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.