Hybridization of burrfish between Chilomycterus antillarum and Chilomycterus schoepfii in captivity revealed by AFLP and mtDNA sequence analyses

The genus Chilomycterus (Diodontidae) includes five species and two subspecies, of which the bridled burrfish Chilomycterus antennatus (Fig. 1a), web burrfish Chilomycterus antillarum (Fig. 1b) and striped burrfish Chilomycterus schoepfii (Fig. 1c) are endemic to the Western Central Atlantic Ocean (Leis 2002; Froese and Pauly 2014). Fertilized epipelagic eggs were collected in May 2008 from an exhibition aquarium (1.1 m) in the Shimonoseki Marine Science Museum, in which a female C. antennatus about 17 cm standard length (SL) (from 2004), a male C. antillarum about 18 cm SL (from 2002) and a female C. schoepfii about 23 cm SL (from 2002) were maintained at 24 C, subject to cyclic 11 hour of light and 13 hour of dark periods. The eggs were subsequently reared, fed with Brachionus plicatilis initially, and three individuals survived for more than two years (about 12 to 13 cm SL). Clearly, the individuals represented hybrids of C. antillarum and the females present. The maternal origin of a hybrid individual was determined by amplified fragmental length polymorphism (AFLP) analysis (Vos et al. 1995) and mtDNA cytochrome oxidase subunit I sequence (COI) (Ward et al. 2008). The three parental candidates as well as an individual of longspined porcupinefish Diodon holocanthus (outgroup) were also analyzed. The present report is the first to provide information on diodontid hybridization. For AFLP analyses, the restriction enzymes EcoRI and MseI were used for digestion of genomic DNA. AFLP fingerprints were produced for each of 24 EcoRI/MseI selective primer combinations with the following selective nucleotides: AAC/CAT, AAG/CAT, AGT/CAT, AGA/ CAT, ACT/CAT, ATC/CAT, AAC/CTA, AAG/CTA, AGT/CTA, AGA/CTA, ACT/CTA, ATC/CTA, AAC/ CAA, AAG/CAA, AGT/CAA, AGA/CAA, ACT/CAA, ATC/CAA, AAC/CTT, AAG/CTT, AGT/CTT, AGA/CTT, ACT/CTT and ATC/CTT. All the EcoRI selective primers were labeled at the 5’-end with fluorescein dye (supplied by Applied Biosystems, Foster City, CA, USA). Each selective PCR product was mixed with GeneScan 500 ROX size standard and Hi-Di-Formamide (Applied Biosystems) and separated electrophoretically using an ABI 3130 Genetic Analyzer (Applied Biosystems). Unambiguous loci, ranging from 50 to 500 bp, were scored automatically using GeneMapper software Version 3.7 (Applied Biosystems) and then proofed manually on the basis of reproducibility of fragment size, proximity to other loci and signal intensity. To determine maternal parentage, the 602 bp portion of COI was amplified utilizing universal primers for fish DNA barcoding (Ivanova et al. 2007), and the PCR products were sequenced directly using BigDye Terminator Version Electronic supplementary material The online version of this article (doi:10.1007/s10228-015-0460-0) contains supplementary material, which is available to authorized users.