Genetic and phylogenetic analyses based on DNA sequences are able to reveal species groups and assign unknown individuals to species, as well as infer evolutionary relationships to identify species and understand phylogenetic relationships of ticks.
In this study, we report the genetic characterization of species of ticks based on 12S rRNA and co1 gene loci from Chongming Eco-Island of Shanghai. Homology analyses revealed four species of ticks, including Ha. flava (97.11%), Ha. longicornis (2.61%), and Ha. doenitzi (0.21%), and Ha. japonica (0.07%). Haemaphysalis flava has a broad geographic distribution in Asia [2]. It was mainly distributed in Hubei Province [32], Hunan Province, and Southwest China [33]. Previous studies reported that Rhipicephalus sanguineus and Ha. longicornis were the dominant tick species in Shanghai [34]. However, the number of Ha. flava on Chongming Island accounted for the largest proportion in this study. Haemaphysalis flava can cause lesions, dermatitis, blood loss, weight loss, and even death as a result of direct bites. It is also a vector of pathogens such as Borrelia burgdorferi [35, 37], severe fever with thrombocytopenia syndrome virus [38], and tick-borne encephalitis virus [39], making it of great medical and veterinary importance in China and worldwide. An epidemiological survey of ticks and tick-borne pathogens in pet dogs in southeastern China reported Ha. longicornis [35], which is by far the most widely distributed and influential tick species, exposing over 40% of the nation’s population in 1140 counties of eastern and northeastern China. Babesia gibsoni is generally transmitted by Ha. longicornis. The species is regarded as the vector of severe fever with thrombocytopenia syndrome, an emerging tick-borne zoonosis [36] carrying Huaiyangshan viruses, a novel tick-borne Bunyavirus discovered in China and later in South Korea and Japan [37]. The geographical area of the distribution of Ha. doenitizi includes China, Japan, Myanmar, Laos, India, Thailand, Malaysia and Australia [38]. The identification of Ha. doenitizi on Chongming Island in this study is consistent with findings from previous studies across China, which found that it was mainly distributed in Fujian, Yunnan, and Taiwan [35]. Haemaphysalis doenitizi can transmit a variety of pathogens, such as Babesia, posing a threat to humans and wildlife [36]. This study also identified on Chongming Island a tick species never reported before in Shanghai, Ha. japonica. Haemaphysalis japonica is mainly distributed in northeastern of China, including Heilongjiang, Jilin, Liaoning, Gansu, Ningxia, Hebei, Shanxi, and Inner Mongolia [35], and was suspected to transmit Lyme borreliosis in humans in Korea [37]. The intraspecies identity for the Ha. flava and Ha. longicornis species (99.11% and 100% identity, respectively) identified in this work is similar to that reported in previous studies [32–35]. However, the single species of Ha. japonica identified in this study had only 83.48% identity compared to those previously reported, especially in Heilongjiang, Jilin, Liaoning, Gansu, Ningxia, Hebei, Shanxi, and Inner Mongolia [35].
Phylogenetic analyses based on the 12S rRNA and co1 gene loci showed species specificity of the Chongming ticks, which clustered with homologous sequences from China and worldwide, and revealed their relationship with orthologous sequences in Ha. cornigera, Ha. hystricis, Ha. concinna, and Ha. verticalis. There are six subgenera under the category of structurally primitive and advanced subgenera of Haemaphysalis, and Ha. longicornis and Ha. hystricis belong to the same group of Kaiseriana subgenus [39, 40]. Within the first phylogroup based on the 12S rRNA gene, the Chongming longicornis ticks identified in this study were closely related to those from Hebei Province (MK439888) and Guangdong Province (MW642389). They formed a lineage and had a closer relationship with Ha. cornigera and Ha. doenitzi than with Ha. flava. In the second phylogenetic group, Ha. cornigera and Ha. doentizi formed a lineage, and the Chongming doentizi ticks were close to Ha. doentizi from Hubei and Sichuan. This finding aligns with the geographical areas of the distribution of Ha. doenitizi in China [41]. The third phylogenetic group included Ha. japonica and Ha. concinna, which clustered into one taxon. The Chongming japonica tick was found to be close to that from Heilongjiang. Within the fourth phylogroup, Ha. flava grouped merely in consistency with homologs from China and worldwide.
The primary function of DNA barcoding is accurate species identification. Over the years, DNA barcoding has been used to identify specimens and discern species. DNA barcoding, using a universal gene region and a routine analytical procedure, has greatly promoted the identification of species and the discovery of new species in a wide variety of animal taxa [42, 43]. Mitochondrial DNAs (mtDNAs) have been widely employed in medically important parasites and vectors for species identification and diversity [44–46]. We found a substantial overlap between the intra- and interspecific K2P distances in Ha. japonica, while the other species showed clear boundaries of barcoding gap (range 0.0040–0.0723). Similar findings in mosquito species Anopheles annulipes have been reported previously, where an overlap between the intra- and interspecific K2P divergences of congeneric sequences based on co1 sequences was observed [47, 48]. In this study, the overlapping region in Ha. japonica may be due to the limited number of japonica tick samples or limitation in the quality of sequencing reads. However, more than 96% of 12S rRNA fragments had clear interspecific boundaries, suggesting that 12S rRNA is a good biomarker to identify interspecific relationships during biological evolution better than intraspecific relationships.
Genetic distance refers to the degree of genetic differences between different populations or species. The size of the genetic diversity of a species is the product of long-term evolution and is the premise of its survival, adaptation and development [49]. In this study, the pairwise distance between Ha. japonica and Ha. doentizi and Ha. longicornis were 0.1728 and 0.1666, respectively. The K2P distance between Ha. japonica and Ha. flava was 0.1236, and the two species clustered into one taxon in the phylogenetic tree. In the 12S rRNA topology, Ha. japonica clustered separately from the other three species, suggesting that 12S rRNA may not be a suitable biomarker for Ha. japonica identification. The genetic diversity indicines based on the 12S rRNA gene loci revealed that Ha. japonica has the largest number of polymorphic loci (n = 56) and the highest nucleic acid diversity (Pi) estimated at 0.0833. Overall, the high genetic diversity observed in the tick species involved in this study suggests evidence of directional selection of tick species [50]. This is consistent with the evidence that the higher the genetic diversity or the richer the genetic variation of a species is, the stronger the ability to adapt to environmental changes, making it easy for the species to expand distribution and open up new environments. Neutrality tests for all the tick species, reflected in Fu and Li’s D and Tajima’s D values, were all negative (p > 0.05). This indicates that the Chongming ticks might have experienced population expansion or genetic hitchhiking [50, 51], which is proportional to the magnitude of their genetic variation.