|Other Abstract||Copepods in the Ergasilidae (Copepoda: Poecilostomatoida) and Lernaeidae (Copepoda: Cyclopoida) are two major groups of parasitic copepods on freshwater fish. The present study aimed to illustrate the phylogenetic relationship among species in the families Ergasilidae and Lernaeidae, and to understand the phylogenetic relationship among three species in the Sinergasilus (Ergasilidae) with the analysis of the Sinergasilus-host co-evolutionary relationship.
A total of 14 species belonging to 4 genera in the Ergasilidae and 3 species to 2 genera in the Lernaeidae were obtained from 24 species of fish in China, and the 18S, 28S rDNA sequences were cloned from the collected parasitic copepods. Phylogenetic analyses using partial 18S and partial 28S rDNA sequences and combined 18S-28S rDNA sequences suggested that both Ergasilidae and Lernaeidae are monophyletic. In the Ergasilidae, the genera Sinergasilus and Paraergasilus are monophyletic, and Ergasilus is paraphyletic. Except the uncertain position of E. scalaris and E. yaluzangbus, copepods in the Ergasilidae can be further separated into three main clades, i.e. the Sinergasilus-Eragsilus clade including species in the Sinergasilus and E. anchoratus in the Ergasilus, the Pseudergasilus-Ergasilus clade including P. parasiluri in the Pseudergasilus and some in the Ergasilus, and the Paraergasilus-Ergasilus clade including species of Paraergasilus and E. peregrinus in the Ergasilus. All the three trees generated by using maximum parsimony, neighbor-joinning and Bayesian inference methods support the Pseudergasilus-Ergasilus clade has a closer phylogenetic relationship with the Sinergasilus-Eragsilus clade based on 18S rDNA，and a closer relationship with the Paraergasilus-Ergasilus clade based on 28S rDNA sequences. The evolutionary rule of parasitic copepods always from half-parasitism to complete parasitism supported the results based on 18S rDNA. Kishino-Hasegawa tests based on either likelihood method or parsimony method supported the null hypotheses that E. scalaris and E. peregrinus were confined to group Pseudergasilus-Ergasilus, and rejected the hypothesis that E. anchoratus was confined to group Pseudergasilus-Ergasilus. In the Lernaeidae, the subfamily Lamprogleninae was monophyletic, as revealed by the maximum parsimony, neighbor-joining and Bayesian inference methods based on 28S rDNA sequences, which is consistent with the morphology taxonomy.
The phylogenetic relationship was also examined between parasitic copepods and free-living copepods from five orders in the subclass Copepoda based on partial 18S rDNA sequences. The cladistic analysis based on morphological characters showed that Siphonostomatoida formed a sister-lineage with Monstrilloida, then clustered with Poecilostomatoida, while Cyclopoida showed no closer relationship with any of the three orders, while the present study supported the sister-lineage of Poecilostomatoida and Cyclopoida, which then were clustered with Siponostomatoida. It is suggested that the Poecilostomatoida and Cyclopoida have a closer relationship, furthermore, Poecilostomatoida, Cyclopoida and Siphonostomatoida might have originated from a common ancestor. Phylogenetic study also showed that Lernaeidae and Caligiade should belong to Cyclopoida and Siponostomatoida, respectively.
In order to resolve the phylogenetic relationship among the three species in the Sinergasilus and the mechanisms involved in their speciation on their nine species of fish hosts, three species in the Sinergasilus species were obtained to construct the phylogenetic trees based on the internal transcribed spacer (ITS) sequences, i.e. S. polycolpus, S. major and S. undulatus, from two speices of host fish, i.e. Hypophthalmichthys molitrix, Aristichthys nobilis, five speices of fish, i.e. Ctenopharyngodon idellus, Mylopharyngodon piceus, Elopichthys bambusa, Squaliobarbus curriculus, Opsariichthys bidens, and two species Cyprinus carpio, Carassius auratus, respectively. All the trees generated showed that S. polycolpus and S. major had a closer relationship. Meanwhile, thirty specimens each for the three species in the Sinergasilus were obtained from C. idellus, H. molitrix and C. carpio in a same lake. It is revealed that the variation in the ITS sequences was among 0.28~0.51% for individuals within a given species, while the variation ranged among 9.65~10.81% at the species level based on the combined ITS1 and ITS2 sequences. Phylogenetic relationship of the 90 copepod individuals also supported that S. polycolpus and S. major had a closer relationship. The coevolutionary trees were constructed by using partial cyt b sequences for the nine species of fish and by using whole ITS sequences for the three species of copepods. It is demonstrated that S. undulatus might have cospeciated with their Cyprininae hosts earlier, and S. polycolpus and S. major, might cospeciate with their Hypophthalmichthyinae and Leuciscinae hosts, respectively later.
In addition, during the investigation of parasites in freshwater fish in Danjiangkou Reservoir, Hubei Province, China, a new species named as Ergasilus danjiangensis was found on gills of Opsariichthys bidens and Zacco platypus. Morphological observation shows that E. danjiangensis is characteristic of prominent arc outer edge on the second segment of antenna, which is similar to E. tumidus. Molecular studies based on ITS and 28S rDNA sequences also support that E. danjiangensis is an independent and new species, although the divergence level between E. danjiangensis and E. braini (4.6% for ITS; 1.6% for 28S rDNA) is not high, nor between E. danjiangensis and E. tumidus (5.4% for ITS; 1.8% for 28S rDNA).|