Complete mitochondrial genome of a subspecies of the great cormorant, Phalacrocorax carbo hanedae (Kuroda, 1925) (Suliformes: Phalacrocoracidae)

Abstract We determined the complete mitochondrial DNA sequence of a subspecies of the great cormorant, Phalacrocorax carbo hanedae (Kuroda, 1925) using long PCR and primer walking methods. The mitochondrial genome was 19,020 bp in length and contained 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, and two control regions. It is basically consistent with the characteristics of the mitochondrial genomes of other Suliformes species. Phylogenetic analysis using 12 species of Suliformes based on the sequences of 13 concatenated protein-coding genes confirmed the monophyly of P. carbo ssp.


Introduction
The great cormorant Phalacrocorax carbo (Linnaeus, 1758) consists of six subspecies distributed worldwide, excepting only South America and the Antarctic. The subspecies P. carbo hanedae (Kuroda, 1925) is endemic to Far East Asia, ranging over Taiwan, Korea, and Japan to Sakhalin (del Hoyo et al. 1992; Figure 1). The recent range expansion and population explosion of P. carbo hanedae in Japan has caused serious damage to freshwater fisheries. Genetic information on this subspecies is essential to promote its proper management while preserving its genetic diversity. This warrants the development of genetic markers with varying evolutionary rates to infer the genetic population structure of P. carbo hanedae. However, the mitogenome of this species has not previously been investigated.

Materials and methods
A liver specimen was obtained from a dead individual of a subspecies of the great cormorant, P. carbo hanedae, collected by the Gyotoku Nature Conservation Club NPO in Ichikawa City, Chiba Prefecture, Japan (35 40 0 25.7 00 N, 139 55 0 26.0 00 E) on July 24, 2015. The genomic DNA was extracted using a DNeasy Tissue and Blood Kit (Qiagen, Hilden, Germany). The DNA specimen was deposited at Hirosaki University (Dr. Atsushi Sogabe, e-mail: atsushi.sogabe@hirosaki-u.ac.jp) under voucher number HUA2103162. Primer walking for the five long PCR products covering whole mitochondrial DNA was used to determine the complete mitogenome sequence (see Tables S1 and S2 for primers used in the study and Figure S1 for PCR gel image). DNA sequencing was conducted in an automated DNA analyzer ABI 3500 (Life Technologies, Carlsbad, CA). The sequence fragments were then assembled using the GeneStudio Professional version 2.2.0.0 (GeneStudio, Inc., Suwanee, GA). The assembled mitogenome sequence was annotated using MITOS web server (Bernt et al. 2013).
Phylogenetic analysis based on BI and ML yielded identical phylogenetic trees (Figure 3). The overall topology of the order Suliformes was consistent with that previously reported (Gibb et al. 2013). We also confirmed the monophyly of P. carbo ssp. This study is expected to contribute to our understanding of the population genetic structure of P. carbo hanedae and the phylogenetic relationships among its subspecies.

Ethical approval
All procedures for collecting samples were performed in accordance with the ethical standards of the Act on Welfare and Management of Animals 1973, with permission from Chiba Prefecture, the municipality in which the dead bird was found, to collect them (no specific permit number was issued). All experimental procedures followed the Hirosaki University guidelines for the ethical treatment of animals.

Author contributions
Rina Honda and Atsushi Sogabe were involved in the conception and design of the study; Mizue Inumaru and Yukita Sato collected the sample; Atsushi Sogabe analyzed the data; Rina Honda, Mizue Inumaru and Yukita Sato were involved in interpretation of the data; Rina Honda and Atsushi Sogabe drafted the paper; Mizue Inumaru and Yukita Sato revised it critically for intellectual content. All authors approved the final manuscript and agreed to be accountable for all aspects of the work.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
No funding was received.

Data availability statement
The data that support the findings of this study are available in DDBJ at https://www.ddbj.nig.ac.jp/index-e.html under the accession no. LC715365. Gel image of long PCR products and Sanger sequencing results (ab1 files) are provided as electronic supplementary materials ( Figure S1 and Data S1-S27).