Research Article - (2016) Volume 5, Issue 1
The phylogenetic analysis of a nuclear gene, 28S ribosomal RNA of two determined and three undetermined species of Ectomocoris Mayr and the mitochondrial gene, cytochrome C of three determined and one undetermined species of Ectomocoris and one species of Catamiarus (Serville) from Australia and Asia was made. It reveals the interspecific and intrageneric phylogenetic affinity among Ectomocoris and intergeneric affinity between Ectomocoris and Catamiarus. Moreover, the Cty c sequence analysis warrant further studies on the undetermined species of Ectomocoris and the validation of Catamiarus as genus or subgenus of Ectomocoris. The study also proves the usefulness of 28S rRNA and Cyt c oxidase subunit I genes as useful molecular markers in the phylogenetic analysis of the peiratine genera Ectomocoris and Catamiarus.
Keywords: Ectomocoris; 28S rRNA; COI; Peiratinae; Biocontrol agents; Intrageneric and intergeneric molecular biosystematics
Some assassin bugs have different morphs, biotypes, and ecotypes with various colours and shapes which often mislead a museum entomologist in recognizing the morphs and ecotypes of a particular species.
Hence, classifications of Reduviidae based on morphological characters [1-3] may at times become insufficient, and there is an urgent need for a cohesive meaningful classification of Reduviidae based on ecological, morphological, behavioural, cytological, and biochemical data [4-6]. Moreover, a multidisciplinary biosystematics is imperative to accurately identify reduviids and employ them against a particular insect pest [4-7]. Literature available on multidisciplinary biosystematics of Reduviidae including molecular tools is very meagre [6,8-10].
Though assassin bugs of subfamily Peiratinae are abundant and have worldwide distribution with about 32 genera and 300 species [3,11-17] only a few taxa have been studied in detail [18]. Cai and Lu [19] studied the Chinese species of Ectomocoris . But no such work is available on the Ectomocoris and Catamiarus species of other geographical regions. Since classification of Peiratinae is being subjected to constant changes an attempt is made here to understand the intrageneric phylogenetic relationship of species of Ectomocoris from Australia and Asia and their relationship with Catamiarus , represented by only one species Catamiarus brevipennis (Serville) endemic to India.
This study was undertaken based on the available a mitochondrial gene, COI and a nuclear gene, 28S rRNA of ten peiratine species: nine species of Ectomocoris Mayr and one species of Catamiarus (Serville) [Table 1]. The inclusion of Ectomocoris species from Asian countries and Australia, two continents further enhances the scope of the work at the intraspecific level and the understanding on the role of geographical isolation in biosystematics.
Species | Distribution | Reference |
---|---|---|
Catamiarus brevipennis (Seville) | India | 3 |
Ectomocoris atrox (Stål) | Bhamao Island, Burma, Cambodia, China, India, Indonesia (Borneo, Celebes, Java and Sumatra), Malaysia, Philippines, Sri Lanka | 3, 24 |
Ectomocoris ornatus (Stål) | Australia | 3, 25 |
Ectomocoris cordigerStål | India, Iraq (Hinaidi, near Baghdad), Iran and Sri Lanka | 3 |
Ectomocoris quadriguttatus (Fabricius) | India | 3 |
Ectomocoris tibialis Distant | India | 3 |
Ectomocoris sp. | Australia | 26 |
Ectomocoris sp.1 | Australia | |
Ectomocoris sp.2 | Australia | |
Ectomocoris sp.3 | Australia |
Table 1: Ten species of Peiratinae: nine species of Ectomocoris and Catamiarus brevipennis subjected to phylogenetic analyses with their distribution.
Taxon sampling
To understand the intrageneric and intergeneric biosystematics and phylogenetics, the sequences of one mitochondrial gene, Cyt c oxidase subunit I gene of four Ectomocoris species and of Catamiarus brevipennis (Serville) and one nuclear gene, 28S rRNA of five species of Ectomocoris [Tables 1 and 2] were subjected to phylogenetic analysis. The genes of species of Ectomocoris viz., E. cordiger Stal, E. quadriguttatus (Fabricius) and E. tibialis Distant and C. brevipennis were sequenced by the authors and the remaining sequences were obtained from the GenBank.
Genes | Species | Genbank Accession Number |
---|---|---|
28S ribosomal RNA | Ectomocorisatrox (Stål) | KP236926.1 |
Ectomocoris ornatus(Stål) | FJ230595.1 | |
Ectomocorissp.1 | JQ942214.1 | |
Ectomocorissp.2 | FJ230761.1 | |
Ectomocorissp.3 | FJ230682.1 | |
Cytochrome c oxidase subunit I | Ectomocoris cordigerStål | KF056933.1 |
Ectomocoris quadriguttatus (Fabricius) | KF056934.1 | |
Ectomocoris tibialis Distant | KF056932.1 | |
Ectomocorissp. | GU198517.1 | |
Catamiarus brevepennis (Serville) | KF056931.1 |
Table 2: Gene sequences of ten species of Peiratinae: nine species Ectomocoris species and Catamiarus brevipennis with their GenBank accession number.
Phylogenetic analysis
The gene sequences were subjected into pairwise distance analysis and the phylogenetic trees were constructed based on maximum likelihood and neighbor-joining, maximum evolution, UPGMA and maximum parsimony methods with MEGA 5 software [20]. The five different methods were used to understand the utility of each method in the biosystematics.
Pairwise alignment
Pairwise distances were carried out with gap opening penalty 15 and gap extension penalty 6.66 (Clustal W) [21].
Maximum parsimony
The maximum parsimony analyses were analysed with MEGA5 (20). Bootstrap method was used with 100 replications and gap/ missing data treatment by complete selection and substitution based on nucleotide sequences [22]. The maximum parsimony tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm [23] with search level 1 [Table 1].
Maximum likelihood
The evolutionary history was inferred based on the Tamura- Nei model [27]. Initial tree for the heuristic search was obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach and then selecting the topology with superior log likelihood value [Table 2].
Neighbor-joining
The evolutionary history was inferred using the neighbor-joining method [28]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) was used [22]. The evolutionary distances were computed using the Tajima-Nei method [29].
Minimum evolution
The evolutionary history was inferred using the minimum evolution method (30). The optimal tree with the sum of branch length =8.45674115 is shown. The confidence probability (multiplied by 100) was estimated using the bootstrap test [30,31].
UPGMA
The evolutionary history was inferred using the UPGMA method [32]. The optimal tree with the sum of branch length =8.42786450 is shown.
The substitution type based nucleotide sequences and the codon positions included were 1st+2nd+3rd+Noncoding and all the positions containing gaps and missing data were eliminated in all the five methods. Five phylograms were thus constructed based on maximum likelihood (ML), neighbor-joining (N-J), maximum evolution (ME), UPGMA and maximum parsimony (MP) methods for one mitochondrial gene, Cyt c oxidase subunit I and one nuclear gene, 28S rRNA. The trees were analyzed based on the arrangement of each species in the tree.
28S rRNA
The ML tree based on 28S rRNA sequence shows a major cluster and two separate minor branches of the first cluster shows the affinity between E. atrox, E. ornatus and Ectomocoris sp.1 whereas the Ectomocoris sp.3 stands distinctly as a separate branch but closer to the major cluster. The Ectomocoris sp.2 also stands as a separate branch but distinctly away from other species (Figure 1).
The NJ (Figure 2) and ME trees based on 28S rRNA sequences almost replicate the affinity observed in ML tree. But Ectomocoris sp.2 took the position of Ectomocoris sp.3 and vice versa. However, the intergeneric affinity among E. atrox, E. ornatus and Ectomocoris sp.1 is maintained (Figure 3).
Though the UPGMA tree based on 28S rRNA exhibits a similar affinity between E. atrox, E. ornatus and Ectomocoris sp.1 the uniqueness of E. atox and the closer affinity between E. ornatus and Ectomocoris sp.1 by forming a cluster with E. ornatus and Ectomocoris sp.1 and a separate branch of E. atrox are visible. Moreover, the affinity between these three species with Ectomocoris sp.2 and sp.3 is similar to that exhibited by ML tree (Figure 4).
The MP tree based on 28S rRNA also shows the intrageneric affinity of E. atrox, E. ornatus and Ectomocoris sp.1 (Figure 5) as observed in the ML, NJ, ME and UPGMA trees but the closer affinity exhibited between E. ornatusand Ectomocoris sp.1 is revealed.
Two species of Ectomocoris viz., E. ornatus and Ectomocoris sp.1 from Australia form the first major cluster with E. atrox from Asia where as the remaining two species from Australia diversified into two separate lines. The results reveal monophyly though they belong to two continents as observed by Liu et al., [33] in Coranus (Reduviidae: Harpactorinae), Cui and Huang [34] in Orthoptera and Ambrose et al., [6] in Rhynocoris Kolenati species of Reduviidae from India. They exhibit affinity despite their geographical isolation as observed by Mahendran et al., [35] in silk-producing insects and Ambrose et al., [6] in R. fuscipes of India with R. segmentarius (Germar) of South Africa. However, we admit that it is premature to suggest the rold of geographical isolation without knowing the molecular characteristics such as number of segregating sites, nucleotide diversity and haplotype diversity and the geographical genetic structure. Although Liu et al., [33] reported that 28S rRNA is highly conserved gene and may not be an optimum molecular marker for phylogenetic studies on a Harpactorine genus, Coranus Curtis, the present analysis contradicts their view and suggests its usefulness in the phylogenetic analysis of Ectomocoris .
Cyt c
The five phylograms (Figure 6-10) of Cyt c gene of three determined species of Ectomocoris viz., E. cordiger , E. quadriguttatus and E. tibialis from Asia, one undetermined species of Ectomocoris from Australia and C. brevipennis an endemic species from India revealed close affinity among them. The Asiatic E. quadriguttatus is closely allied with the other two Asiatic species viz., E. cordiger and E. tibialis but interestingly distantly related to them than to C. brevipennis . This is evidenced by the existence of a separate diversified lineage of E. quadriguttatus in ML, NJ and ME trees based on Cyt c gene sequences. In the UPGMA tree, in addition to the closer affinity exhibited between E. quadriguttatus and E. tibialis and these two species are distantly aligned with E. cordiger . Here again, C. brevipennis forms a diversified branch but closer to all the three Asian Ectomocoris species and clearly demarcated from the Ectomocoris sp. of Australia. Interestingly, C. brevipennis and E. cordiger diversified as a separate cluster and E. tibialis form a separate branch. Moreover, E. quadriguttatus from Asia clustered with Ectomocoris sp. of Australia suggesting intrageneric affinity despite geographical isolation. However, we admit that it is premature to suggest the role of geographical isolation without knowing the molecular characteristics such as the number of segregating sites, nucleotide diversity and haplo type diversity and the geographical genetic structure. Moreover, the quantity of sampling is too small. Except in MP tree, the undetermined Australian Ectomocoris sp. stands as a separate line away from the three Asian Ectomocoris species and the lone Indian endemic Catamiarus brevipennis suggesting the requirement of further studies to validate the taxonomic position of Australian Ectomocoris . Similarly, the genetic status of Catamiarus has to be validated either as a separate genus as it now exists or to place it as a subgenus of Ectomocoris .
Thus, intrageneric phylogenetic affinity among the four Ectomocoris species despite geographical isolation as well as intergeneric affinity between Ectomocoris and Catamiarus are revelaed. Baskar [36], Baskar et al., [37-39] and Ambrose et al., [6] reported genetic diversity among four harpactorine reduviid species of Rhynocoris viz., R. kumarii Ambrose and Livingstone, R. marginatus (Fabricius), R. longifrons (Stal) and R. fuscipes (Fabricius) based on mitochondrial genes and the present results corroborate with their findings. Hence, the existence of genetic diversity, with low level of gene flow might be operating among Ectomocoris and Catamiarus species as Zhao and Zhu [40] observed in Branchiostoma japanicum Lonnberg. Moreover, these observations again support the monophyly of family Reduviidae as observed by Liu et al., (33) and Ambrose et al., [6]. However, these observations are contrary to those of Giordano et al., [41] in a haematophagous reduviid, Triatoma infestans (Klug). This contradiction might be the result of the non-dispersal haematophagous feeding behaviour of Triatoma in contrast to the dispersal predatory behavior of Ectomocoris and Catamiarus . The findings further suggest that the Cyt c oxidase subunit I gene is a useful marker to understand the intrageneric and intergeneric affinity [42].
The results obtained not only have enriched the knowledge on biosystematics of Ectomocoris and Catamiarus but also supplemented multidisciplinary data of the two genera. The results further reveal the utility of mitochondrial gene Cyt c oxidase subunit I and nuclear gene 28S rRNA sequence in phylogenetic analysis in Ectomocoris and Catamiarus , i.e., as useful markers to understand the intrageneric affinity of Ectomocoris and its intergeneric affinity with Catamiarus . The findings further suggest intraspecific and interspecific phylogenetic affinity of Ectomocoris species from two continents. However, our study based on the available genetic sequences of about 10 per cent of the total number of Ectomocoris species emphasizes the need of further studies on the genetic sequencing and analysis of the genus Ectomocoris . Further analyses with more number of species will reveal their better phylogenetics.
The authors are grateful to the authorities of St. Xavier’s College (Autonomous), Palayamkottai, Tamil Nadu, India for facilities. We are grateful to the Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi, for financial assistance (Ref. No. 21(0865)/11/EMR-II, 2012-2013 dated 28.12.2011).