Species delimitation
The haplotype analysis result show that all the 222 sequences obtained
was divided into 95 haplotypes, of which the widely distributed species
such as M.nipponensis has greater genetic differentiation
(FIG.S2). The NJ tree based on the p-distance model was not shown
because of the same topology and similar bootstrap values to those of
the K2P model (FIG.2). The NJ tree reveals the phylogenetic clusters
formed by the specimens and taxonomic assignment based on morphology.
All of the sequences were divided into 11 groups, among which theM.maculatum , N.davidi and P.modestus were further
subdivided. By computation, the maximum K2P distances of all species
were less than 2%, with the exception ofP.modestus ,M.maculatum and M.nipponense which were 2.6%, 2.3% and
2.0% respectively. The maximum p-distances of all species were also
less than 2%, with the exception of P.modestus , andM.maculatum , which were 2.5% and 2.3%, respectively. Mean K2P
distances and p-distances within species were 1.6% and1.5%. The
results show that both the K2P and the p-distances have similar results
in genetic distance and phylogenetic analysis. In addition, the result
of ABGD analysis and bPTP analysis shown that, the 222 original
sequences were identified as 8 and 9-17 OTUs respectively. However, the
222 sequences were divided into 12 and 8-12 OTUs respectively when the
downloaded reference sequences were introduced (FIG.3). All the above
analyses supported that P.modestus and M. sp.’qilianensis’were a distinct monophyletic clade, and ABGD identified them as one
species. Due to the failure to identify enough morphological
differentiation on the M.sp. ’qilianensis’ , and there are not
sufficient description and evidence in the relevant references and
original literature, we tentatively infer that M.
sp.’qilianensis’ may be an invalid species and it may be the synonym of
the P.modestus . In addition, there is only one sample and the
morphology of them are damaged so that it cannot be effectively
identified by morphological identification. So, the existence ofN.ikiensis and N.palmata need to be further collected and
confirmed.
The comprehensive results of morphological characteristics and molecular
delimitation indicate that there are at least 9 freshwater shrimps in
Henan Province, among which the species of the Palaemon ,Macrobrachium and Neocaridina have a certain degree of
morphological and genetic variation, the Macrobrachium was most
obvious in them. Specifically, the differentiation of P.modestus ,M.maculatum and M.nipponense is more obvious. Because of
the changeable morphology and blurred boundaries, they are only
identified to the species level, and further research is needed to
refine intraspecies classification. It is possible that these species
may be in the middle of the differentiation process that from a species
to a new species or there are some cryptic species which have not been
discovered. In short, more evidence and studies are needed to determine
whether the intermediate process of a species’ differentiation to a new
species is sufficient to form a new species, and is there a cryptic
species.