Mediterranean. Vol. 2, UNESCO, Paris. pp. 883-907.). Over the last few decades kyphosid catch records for the Atlantic and Mediterranean have experienced an increased number of observations. For the Mediterranean Sea, new records of K. sectatrix (Ligas et al. 2011Ligas A., Sartor P., Sbrana M., et al. 2011. A new record of Kyphosus saltatrix (Pisces: Kyphosidae) along the Italian coasts (north-western Mediterranean). Mar. Biodivers. Rec. 4: e6., Kiparissis et al. 2012Kiparissis S., Loukovitis D., Batargias C. 2012. First record of the Bermuda sea chub Kyphosus saltatrix (Pisces: Kyphosidae) in Greek waters. Mar. Biodivers. Rec. 5: e11.) and K. incisor (Azzurro et al. 2013Azzurro E., Peñas-Rivas L., Lloris D., et al. 2013. First documented occurrence of Kyphosus incisor in the Mediterranean Sea. Mar. Biodivers. Rec. 6: e98. ) have been reported successively. For the European Atlantic waters, specimens of K. sectatrix have been reported sporadically in the Macaronesian islands, south of Portugal, northwest of Spain and in the Bay of Biscay (Bañón et al. 2010Bañón R., Villegas-Ríos D., Serrano A., et al. 2010. Marine fishes from Galicia (NW Spain): an updated checklist. Zootaxa 2667: 1-27., Canas et al. 2005Canas A., Vasconcelos P., Lino P.G., et al. 2005. Northernmost record of Kyphosus sectator (Osteichthyes: Perciformes: Kyphosidae) in the north-eastern Atlantic. J. Mar. Biol. Assoc. U.K. 85: 1535-1537., Quéro et al. 2009Quéro J.-C., Jerome S., Vayne J.-J., et al. 2009. Observations ichtyologiques effectuées en 2008. Ann. Soc. Sci. Nat. Charente-Marit. 9: 932-940.).

Two taxonomic views on Kyphosus with different perspectives have recently been published. Knudsen and Clements (2013)Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101. re-examine this genus within a world revision of the family based on both morphological and molecular characteristics, recognizing 12 valid species. According to these authors, four species are present in the Atlantic Ocean and the Mediterranean Sea: the brown chub Kyphosus bigibbus Lacepède, 1801, the brassy chub Kyphosus vaigiensis (Quoy and Gaimard, 1825), the blue sea chub Kyphosus cinerascens (Forsskål, 1775) and K. sectatrix. These authors consider K. incisor a junior synonym of K. vaigiensis.

A different taxonomic view of Kyphosus in the Atlantic and eastern Pacific Oceans, and using only morphologic characteristics, was provided by Sakai and Nakabo (2014)Sakai K., Nakabo T. 2014. Taxonomic review of Kyphosus (Pisces: Kyphosidae) in the Atlantic and Eastern Pacific Oceans. Ichthyol. Res. 61: 265-292., who only recognize 11 species, and share only 6 species with the revision presented by Knudsen and Clements (2013)Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101., stating that only three of them are present in the Atlantic Ocean and the Mediterranean Sea: Kyphosus atlanticus Sakai and Nakabo, 2014, Kyphosus bosquii (Lacepède, 1802) and K. incisor. According to Sakai and Nakabo (2014)Sakai K., Nakabo T. 2014. Taxonomic review of Kyphosus (Pisces: Kyphosidae) in the Atlantic and Eastern Pacific Oceans. Ichthyol. Res. 61: 265-292., K. sectatrix in the Atlantic Ocean is found to represent two species, K. bosquii and a new species, K. atlanticus.

Many of the morphological discrepancies between the two mentioned revisions have been recently reported by Gilbert (2015)Gilbert C.R. 2015. Designation of a neotype for the kyphosid fish Kyphosus sectatrix (Linnaeus, 1758). Zootaxa 3999: 295-297., who considers K. atlanticus a junior synonym of K. sectatrix, following Knudsen and Clements (2013)Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101..

The practice of an integrative taxonomy approach (Dayrat 2005Dayrat B. 2005. Towards integrative taxonomy. Biol. J. Linn. Soc. 85: 407-415.) that gathers information from different sources is advisable when identifications based on morphology alone are inadequate for distinguishing between species. DNA barcoding has been considered an efficient aid to traditional taxonomy and is designed to facilitate fast and accurate species identification from a short, standardized DNA sequence (Hebert et al. 2003Hebert P.D.N., Cywinska A., Ball S.L., et al. 2003. Biological identifications through DNA barcodes. Proc. R. Soc. Lond. B 270: 313-321. ). In its strictest sense, DNA barcoding addresses only a limited aspect of the taxonomic process, by matching DNA sequences to “known” species, the latter being delimited with traditional (e.g. morphological) methodologies. In this context, the role of barcodes is to provide a methodology for assigning unidentified specimens to already characterized species (Hebert et al. 2003Hebert P.D.N., Cywinska A., Ball S.L., et al. 2003. Biological identifications through DNA barcodes. Proc. R. Soc. Lond. B 270: 313-321. ). This is of great help to the end users of taxonomy, and is helping to make more rapid progress in species identification and delimitation of species groups (Ratnasingham and Hebert 2007Ratnasingham S., Hebert P.D.N. 2007. BOLD: The Barcode of Life Data System. Mol. Ecol. Notes 7: 355-364.). Where species are simply unknown or no attempts have been made to delimit them, the barcode approach as originally intended is inadequate in its applicability and should be applied with precaution. It is generally assumed for the majority of vertebrate species that DNA markers such as mtDNA-COI can be used to distinguish between species, and therefore the barcoding approach is based on the assumption that the variation within species of vertebrates is smaller than between species (Ratnasingham and Hebert 2007Ratnasingham S., Hebert P.D.N. 2007. BOLD: The Barcode of Life Data System. Mol. Ecol. Notes 7: 355-364.). As a consequence, DNA barcoding has the potential to aid taxonomic studies and help to clarify cases of potential synonymy and delimitation of cryptic species (Grant et al. 2011Grant R.A., Griffiths H.J., Steinke D., et al. 2011. Antarctic DNA barcoding; a drop in the ocean? Polar Biol. 34: 775-780.). Limitations of using mtDNA to infer species boundaries include: retention of ancestral polymorphism, male-biased gene flow, selection on any mtDNA nucleotide, introgression following hybridization, and paralogy resulting from transfer of mtDNA gene copies to the nucleus (Moritz and Cicero 2004Moritz C., Cicero C. 2004. DNA Barcoding: Promise and Pitfalls. PLoS Biol. 2: e354.). Despite their benefits and pitfalls, mtDNA-COI barcode sequences and their ever increasing taxonomic coverage have been considered an unprecedented resource for taxonomy and systematics studies and also function as a diagnostic tool.

DNA barcoding is recognized as an important new tool that can be usefully applied to help resolve taxonomic issues in fishes (Ward et al. 2009Ward R.D., Hanner R., Hebert P.D.N. 2009. The campaign to DNA barcode all fishes, FISH-BOL. J. Fish Biol. 74: 329-356.), based on the development of a reference library of barcode sequences from vouchered specimens. The analysis of validated DNA barcodes for cluster recognition provides an efficient approach for recognizing putative species (operational taxonomic units, OTU). The Barcode Index Number (BIN) system is a persistent registry for animal OTUs recognized through sequence variation in the mtDNA-COI barcode region (Ratnasingham and Hebert 2013Ratnasingham S., Hebert P.D.N. 2013. A DNA-based registry for all animal species: the Barcode Index Number (BIN) System. PLoS ONE 8: e66213.).

Molecular data including DNA sequences of Kyphosus species are scarce in the scientific literature. Markers like mitochondrial cytochrome b, 12S and 16S rDNAs have been used to identify K. sectatrix in Greek waters (Kiparissis et al. 2012Kiparissis S., Loukovitis D., Batargias C. 2012. First record of the Bermuda sea chub Kyphosus saltatrix (Pisces: Kyphosidae) in Greek waters. Mar. Biodivers. Rec. 5: e11.). Currently 45 records of mtDNA-COI from Kyphosus are available from the BOLD reference database (April 2016). Of these, 42 are assigned to species level and represent 5 species. Some barcodes from K. vaigiensis were obtained during the barcoding identification of marine fishes from Japan (Zhang and Hanner 2011Zhang J.-B., Hanner R. 2011. DNA barcoding is a useful tool for the identification of marine fishes from Japan. Biochem. Syst. Ecol. 39: 31-42.) and in the identification of K. vaigiensis from the Mediterranean Sea (Mannino et al. 2015Mannino A.M., Balistreri P., Iaciofano D., et al. 2015. An additional record of Kyphosus vaigiensis (Quoy and Gaimard, 1825) (Osteichthyes, Kyphosidae) from Sicily clarifies the confused situation of the Mediterranean kyphosids. Zootaxa 3963: 45-54.). Sequences of mtDNA-COI from K. cinerascens and K. vaigiensis have been obtained in relation studies aimed at resolving cryptic diversity in Indo-Pacific coral-reef fishes (Hubert et al. 2012Hubert N., Meyer C.P., Bruggemann H.J., et al. 2012. Cryptic diversity in Indo-Pacific coral-reef fishes revealed by DNA-barcoding provides new support to the centre-of-overlap hypothesis. PLoS ONE 7: e28987.). Barcodes of K. sectatrix and K. incisor have been used to identify marine fishes of Sao Paulo State in Brazil (Ribeiro et al. 2012Ribeiro A.O., Caires R.A., Mariguela T.C., et al. 2012. DNA barcodes identify marine fishes of Sao Paulo State, Brazil. Mol. Ecol. Resour. 12: 1012-1020.).

The aim here is to identify specimens of Kyphosus caught in European Atlantic Galician waters by combining morphological identifications in relation to their mtDNA-COI sequences. Since OTUs often tend to show high concordance with vertebrate species delimitations, this approach can be used to support species identifications based on morphological characters (Ratnasingham and Hebert 2013Ratnasingham S., Hebert P.D.N. 2013. A DNA-based registry for all animal species: the Barcode Index Number (BIN) System. PLoS ONE 8: e66213.).

MATERIAL AND METHODSTop

Sample collection and specimen assignation

Three specimens belonging to the genus Kyphosus were recorded on the Galician coasts (NW Spain) in 2013 and 2014 by recreational spear fishermen and professional artisanal fishers. Specimens were first preserved frozen and later thawed and measured to the nearest mm, and meristic characters were determined according to Knudsen and Clements (2013)Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101.. Morphology-based identifications, taxonomical nomenclature and classification were carried out following Knudsen and Clements (2013)Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101.. Afterwards, the fish were fixed in 10% formalin, transferred to 70% ethanol and finally deposited in the fish collection of the Museum Luis Iglesias de Ciencias Naturais (MHNUSC) in Santiago de Compostela (Galicia, Spain).

DNA extraction, polymerized chain reaction (PCR) amplification and nucleotide sequencing

Total DNA was purified from 25 mg of muscle tissue taken from each specimen using the spin-column protocol of the Tissue DNA Extraction Kit (Omega-Biotek). The standard 5´ barcoding region of the COI gene (ca. 650 bp) was amplified by PCR using the primers set C_FishF1t1-C_FishR1t1 (Ivanova et al. 2007Ivanova N.V., Zemlak T.S., Hanner R.H., et al. 2007. Universal primer cocktails for fish DNA barcoding. Mol. Ecol. Notes 7: 544-548.). Polymerase chain reaction was carried out using Phire Green Hot Start II DNA Polymerase (Thermo Scientific, Waltham, MA, USA); mixtures contained a final volume of 50 µL and included 19 reaction buffer, 200 µM of each dNTP, 0.1 µM of each primer and 1 µL of polymerase enzyme; between 50 and 100 ng of template DNA was added. PCR reaction products were visualized on 1.2% agarose gels (Seakem LE Agarose) stained with ethidium bromide and, due to the specificity of the results, purified directly with ExoSAP-IT (USB) following the manufacturer's instructions. DNA sequencing reactions were carried out in the forward and reverse senses using the M13F (-21) and M13R (-27) primers and the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The resulting products were resolved on an ABI 3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA) and the consensus sequences were obtained after removing the primer-bind regions and assembling the direct and reverse traces with SeqScape v2.5 (Applied Biosystems, Foster City, CA,USA).

Sequence alignment and molecular analysis

Forty-three mtDNA-COI sequences assigned to Kyphosus of at least 600 bp were retrieved from BOLD and GenBank, including the sequence FOAJ442-09 from Zhang and Hanner (2011)Zhang J.-B., Hanner R. 2011. DNA barcoding is a useful tool for the identification of marine fishes from Japan. Biochem. Syst. Ecol. 39: 31-42.. The final dataset, including the three Galician specimens, comprised 46 sequences with a total length of 651 nucleotides. The nucleotide and their deduced amino acid alignments were built with the MUSCLE algorithm (Edgar 2004Edgar R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32: 1792-1797.) with pairwise deletion. The specimens employed in the analysis are listed in Table 1.

Table 1. – List of specimens employed in this investigation.

No. BOLD Process ID Species name Country GenBank Acc. No.
1 ABFJ021-06 Kyphosus vaigiensis Japan JF952771
2 ABFJ232-07 Kyphosus vaigiensis Japan JF952770
3 BAHA219-08 Kyphosus sectatrix Bahamas JQ839801
4 BIM473-16 Kyphosus vaigiensis Israel
5 BZLWA537-06 Kyphosus sp. Belize JQ840121
6 BZLWC068-06 Kyphosus sp. Belize JQ840890
7 BZLWE009-08 Kyphosus incisor Belize JQ841613
8 CFSAN073-11 Kyphosus incisor United States KF461190
9 DSFSF676-09 Kyphosus bigibbus South Africa GU804959
10 DSFSG575-11 Kyphosus sectatrix South Africa KF489619
11 DSFSG612-11 Kyphosus sectatrix South Africa KF489620
12 DSLAG090-10 Kyphosus bigibbus South Africa
13 DSLAG091-10 Kyphosus bigibbus South Africa
14 DSLAG1112-11 Kyphosus bigibbus South Africa
15 DSLAG1120-11 Kyphosus vaigiensis South Africa
16 DSLAG1121-11 Kyphosus vaigiensis South Africa
17 DSLAG397-10 Kyphosus bigibbus South Africa
18 DSLAG398-10 Kyphosus vaigiensis South Africa
19 DSLAG684-10 Kyphosus bigibbus South Africa
20 DSLAR424-08 Kyphosus bigibbus South Africa
21 DSLAR425-08 Kyphosus bigibbus South Africa
22 DSLAR426-08 Kyphosus bigibbus South Africa
23 DSLAR427-08 Kyphosus bigibbus South Africa
24 DSLAR428-08 Kyphosus bigibbus South Africa
25 FOAJ442-09 Perciformes Indonesia GU674403
26 GBGCA11901-15 Kyphosus vaigiensis Spain KP116934
27 GBGCA11902-15 Kyphosus vaigiensis Spain KP116935
28 GBGCA11903-15 Kyphosus vaigiensis Italy KR013046
29 LIDMA357-10 Kyphosus bigibbus Belize HQ987871
30 MBFA125-07 Kyphosus vaigiensis French Polynesia JQ431874
31 MFSP091-09 Kyphosus incisor Brazil JX124794
32 MFSP2072-11 Kyphosus incisor Brazil JQ365389
33 MFSP413-10 Kyphosus sectatrix Brazil JQ365390
34 MFSP536-10 Kyphosus sectatrix Brazil JQ365391
35 MLIII601-08 Kyphosus vaigiensis Belize GU224526
36 SBF352-11 Kyphosus cinerascens Madagascar JQ350079
37 TOBA235-09 Kyphosus sectatrix Trinidad and Tobago JQ842912
38 TOBA410-09 Kyphosus sp. Trinidad and Tobago JQ842911
39 TZMSB180-04 Kyphosus vaigiensis South Africa JF493713
40 TZMSC478-05 Kyphosus bigibbus South Africa JF493712
41 TZMSC479-05 Kyphosus vaigiensis South Africa JF493714
42 TZSAL621-13 Kyphosus bigibbus South Africa
43 TZSAL622-13 Kyphosus bigibbus South Africa
44 Kyphosus cinerascens Philippines KF009602
45 Kyphosus cinerascens Japan NC013138
46 Kyphosus sectatrix Spain KT780867

The molecular analysis was conducted using the neighbour-joining (NJ) (Saitou and Nei 1987Saitou N., Nei M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.) method in MEGA 6.0 (Tamura et al. 2013Tamura K., Stecher G., Peterson D., et al. 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 30: 2725-2729.). The nucleotide substitution model employed was p-distance and the confidence limits were tested though a bootstrap procedure (Felsenstein 1985Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791.) with 2000 replicates. The resulting tree was edited using TreeGraph (Stöver and Müller 2010Stöver B.C., Müller K.F. 2010. TreeGraph 2: Combining and visualizing evidence from different phylogenetic analyses. BMC Bioinformatics 11: 7.).

RESULTSTop

Morphological aspects

sm4601fig1.jpg

Full size image

Fig. 1.Kyphosus specimens caught in Galician waters: K. sectatrix, MHNUSC 25018-1, 450 mm total length (A); K. vaigiensis, MHNUSC 25017-1, 482 mm total length (B); K. vaigiensis, MHNUSC 25017-2, 280 mm total length (C).

Kyphosus sectatrix (L., 1758)
(Fig. 1A)

Material examined. MHNUSC 25018-1, 450 mm LT, total length, 16 August 2013, Malpica, 43.325°N 8.810°W, 2-3 m depth.

Description. Body oval and moderately compressed; head short, 4.9 times in TL; mouth small and terminal; snout slightly greater than eye diameter; pelvic fin short, 1.8 times in head length; caudal fin not deeply emarginated. Colour dusky grey in body and fins, lighter ventrally, without yellow lines patents. The main morphometric and meristic characteristics are presented in Table 2.

Table 2. – Morphometric and meristic data of the K. sectatrix specimen caught in Galician waters.

K. sectatrix MHNUSC 25018-1 Knudsen and Clements (2013)Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101.
LT (mm) 450 31-538
LS (mm) 354 26-448
As % of LS:
Head length 25.7 21.0-34.2
Snout length 7.3 5.0-9.0
Postorbital length 11.3 10.6-16.7
Eye diameter 5.1 4.7-11.9
Upper jaw length 7.3 2.6-9.4
Interorbital length 10.2 8.6-13.9
Predorsal length 34.5 31.1-41.2
Preanal length 61.0 55.3-67.3
Prepectoral length 22.6 20.1-31.1
Preventral length 35.6 29.6-40.3
Base dorsal length 48.6 40.3-62.5
Anal base length 22.3 16.2-23.4
Pectoral length 18.9 13.3-24.1
Ventral length 14.4 11.8-19.0
Caudal peduncle length 20.9 12.1-23.8
Caudal peduncle depth 9.9 8.8-14.3
Body depth 42.7 38.7-54.7
Body width 15.8 11.3-20.7
2º anal fin ray length 9.3 8.5-15.2
4º dorsal fin ray length 7.3 4.8-11.9
Meristic features:
Dorsal fin rays XI+12 X-XI+11-12
Anal fin rays III+11 III+10-12
Pectoral fin rays 18 17-20
Ventral fin rays I+5 I+5
Gill rakers 7+17 5-8+14-18
Scales in lateral line 73 63-76
Pored scales in lateral line 58 51-61
Scale rows above lateral line 12 10-14

Habitat and distribution. Adults are found on rocky reefs or reef flats down to 25 m depth. Juveniles are pelagic, associated with flotsam, and can be encountered in the open ocean. Widely distributed in the west Pacific, to at least the Revillagigedo Islands, the east Pacific, the Indian Ocean, the Red Sea, the Atlantic and the Mediterranean (Knudsen and Clements 2013Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101.).

Kyphosus vaigiensis (Quoy and Gaimard, 1825)
(Fig. 1B, C)

Material examined. MHNUSC 25017-1, 482 mm LT, 21 January 2014, between Cape Finisterre and Punta Cabanas; 42.887°N 9.264°W, 4-6 m depth; MHNUSC 25017-2, 280 mm TL, 27 April 2014, “Pedras Negras”, O Grove (Ría de Arousa), 42.887°N 9.264°W, 8 m depth, in a rock crevice.

Description. Body oval and moderately compressed; head short, 5.1 and 5.3 times in TL; mouth small and terminal; snout slightly greater than eye diameter; pelvic fin a little shorter than pectoral fin, 1.6 and 1.4 times in head length; caudal fin not deeply emarginated. Colour dusky grey in body and fins, lighter ventrally; golden yellow horizontal scale rows along body from operculum to caudal fin and head with two gold stripes, one below eye and the other behind eye. The main morphometric and meristic characteristics are presented in Table 3.

Table 3. – Morphometric and meristic data of K. vaigiensis specimens caught in Galician waters.

K. vaigiensis MHNUSC 25017-1 K. vaigiensis MHNUSC 25017-2 Knudsen and Clements (2013)Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101.
LT (mm) 482 280 51-479
LS (mm) 390 220 41-444
As % of LS:
Head length 24.4 24.1 21.5-33.1
Snout length 7.9 6.8 4.4-9.5
Postorbital length 11.8 11.4 11.1-15.2
Eye diameter 5.4 5.9 4.9-11.8
Upper jaw length 7.7 7.3 4.9-11.8
Interorbital length 10.5 11.4 9.6-14.2
Predorsal length 39.7 32.3 30.3-41.9
Preanal length 59.7 56.8 54-63.4
Prepectoral length 23.8 26.8 22.4-33.5
Preventral length 34.6 32.7 31.5-41.6
Base dorsal length 50.3 48.2 41.9-56.1
Anal base length 26.2 23.2 15.1-26.2
Pectoral length 16.7 17.7 14.5-20.8
Ventral length 15.1 17.3 14.1-19.7
Caudal peduncle length 20.5 19.1 15.4-21.5
Caudal peduncle depth 10.0 10.9 8.6-15.1
Body depth 41.8 43.2 35.3-48.9
Body width 16.7 13.2 10.4-23.4
L 2º anal fin ray 10.0 11.4 6.9-14
L 4º dorsal fin ray 6.9 9.1 4.2-12.5
Meristic features:
Dorsal fin rays XI+14 XI+14 X-XI+13-15
Anal fin rays III+13 III+13 III+12-14
Pectoral fin rays 20 19 17-20
Ventral fin rays I+5 I+5 I+5
Gill rakers 10+22 10+22 5-10+16-23
Scales in lateral line 78 78 63-80
Pored scales in lateral line 60 59 52-63
Scale rows above lateral line 13 11 9-15

Habitat and distribution. Adults are usually found close to the shore and the coastline, while juveniles are associated with flotsam and can be encountered in the open ocean close to the surface. Widely distributed in the Pacific, Indian and Atlantic Oceans, and also in the Mediterranean Sea (Knudsen and Clements 2013Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101.).

Molecular analysis

Three COI DNA sequences were obtained from specimens of Kyphosus caught in Galician waters. An alignment of 651 nucleotide positions was built that, when translated, gave in all cases an amino acid sequence of 217 residues. When nucleotide positions were compared in all 46 sequences, a total of 598 conserved and 53 variable positions were found, from which 49 were parsimony-informative.

A graphic representation of divergences among specimens was created in the form of a consensus tree (Fig. 2) that grouped the sequences according to BIN and showed in its general topology all the Kyphosus sequences divided into two highly supported clades. One clade included all the sequences previously assigned to K. sectatrix and K. bigibbus, which clustered in two different, well-supported groups. The first group consisted of 19 sequences, 15 of them identified as K. bigibbus, three as K. sectatrix and one as K. cinerascens. The average genetic distance among them was 0.01% (Table 4), constituting BIN:AAF3652. The second group comprised four voucher specimens that yield a single haplotype identified as K. sectatrix, including KT780867 obtained in this study, corresponding to BIN:ACF1414. The genetic distance between these two BINs varied between 1.50% and 1.70% (Table 4).

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Full size image

Fig. 2. – Neighbour-joining tree of Kyphosus COI sequences. Numbers by the nodes indicate bootstrap supports. Sequences from European Atlantic waters of Galicia specimens are in bold letters. BIN: Barcode Index Number.

Table 4. – Genetic distances (% of p-distances) among COI sequences of kyphosid BIN (range values are shown in brackets).

Within BIN Between BIN
BIN:AAC3456 BIN:ABX5727 BIN:ACF1414
BIN:AAC3456 0.2 (0-0.8)
BIN:ABX5727 0.2 (0-0.3) 2.6 (2.3-3.2)
BIN:ACF1414 0 5.1 (4.9-5.8) 4.5 (4.5-4.6)
BIN:AAF3652 0.1 (0-0.3) 4.7 (4.5-5.5) 4.1 (4.0-4.3) 1.6 (1.5-1.7)

In the second clade, the first node delimited two well-supported groups. The first contained five sequences, two of them previously associated with K. cinerascens, one as Kyphosus sp., and the last one labelled “Perciformes”, with an average genetic distance among them of 0.20% (Table 4), constituting BIN:ABX5727. The second group comprised a total number of 18 sequences that together constitute BIN:AAC3456, from which 12 were identified as K. vaigiensis, including the two Galician specimens (GBGCA11901-15 and GBGCA11902-15). In addition, four sequences were named as K. incisor and two as Kyphosus sp. The average genetic distance among them was 0.20% (Table 4).

The barcoding results thereby support the identification of the specimens made by morphological examination.

DISCUSSIONTop

Morphologically, the examined K. sectatrix and K. vaigiensis specimens could be differentiated mainly by dorsal and anal soft ray counts (14 dorsal fin and 13 anal fin soft rays in K. vaigiensis compared with 12 dorsal fin and 11 anal fin soft rays in K. sectatrix) and the number of gill rakers (32 in K. vaigiensis and 24 in K. sectatrix), which agree with previous taxonomical reports (Knudsen and Clements 2013Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101., Mannino et al. 2015Mannino A.M., Balistreri P., Iaciofano D., et al. 2015. An additional record of Kyphosus vaigiensis (Quoy and Gaimard, 1825) (Osteichthyes, Kyphosidae) from Sicily clarifies the confused situation of the Mediterranean kyphosids. Zootaxa 3963: 45-54.).

In the NJ tree constructed from COI DNA sequences from Kyphosus, each of the three Galician specimens was placed within well-defined clades together with individuals from other geographical areas. The Galician sequences of K. vaigiensis grouped with sequences previously obtained from specimens of K. incisor and K. vaigiensis of Atlantic, Mediterranean and Indo-Pacific areas, including BIN:ACC3456, thereby supportive of considering K. incisor a synonym of K. vaigiensis (Knudsen and Clements 2013Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101., Mannino et al. 2015Mannino A.M., Balistreri P., Iaciofano D., et al. 2015. An additional record of Kyphosus vaigiensis (Quoy and Gaimard, 1825) (Osteichthyes, Kyphosidae) from Sicily clarifies the confused situation of the Mediterranean kyphosids. Zootaxa 3963: 45-54.). The K. sectatrix sequence obtained formed a robust clade with another three sequences from South Africa and the western Atlantic assigned to the same species under the designation BIN:ACF1414, producing all together a single haplotype.

The NJ tree obtained showed a number of probably wrongly assigned individuals, in agreement with the complexity described for Kyphosus (Knudsen and Clements 2013Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101.). As an example, Zhang and Hanner (2011)Zhang J.-B., Hanner R. 2011. DNA barcoding is a useful tool for the identification of marine fishes from Japan. Biochem. Syst. Ecol. 39: 31-42. obtained two barcodes of K. vaigiensis during the identification of marine fishes from Japan. They remarked that the K2P genetic distance between the specimens ABFJ021-06 (identified as K. vaigiensis) and the BOLD specimen FOAJ442-09 (identified as K. cinerascens) was 0.2%, which is in stark contrast with the 2.7% intraspecific genetic value found in their K. vaigiensis group. However, their specimen ABFJ021-06 (K. vaigiensis) grouped with K. cinerascens individuals in BIN:ABX5727, while ABFJ232-07 (K. vaigiensis) grouped with K. vaigiensis/K. incisor individuals (BIN:AAC3456). The short genetic distances between ABFJ021-06 and FOAJ442-09 can be explained by misidentification of the former, or by a hybridization phenomenon. Therefore, the high intraspecific distance reported for K. vaigiensis (2.7%) could be due to misidentification or a case involving cryptic species.

Mannino et al. (2015)Mannino A.M., Balistreri P., Iaciofano D., et al. 2015. An additional record of Kyphosus vaigiensis (Quoy and Gaimard, 1825) (Osteichthyes, Kyphosidae) from Sicily clarifies the confused situation of the Mediterranean kyphosids. Zootaxa 3963: 45-54. published an NJ tree of Kyphosus COI sequences constructed with a K2P model using data from GenBank to form a 627 bp alignment, which included sequence DSFFSG612-15, assigned to K. bigibbus. In the present work, the latter grouped with three K. sectatrix specimens (BIN:ACF1414), sharing the same haplotype. In fact, this sequence is now reassigned as K. sectatrix in the BOLD database. Two K. sectatrix sequences (BAHA219-08 and MFSP413-10) mentioned in the study by Mannino et al. (2015)Mannino A.M., Balistreri P., Iaciofano D., et al. 2015. An additional record of Kyphosus vaigiensis (Quoy and Gaimard, 1825) (Osteichthyes, Kyphosidae) from Sicily clarifies the confused situation of the Mediterranean kyphosids. Zootaxa 3963: 45-54. shared the same haplotype found in the present study with another 12 sequences identified as K. bigibbus (BIN:AAF3652).

The barcoding technique has been successfully integrated with the traditional morphological analysis in the systematic study of fishes in the context of an integrative taxonomy (Dayrat 2005Dayrat B. 2005. Towards integrative taxonomy. Biol. J. Linn. Soc. 85: 407-415.). Application of a combination of both morphological and molecular barcoding identification of species is recommendable in all taxonomic studies of fishes, and especially for problematic groups like Kyphosus, with a rather uniform morphology and only subtle variations among species. The result of barcoding allowed the species assignation of these north Atlantic specimens and supported the hypothesized assignment based on their morphological identification, from an integrative taxonomy point of view.

Both Kyphosus described here are warm-water species found northwards of their usual distribution ranges, with K. vaigiensis being the northernmost confirmed occurrence in the eastern Atlantic. The presence of Kyphosus species in the Mediterranean and the European Atlantic has been related to the warming of waters (Bañón 2004Bañón R. 2004. New records of two southern fish in Galician waters (NW Spain). Cybium 28: 367-368., Canas et al. 2005Canas A., Vasconcelos P., Lino P.G., et al. 2005. Northernmost record of Kyphosus sectator (Osteichthyes: Perciformes: Kyphosidae) in the north-eastern Atlantic. J. Mar. Biol. Assoc. U.K. 85: 1535-1537.). A rise of 0.24°C per decade has been observed in the Galician sea waters since 1974 (Gómez-Gesteira et al. 2011Gómez-Gesteira M., Gimeno L., de Castro M., et al. 2011. The state of climate in NW Iberia. Climate Res. 48: 109-144.), with a decrease in the extension and intensity of the upwelling seasons, responsible for the presence of colder coastal surface waters in summer, and an increase in the extension and intensity of the downwelling seasons, which favour the poleward current (Álvarez-Salgado et al. 2008Álvarez-Salgado X.A., Labarta U., Fernández-Reiriz M.J., et al. 2008. Renewal time and the impact of harmful algal blooms on the extensive mussel raft culture of the Iberian coastal upwelling system (SW Europe). Harmful Algae 7: 849-855.).

Relationships between water temperature and Kyphosus abundance and distribution have also been found at other latitudes, supporting the presence and abundance of these species as indicators of global warming at temperate latitudes. In western Japan, where this genus is more common, it has been observed that the number of caught specimens decreases as the water temperature decreases (Yamaguchi et al. 2010Yamaguchi A., Furumitsu K., Yagishita N., et al. 2010. Biology of herbivorous fish in the coastal areas of Western Japan. In: Ishimatsu A., Lie H.J., (eds), Coastal environmental and ecosystem issues of the east China Sea. Nagasaki University: TERRAPUB. pp. 181-190.). In southeastern Australia, the silver drummer Kyphosus sydneyanus (Günther, 1886) has expanded its distribution range and abundance in response to climate change (Last et al. 2011Last P.R. White W.T., Gledhill D.C., et al. 2011. Long-term shifts in abundance and distribution of a temperate fish fauna: a response to climate change and fishing practices. Glob. Ecol. Biogeogr. 20: 58-72.).

As occurred with other warm-water affinity fishes found in Galician waters, K. vaigiensis was previously recorded in the Mediterranean Sea, where it was reported under the synonym, K. incisor (Azzurro et al. 2013Azzurro E., Peñas-Rivas L., Lloris D., et al. 2013. First documented occurrence of Kyphosus incisor in the Mediterranean Sea. Mar. Biodivers. Rec. 6: e98. ), a misidentification of K. sectatrix (Ligas et al. 2011Ligas A., Sartor P., Sbrana M., et al. 2011. A new record of Kyphosus saltatrix (Pisces: Kyphosidae) along the Italian coasts (north-western Mediterranean). Mar. Biodivers. Rec. 4: e6.) finally clarified by Knudsen and Clements (2013)Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101., or with its current name (Mannino et al. 2015Mannino A.M., Balistreri P., Iaciofano D., et al. 2015. An additional record of Kyphosus vaigiensis (Quoy and Gaimard, 1825) (Osteichthyes, Kyphosidae) from Sicily clarifies the confused situation of the Mediterranean kyphosids. Zootaxa 3963: 45-54.). This seems to confirm a general and gradual northward displacement of these species in the eastern Atlantic, using the Gibraltar Strait as an escape valve in this migration to the north. This is a general trend also noted in other tropical and subtropical species such as Pisodonophis semicinctus (Richardson, 1848) and the lesser amberjack Seriola fasciata (Bloch, 1793) (Bañón et al. 2010Bañón R., Villegas-Ríos D., Serrano A., et al. 2010. Marine fishes from Galicia (NW Spain): an updated checklist. Zootaxa 2667: 1-27.), which have also been recently found in Galician waters. K. vaigiensis is one of the most widely distributed species of sea chubs, being present across the Pacific, Indian and Atlantic oceans, and also the Mediterranean Sea (Knudsen and Clements 2013Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101.), which is in agreement with the present results.

An environmental tropicalization could increase the Kyphosus population in Galician waters, which would adversely affect the seaweed abundance (Vergés et al. 2016Vergés A., Doropoulos C., Malcolm H.A., et al. 2016. Long-term empirical evidence of ocean warming leading to tropicalization of fish communities, increased herbivory, and loss of kelp. PNAS 113: 13791-13796.). The family Kyphosidae sensu Knudsen and Clements (2013)Knudsen S.W., Clements K.D. 2013. Revision of the fish family Kyphosidae (Teleostei: Perciformes). Zootaxa 3751: 1-101. is a strictly herbivorous family, with morphological and physiological traits suited for consumption of algae, such as the ability to perform microbial fermentation in their guts. Herbivorous fishes have a significant effect on macroalgal vegetation, not only in tropical but also in warm temperate waters, and Kyphosus species are very important for understanding the feeding damage inflicted on seaweed beds by herbivorous fishes (Yamaguchi et al. 2010Yamaguchi A., Furumitsu K., Yagishita N., et al. 2010. Biology of herbivorous fish in the coastal areas of Western Japan. In: Ishimatsu A., Lie H.J., (eds), Coastal environmental and ecosystem issues of the east China Sea. Nagasaki University: TERRAPUB. pp. 181-190.).

The poleward-flowing boundary currents are creating ocean warming hotspots around the globe, enabling the range expansion of tropical species and increasing their grazing rates in temperate areas (Vergés et al. 2014Vergés A, Steinberg P.D., Hay M.E., et al. 2014. The tropicalization of temperate marine ecosystems: climate-mediated changes in herbivory and community phase shifts. Proc. R. Soc. Lond., B, Biol. Sci. 281: 8-46.). For example, Franco et al. (2015)Franco J.N., Wernberg T., Bertocci I., et al. 2015. Herbivory drives kelp recruits into ‘hiding’ in a warm ocean climate. Mar. Ecol. Prog. Ser. 536: 1-9. found 45 times more herbivorous fishes in a “warm” than in a “cool” region in the neighbouring Portuguese coast. Further research effort is needed in order to reveal the ecological consequences of Kyphosus species as “natural invaders” of new temperate habitats.

ACKNOWLEDGEMENTSTop

We thank the spear fisherman Antonio Barreiro and the secretary of A.D.C. Raspa, V. Pérez Quintela, for their kind donation of the specimens. Thanks also to Dr S. Knudsen and K. Clements (School of Biological Sciences, University of Auckland, New Zealand) for their useful comments in the identification of specimens. This study was financed by the agreement between the CSIC and the Xunta de Galicia to analyse fisheries-dependent data from the monitoring programme of small-scale fisheries in Galicia (ref 20164040). These results will partially fulfil the PhD requirements of DBG at the University of Vigo.

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