Research Article - (2016) Volume 5, Issue 1

Intrageneric and Intergeneric Phylogenetics Based on Available Mitochondrial Genes and Nuclear Gene Variation among Ten Peiratine Species: Nine Species of Ectomocoris Mayr and One Species of Catamiarus (Serville) (Hemiptera: Reduviidae: Peiratinae)

Muthumari Manimuthu, Arumugam Ganesh Kumar and Dunston Philamon Ambrose*
Entomology Research Unit, St. Xavier’s College (Autonomous), Palayamkottai-627 002, Tamil Nadu, India
*Corresponding Author: Dunston Philamon Ambrose, Entomology Research Unit, St. Xavier’s College (Autonomous), Palayamkottai-627 002, Tamil Nadu, India, Tel: 0462 2560744 Email:

Abstract

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

Introduction

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.

Materials and Methods

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.

Results and Discussion

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).

gene-technology-tree-gene-variations

Figure 1: ML tree based on 28S rRNA gene variations showing the relationships of five Ectomocoris species.

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).

gene-technology-tree-rRNA-gene

Figure 2: NJ tree based on 28S rRNA gene variations showing the relationships of five Ectomocoris species.

gene-technology-ME-rRNA-relationships

Figure 3: ME tree based on 28S rRNA gene variations showing the relationships of five Ectomocoris species.

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).

gene-technology-UPGMA-method-tree

Figure 4: UPGMA method tree based on 28S rRNA gene variations showing the relationships of five Ectomocoris species.

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.

gene-technology-method-rRNA-variations

Figure 5: MP method tree based on 28s rRNA gene variations showing the relationships of five Ectomocoris species.

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 .

gene-technology-Cyt-subunit-species

Figure 6: ML tree based on Cyt c subunit like 1 gene variations showing the relationships of five Ectomocoris species.

gene-technology-tree-subunit-gene-variations

Figure 7: NJ tree based on Cyt c subunit like 1 gene variations showing the relationships of five Ectomocoris species.

gene-technology-Minimum-Evolution-Method

Figure 8: Minimum Evolution Method Tree Based On Cyt C Subunit Like 1 Gene Variations Showing The Relationships Of Five Ectomocoris Species.

gene-technology-Upgma-Method-Tree

Figure 9: Upgma Method Tree Based On Cyt C Subunit Like 1 Gene Variations Showing The Relationships Of Five Ectomocoris Species.

gene-technology-method-relationships-species

Figure 10: MP method tree based on Cyt c subunit like 1 gene variations showing the relationships of five Ectomocoris species.

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].

Conclusion

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.

Acknowledgements

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).

References

  1. Usinger RL (1943) A revised classification of the Reduvioidea with a new subfamily from S. America. Annals of the Entomological Society of America36:602-618.
  2. Putshkov PV, Putshkov VG (1985) A Catalogue of the Assassin Bug Genera of the World (Heteroptera: Reduviidae). Viniti, Lyubertsy1-138.
  3. Maldonado JC (1990) Systematic catalogue of the Reduviidae of the world (Insecta: Heteroptera) Special Edition of Caribbean Journal of Science1-694.
  4. Ambrose DP (1999) Assassin bugs. Science Publishers, New Hampshire, USA and Oxford and IBH Publishing Company Private Limited, New Delhi, India p: 337.
  5. Ambrose AD, Ambrose DP(2009) Predation, copulation, oviposition and functional morphology of tibia, rostrum and eggs as tools in the biosystematics of Reduviidae (Hemiptera) Indian Journal of Entomology71:1-17.
  6. Ambrose DP, et al.(2014) Intrageneric phylogenetics based on mitochondrial DNA variation among fifteen harpactorine assassin bugs with four ecotypes and three morphs (Hemiptera: Reduviidae: Harpactorinae). Zootaxa.3779:540-550.
  7. Ambrose AD, Ambrose DP (2003) Linear regression coefficient (r) of postembryonic developmental morphometry as a tool in the biosystematics of Reduviidae (Insecta: Hemiptera) Shaspa10:57-66.
  8. Weirauch C (2008) Cladistic analysis of Reduviidae (Heteroptera: Cimicomorpha) based on morphological characters. Systematic Entomology33:229-274.
  9. Patterson JS, Gaunt MW (2010) Phylogenetic multi-locus codon models and molecular clocks reveal the monophyly of haematophagousreduviid bugs and their evolution at the formation of South America. Molecular Phylogenetics and Evolution56:608-621.
  10. Hwang WS, Weirauch C (2012)Evolutionary history of assassin bugs (insecta: hemiptera: Reduviidae): insights from divergence dating and ancestral state reconstruction.PLoS One7:e45523.
  11. Coscaron MC, Morrone JJ (1995) Systematic, cladistic, and biogeography of the Peiratescollarti and P. lepturoides groups (Heteroptera: Reduviidae, Peiratinae) EntomologicaScandinavica26:191-228.
  12. Coscarón MC (1995) A new Neotropical genus of Peiratinae (Reduviidae, Heteroptera) The RevistaBrasileira de Entomologia39:453-457.
  13. Coscarón MC (1996) Synonimizing Spilodermus Stål under Peirates Serville, and revision of the resulting P. quadrinotatus species group (Heteroptera: Reduviidae: Peiratinae) Proceedings of the Entomological Society, Washington98:500-508.
  14. Coscarón MC (1997)Froeschnerisca nom. nov.for the junior homonym of FroeschnerielliaCoscarón (Heteroptera: Reduviidae) Physis Section C54:49.
  15. Gil-Santana HR, Costa LAA (2003) Um novo gênero e espécie de Peiratinae de Nova Friburgo, Rio de Janeiro, Brasil (Hemiptera, Heteroptera, Reduviidae) RevistaBrasileira de Zoologia21:3-8.
  16. Cai W, Tomokuni M (2004) Taxonomic notes on Vietnamese Peiratinae, with description of a new species (Hemiptera: Reduviidae) Oriental Insects38:85-94.
  17. Cai W, Taylor JS (2006) Lentireduvius, a new genus of Peiratinae from Brazil, with a key to the New World genera (Hemiptera: Reduviidae) Zootax1360:51-60.
  18. Chlond D (2007) Classification of the true bugs of the subfamily Peiratinae (Heteroptera: Reduviidae) Proceedings of the 8th Conference of the Polish Taxonomical Society, Wiechlice14:67-69.
  19. Cai W,Lu J (1990) A taxonomic revision of the Chinese species of SirtheneaSpinola (Hemiptera: Reduviidae: Peiratinae) Entomotaxonomia12:85-96.
  20. Tamura K, et al. (2011) MEGA5: Molecular Evolutionary Genetic Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution 28:2731-2739.
  21. Thompson JD, et al (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research22:4673-4680.
  22. Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783-791.
  23. Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics. Oxford University Press, New York p: 333.
  24. 25.http://www.gbif.org/occurrence/search?taxon_key=4774300&HAS_COORDINATE=true&HAS_GEOSPATIAL_ISSUE=false
  25. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees.Mol Biol Evol 10:512-526.
  26. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees.Mol Biol Evol4:406-425.
  27. Tajima F, Nei M (1984) Estimation of evolutionary distance between nucleotide sequences.Mol Biol Evol 1: 269-285.
  28. Rzhetsky A, Nei M (1992) A simple method for estimating and testing minimum evolution trees. Molecular Biology and Evolution 9: 945-967.
  29. Dopazo J (1994) Estimating errors and confidence intervals for branch lengths in phylogenetic trees by a bootstrap approach.J Mol Evol 38: 300-304.
  30. Sneath PHA, Sokal RR (1973) Numerical Taxonomy. Freeman, San Francisco.
  31. Liu H, Zhao P, Li S, Cai W (2009) Taxonomic status of Velinoides Matsumura (Hemiptera: Reduviidae: Harpactorinae) inferred from mitochondrial and nuclear genes. Zootaxa 2080: 55-68.
  32. Cui AM, Huang Y (2012) Phylogenetic relationships among Orthoptera insect groups based on complete sequences of 16S ribosomal RNA.Yi Chuan 34: 597-608.
  33. Mahendran B, Ghosh SK, Kundu SC (2006) Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes.J Genet 85: 31-38.
  34. Baskar A (2010) Molecular Genetic Variation and Phylogeny of Rhynocoris spp. Based on Mitochondrial Gene Analysis (Heteroptera: Reduviidae) Ph.D. Thesis, University of Madras.
  35. Baskar A, Ambrose DP, Tirumurugan KG, Fleming AT (2012a) Ecotypic diversity in the assassin bug, Rhynocoris marginatus Fabricius (Heteroptera: Reduviidae) Hexapoda 19: 38-46.
  36. Baskar A, Ambrose DP, Tirumurugan KG (2012b) Ecotypic diversity in the assassin bug, Rhynocoris kumarii Ambrose and Livingstone (Heteroptera: Reduviidae) Journal of Entomological Research 36: 369-375.
  37. Baskar A, Ambrose DP, Tirumurugan KG, Fleming AT (2012c) Ecotypic diversity in the assassin bug, Rhynocoris fuscipes Fabricius (Heteroptera: Reduviidae) Journal of Advanced Zoology 33: 133-121.
  38. Zhao Q, Zhu Q (2011) Taxonomic and genetic status of lancelet in Weihai coastal waters based on mitochondrial DNA sequence. Chinese Jour nal of Oceanology and Limnology 29: 623-32.
  39. Giordano R, Cortez JCP, Paulk S, Stevens L (2005) Genetic diversity of Triatomainfestans (Hemiptera: Reduviidae) in Chuquisaca, Bolivia based on the mitochondrial cytochrome b gene. Memórias do InstitutoOswaldo Cruz 100: 753-760.
  40. Naranjo BM, Zuriaga MA, Azofeifa G, Zeledon R, Bargues MD (2010) Molecular evidence of intraspecific variability in different habitat-related populations of Triatomadimidiata (Hemiptera: Reduviidae) from Costa Rica. Parasitological Research 106: 895-905.
Citation: Manimuthu M, Kumar AG, Ambrose DP (2016) Intrageneric and Intergeneric Phylogenetics Based on Available Mitochondrial Genes and Nuclear Gene Variation among Ten Peiratine Species: Nine Species of Ectomocoris Mayr and One Species of Catamiarus (Serville) (Hemiptera: Reduviidae: Peiratinae). Gene Technology 5:133.

Copyright: © 2016 Manimuthu M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.