Barcoding success
It is well known that taxonomic identification and species identification of organisms are the most fundamental and important task of all biological research (Luo et al., 2018 ). The early classification identification is mainly based on the detailed morphological characteristics observation and anatomical structure verification by professional taxonomists. However, it takes a lot of time, energy, and has very high requirements for researchers and experimental specimens (Carvalho et al., 2011 ; P. D. Hebert, Cywinska, et al., 2003 ; Shen et al., 2016 ). In addition, there is always the demise of existing species and the emergence of new species in the biological world. With the rapid development of science and technology, more and more new species have been discovered one after another, and specialists in alpha taxonomy are not enough to carry out extensive and complex morphological identification (Coleman, 2015 ). Therefore, it is not only prone to misjudgment but also not conducive to widespread implementation. With the development of modern technology and the arrival of the molecular era, traditional morphological identification is gradually replaced by molecular biological identification.
Compared with traditional morphological identification, barcode technology has many advantages and applicability, for example: First, DNA is more stable than morphological characteristics because DNA characters are constant throughout development. However, morphological characteristics vary with age, developmental stage, environment and objective factors. For example, molecular identification of deformed and underdeveloped shrimp larvae has absolute advantages over morphological identification (Burghart et al., 2014 ; Lee & Kim, 2014 ); Secondly, it can obtain sample DNA through some small parts of tissues, secretions, and even its living environment (Pont et al., 2018 ) of the biological body, which reduces the requirements of the sample (Chang, Lin, Ren, Lin, & Shao, 2016 ); More importantly, it is very friendly to operators and researchers. It is easy to operate, fast and efficient. It can be identified in batches, and requires less professional knowledge (Takahara, Minamoto, & Doi, 2013 ;Tinacci et al., 2018 ). To be specific, the aims of DNA barcoding are identification unkonwn specimens with DNA barcodes of a priori defined taxonomic entities in databases (Merckelbach & Borges, 2020 ). It is being increasingly utilized to tackle many issues including illegal species exploitation, food fraud and biological invasions, and for biodiversity monitoring(Bohmann et al., 2014 ; Collins, Armstrong, Holyoake, & Keeling, 2013 ;Frantine-Silva, Sofia, Orsi, & Almeida, 2015 ; Goncalves, Oliveira-Marques, Matsumoto, & Miyaki, 2015 ;Hubert, Espiau, Meyer, & Planes, 2015 ; Khaksar et al., 2015 ;Xiong et al., 2016 ). Of course, as mentioned above, barcode technology also has some drawbacks, but these deficiencies are gradually being improved and overcome. Since the first use of cytochrome C oxidase subunit I(COI) for species identification, it has been shown that this gene fragment can be used as a ”DNA barcoding” for biological authentication in many invertebrates species (Barrett & Hebert, 2005 ;Clare, Lim, Engstrom, Eger, & Hebert, 2007 ; P. D. Hebert, Cywinska, et al., 2003 ; Hendrich et al., 2015 ). The research of Costa (2007), MAR(2018) et al. further proof that barcode technology is efficient and accurate in species identification of freshwater shrimp(Costa et al., 2007 ;Mar, Kang, Mao, & Wang, 2018 ). In our results, 100% of the delimitation of species within freshwater shrimps was in accordance with genetic and morphological definitions. It demonstrating that DNA barcoding can help identify new taxa in complex groups and it can be widely used in the identification of unknown species and cryptic species (Iyiola et al., 2018 ).Phylogenetic relationship
Phylogeny refers to the formation and development process of a certain taxa, which is usually represented by evolutionary trees. In most cases, the topological structure and monophyletic branches of evolutionary trees can intuitively reflect the evolutionary and taxonomic status of each taxa. Thus, a taxon’s taxonomic status may vary due to objective or human factors, but its phylogeny is stable. The taxonomic status of freshwater shrimp has been established as early as the 19th century, but with the development of taxonomy for many years, its taxonomic status has been continuously hit and challenged. As far as Caridea is concerned, Bracken (2009) and Chan (2010) et al. believe that there is a certain controversy in the classification of the current super family (Bracken, De Grave, & Felder, 2009 ; Chan, Lei, Li, & Chu, 2010 ). The systematic placement of the infraorder Caridea within the decapods has been resolved several years ago, but the past studies have relied mainly on morphological features, leading to conflicting patterns of systematic development (Bracken et al., 2009 ). In recent years, studies have reexamined the phylogenetic relationship based on molecular characteristics, and the phylogenetic relationship tends to be stable. Some of these studies also shed new light on the phylogenetic relationships of Caridea (Davis, De Grave, Delmer, & Wills, 2018 ; C. P. Li, De Grave, Chan, Lei, & Chu, 2011 ). The infraorder Caridea are a highly diverse group which had been described more than 3,500 species(Grave & Fransen, 2011 ), with a significant number of additions since then. Among them,approximately amounting to nearly a quarter live in freshwater of global caridean(De Grave et al., 2015 ). Due to the lack of high-level cladistics and genetic studies, there is little describe about the phylogeny of freshwater shrimp Except for Pereira (1997) (De Grave, Cai, & Anker, 2008 ; Pereira, 1997 ).
As one of the most special superfamily in the Caribbean suborder and several taxonomic schemes have been proposed and modified since Palaemonoidea inception. However, its classification is still controversial (Kou, Li, Chan, Chu, & Gan, 2013 ). As most of Palaemoninae are highly conserved in morphological characteristics (Short, 2004 ;Walker & Poore, 2003 ), the previous studies mostly focused on morphological characteristics, so there were big problems in the classification of genus. With the study of Ashelby (2012) and Kou (2013) et al. for phylogenetic relationship based on molecular characteristics, the phylogenetic and evolutionary relationship of Palaemoninae has been preliminarily determined(Ashelby, Page, De Grave, Hughes, & Johnson, 2012 ; Kou et al., 2013 ).. In all of their analyses, Palaemonetes ,Exopalaemon , Coutierella and certain Palaemonrecover as a strongly supported single monophyletic clade, now they’ve been reclassified into the Palaemon (Ashelby et al., 2012) and be widely accepted (Ashelby et al., 2012 ). Our research also fully proves it. The Macrobrachium of Palaemonoidea have been intensively studied because of their rich diversity and wide distribution, it has been the subject of many molecular phylogenetic studies. The results all showed that there was a clear separation of the genetic differentiation of the species within the genus and Macrobrachium from other species of the related genera (Chen et al., 2009 ;Nicholas P. Murphy & Austin, 2003 ; N. P. Murphy & Austin, 2004 ; Nicholas P. Murphy & Austin, 2005 ; Pileggi & Mantelatto, 2010 ; Wowor et al., 2009 ). In this study, the species of the Macrobrachium also showed greater genetic differentiation. In contrast, only a handful of phylogenetic studies have been carried out in the Neocaridina of Atyoidea, which is highly abundant and widely distributed. As the widely distributed species north of the Yangtze River, the taxonomy ofN.davidi has always partially conflicting and unclear (Klotz, Miesen, Hullen, & Herder, 2013 ). With all these taxonomic and nomenclatural uncertainties of it (Cai, 1996 ;Liang, 2004 ;Shih & Cai, 2007 ), as the senior synonym the C. davidi Bouvier, 1904 has clear priority overN. heteropoda Liang, 2002 (article 23 of the ICZN). Thus, hereby we follow Klotz (2013) and retain the name Neocaridina davidi(Klotz et al., 2013 ).
Cryptic species
The investigation results show that there are at least 9 species of freshwater shrimp in Henan province. Compared with the existing studies (F. Wang, 1989 ), we have not been collected the Macrobrachium superbum , Macrobrachium asperulum and Macrobrachium iusulare . Although the existing sampling sites completely cover the original sampling sites and we have carried out targeted sampling on some samples. Unfortunately, these species has not yet been found. This may be because they are mostly distributed in the provinces to the south of the Yangtze River, and less distributed in the north, so it is difficult to collect (X. Li et al., 2007 ); or they may have migrated or even become extinct in the province; of course, it may also be because they are similar to M.maculatum andM.nipponense what has led to the misjudgments in morphological identification. In short, it needs more strong evidence to prove whether they exist in the province.
In this study, a variety of molecular boundary analyses all supported that some species of the Neocaridina , Macrobrachium andPalaemom which are widely distributed and rich in species (De Grave et al., 2008 ), have large genetic distance and more than one OTUs. Although both molecular and morphological characteristics show significant genetic differentiation and morphological differences between some species of these genera, there is no definitive criterion for whether these indicators are sufficient to indicate the emergence of a new species or the existence of an underlying species. In general, phylogenetic tree, genetic distance, haplotype analysis and PTP analysis indicated that the genetic differentiation of M.maculatum , M.nipponense andP.modestus were obvious, all of them had high haplotype diversity and no less than 2% intra-specific genetic distance although each of these groups formed a separate cluster or monophyletic clade. At the same time, most molecular delimitation analyses showed a higher number of species than morphological identification. This suggests that there are likely to be cryptic species within these species that have yet to be identified and described thus potentially explaining the high intraspecific diversity, even if they are not sufficiently differentiated to support the formation of a single new species.
Cryptic species are to a large extent intermediate products or even final products of new speciation. In the process of speciation, the boundaries of new species become more and more obvious over time. However, before the completion of this process (known as gray zone sense), the boundaries between species are often fuzzy and difficult to recognize, which makes the boundaries and identification of new species more subjective and dependent on the concept of species (De Queiroz, 2007 ). It is shown that the ability of DNA barcodes to identify recent speciation and no fully differentiated species is limited. The discovery of new species is usually accompanied by the delineation of molecular species, sometimes referred to as molecular operational taxonomic units (MOTUs) (Blaxter et al., 2005 ). However, many newly discovered species are undescribed, even when the species hypothesis and species delimitation are highly supported by substantial evidence (Pante, Schoelinck, & Puillandre, 2015 ), which hinders taxonomic progress, identification of species and estimation of biodiversity (Schlick-Steiner et al., 2007 ). If a species is marked as merely presumed rather than formally described and therefore fully established, the taxonomy is still incomplete. In many cases, the transition from species delimitation to species description is a major task to be accomplished (Jorger & Schrodl, 2013 ;Merckelbach & Borges, 2020 ).
Acknowledgements
We are grateful to the anonymous reviewers for their constructive comments. We thank for Xi Wang, Hui-hui Wu, Ru-yao Liu, and Xue-meng Yan for assistance with field work and their help.
In addition, this work was supported by the following funding: the National Natural Science Foundation of China (31872199, U2004146), this study support was provided by The High Performance Computing Center of Henan Normal University. Thanks also go to these funds and agencies for their support.