1. Introduction

Tetraodontiformes is a special group of higher teleost fishes, famous for the name of “puffer fish”. It is believed that puffer fish is the general name of fishes in family Tetraodontidae of order Tetraodontiformes. However, there is also a view that all species in the order Tetraodontiformes are called as puffer fish (Wu & Chen, 1981). The species in this order are very abundant and distributed in various habitats, such as freshwater, brackish and coastal waters, open seas and so on (Yamanoue et al., 2008). So far, FishBase (https://www.fishbase.de/)has recorded 446 species of Tetraodontiformes, belonging to 106 genera and 10 families. According to the records of “Fauna Sinica”, there are 131 species in 61 genera of 10 families in China (Su & Li, 2002), one of them is freshwater fish. Tetraodontiformes are distributed in a considerable amount along the coast of China, and occupy an important economic position in Chinese fishery (Su & Li, 2002). Among them, Thamnaconus ,Takifugu and Lagocephalus are with high economic value.
The puffer fish has a long edible history in China and Japan, and it is regarded as a precious aquatic product for its high protein content and extremely delicious taste. Although most of the puffer fishes taste delicious and have high economic value, many of them contain toxic tetrodotxin (TTX) in the ovary, liver, kidney, eyes and blood. If they are not handled properly or eaten by mistake, people will be poisoned or even die. Therefore, it is urgent to screen the species of edible puffer fish and avoid the occurrence of puffer fish poisoning. The taxonomy and identification of puffer fish is the key to solve the above problems.
In the traditional taxonomy, the classification and identification of Tetraodontiformes are mainly based on the morphological characteristics of stripes, spots, body color, and mouth shape, etc. However, the morphology of closely related species of this order is very similar and difficult to distinguish. Whether professionals or non-professionals, it is an extremely difficult task to identify Tetraodontiformes fishes only according to their morphological characteristics. So far, there are a large number of synonyms in the order Tetraodontiformes (Liu et al., 1999; Song et al., 2001; Cui et al., 2005; Reza et al., 2008; Matsuura, 2010; Sakai, Sakamoto, & Yoshikawa, 2021), and the classification and identification of species are confused, which makes it difficult to accurately count the number of species of this order (Chen & Zhang, 2015; Su & Li, 2002). Moreover, most of the commodities of the puffer fish in the market have been processed and lost their original morphological characteristics, which makes it impossible to identify species. DNA barcoding is used to identify species at molecular level, which is not affected by time, region and individual morphology (Hebert, Ratnasingham & Waard, 2003). Combining the morphological characteristics of species and DNA barcodding technology to identify species will make the identification results more objective and reliable.
At present, there have been many reports on the study of puffer fish at the molecular level. However, these studies only focus on one genus or several species of Tetraodontiformes (Song et al., 2001; Elmerot et al., 2002; Ishizaki et al., 2006; Reza et al., 2008; Zhang & He, 2008; Santini, 2013; Chen, 2019), and there is no study on the species identification of the whole order Tetraodontiformes. Therefore, it is urgent to carry out accurate classification and identification research of order Tetraodontiformes, so as to improve technical support for food safety of fishery biological sources and trade regulation of aquatic products.
In order to solve the difficulty of morphological classification of Tetraodontiformes, this study used DNA bar code technology and morphological methods to identify 616 samples of Tetraodontiformes collected from the coast of China, clarified the synonyms of some species of this order, and confirmed the effective species names of these species Building a perfect classification system of DNA barcode for the order Tetraodontiformes, will not only enrich the database of fish DNA barcode, but also promote the rapid development of puffer fish taxonomy and systematics, and provide technical support for aquatic product quality safety and trade supervision.