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.