The sequence of three mitochondrial genes viz., 16S rRNA, Cyt b, COI and one nuclear gene, 28S rRNA of Coranus Curtis species downloaded from the GenBank were subjected to phylogenetic analyses to understand the intrageneric and intraspecific variations and the role of geographical isolation on speciation using CLUSTAL W in MEGA version 5.6. This analysis includes thirteen species of Coranus Curtis and probably two ecotypes of Coranus callosus Stål from four countries viz., Australia, Brunei, China and Nigeria of three continents viz., Africa, Asia and Australia. The pairwise genetic distances were calculated and phylograms were constructed using maximum likelihood, neighbor-joining, minimum evolution, UPGMA and maximum parsimony methods. These preliminary analyses not only demarcated the thirteen species of Coranus and the two ecotypes of C. callosus but also revealed phylogenetic relationships and the role of geographical isolation on speciation.

Keywords: Coranus; 16S rRNA; Cyt b; COI and 28S rRNA; Harpactorinae; Biocontrol Agents; Intrageneric Molecular Biosystematics; Speciation; Ecotypes; Geographical Isolation;

Some assassin bugs have different morphs, biotypes, and ecotypes with various colors 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 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 [1,2,3,4,5]. Moreover, a multidisciplinary biosystematics is imperative to accurately identify reduviids and employ them against a particular insect pest [4,5,6,7]. Though the literature available on multi-disciplinary biosystematics of Reduviidae including molecular tools is available at family or species level it is very meager [5,8,9,10,11,12,13,14].

Curtis established the genus Coranus with Cimex subapterus De Geer as the type species. Coranus is one of the largest genera of subfamily Harpactorinae in the family Reduviidae with 100 known species worldwide [2,3,4,15]. The members of Coranus are widely distributed and occur throughout the Eastern Hemisphere with 30 Palaearctic, 21 Oriental, 41 Ethiopian and 17 Australian species [11]. Malipatil revised the Australian Coranus with redescription of seven species, description of eight new species and formulated key to identify the fifteen species [16]. Liu . inferred the phylogenetic relationship of the harpactorine genus Velinoides Matsmura with Coranus Curtis based on three mitochondrial genes (cyt b, CoI and 16S rRNA) and one nuclear (28S rRNA) gene [11]. Since they found molecular affinity between these two genera supported with morphological and cytogenetic characteristics they validated the status of genus Velinoides and its phylogenetic affinity with the genus Coranus. They suggested that these two genera could be two subgenera of the genus Coranus. They further reported that the 28S rDNA gene alone might not be an optimal marker for the phylogeny of the genus Coranus. Though twelve species of Coranus have been recorded from India none of its gene sequence is available. Except the work of Liu et al. no work on the molecular phylogenetics of the genus Coranus is available [4,11].

Hence, this study was undertaken based on the sequences of three mitochondrial genes, 16S rRNA, Cyt b, COI and one nuclear gene, 28S rRNA of thirteen species of Coranus Curtis and probably two ecotypes of Coranus callosus Stål from four countries viz., Australia, Brunei, China and Nigeria of three continents viz., Africa, Asia and Australia and probably two ecotypes of C. callosus Stål from western Australia downloaded from the GenBank (Table 1). The inclusion of Coranus species from four countries of three continents further enhances the scope of the work at the intraspecific level and the understanding on the role of geographical isolation in biosystematics.

To understand the intrageneric biosystematics and phylogenetics the sequences of three mitochondrial genes, 16S rRNA, Cyt b, Cyt c oxidase subunit I gene and one nuclear gene, 28S rRNA of thirteen species of Coranus Curtis and probably two ecotypes of C. callosus Stål (Tables 1,2) downloaded from the GenBank were subjected to 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 [17]. The five different methods were used to understand the utility of each method in the biosystematics.

Pairwise distances were carried out with gap opening penalty 15 and gap extension penalty 6.66 (Clustal W) [18].

The maximum parsimony analyses were analysed with MEGA5 [17]. Bootstrap method was used with 100 replications and gap/missing data treatment by complete selection and substitution based on nucleotide sequences [19]. The maximum parsimony tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm with search level 1 (Table 1) [20].

The evolutionary history was inferred based on the Tamura-Nei model [21]. 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).

The evolutionary history was inferred using the neighbor-joining method [22]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) was used [19]. The evolutionary distances were computed using the Tajima-Nei method [23].

The evolutionary history was inferred using the minimum evolution method [24]. 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 [24,25].

The evolutionary history was inferred using the UPGMA method [26]. 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 1^st+2^nd+3^rd+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 three mitochondrial genes, 16S rRNA, Cyt b and 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.

The ML tree constructed for the 16s rRNA gene of three undetermined and ten determined Coranus species has two major clusters (Figure 1). The first major cluster divides into two subclusters; with Coranus hammarstroemi Reuter that evolved as a separate lineage; the other species in this subcluster further divides into two minor clusters. One such minor cluster has two species viz., Coranus subapterus (De Geer) and Coranus emodicus Kiritschenko. The minor cluster further diversified into two clusters one with Coranus spiniscutis Reuter and Coranus lativentris Jakovlev and another with Coranus dilatatus (Matsumura) as a separate lineage and was distant to all other Coranus species [11]. Another cluster forms with two species viz., Coranus fuscipennis Reuter and Coranus marginatus Hsiao while maintaining its affinity with C. dilatatus on one hand and with the minor cluster of C. subapterus and C. emodicus on the other hand [11]. The second major cluster diversified into Coranus sichuensis Hsiao & Ren as a separate lineage as C. hammarstroemi of the first major cluster and further diversified into two subclusters as observed by Liu et al. [11]. The first cluster has only Coranus callosus Stål and second cluster has two undetermined

species Coranus sp.₁ and a further diversified cluster with two undetermined species Coranus sp.₃ and Coranus sp.₂.

The NJ (Figure 2) and ME trees (Figure 3) replicate the second major cluster as in ML tree. However, in the first major cluster, the positions of C. dilatatus and C. hammarstroemi vary. Though almost a similar kind of phylogeny is observed for UPGMA (Figure 4) and MP methods (Figure 5) slight deviations were found in relation to C. dilatatus in UPGMA and C. hammarstroemi and C. callosus in MP tree.

Eight species of Coranus from China form the first major cluster where as the ninth species C. sichuensis either diversified separately or clustered with Coranus of Australia, Brunei and Nigeria. The results reveal monophyly though they belong to four countries and three continents as observed by Cui & Huang in Orthoptera, Liu et al. in Coranus species of China, Ambrose et al. in Rhynocoris Kolenati (Harpactorinae) and Lenin in Acanthaspis Amyot and Serville, Edocla Stål, Empyrocoris Miller and Velitra Stål (Reduviinae) and Manimuthu (Manimuthu et al.,) in Ectomocoris Mayr and Catamiarus (Serville) (Peiratinae) species of Reduviidae from India [5,11,12,13,14,27].

Coranus sp.₂ of Australia instead of clustering with C. callosus of Australia clustered with Coranus sp.₃ of Nigeria and Coranus sp.₁ of Brunei. These species exhibit affinity despite their geographical isolation as observed by Mahendran et al. in silk producing insects and Ambrose et al. in R. fuscipes (Fabricius) of India with R. segmentarius (Germar) of South Africa [5,28].

Cyt b. The five phylograms observed for eight Coranus species of China except C. emodicus formed into two major clusters (Figure 6,7,8,9 and 10). The first cluster had C. spiniscutis, C. hammarstroemi, C. subapterus and C. sichuensis and the second cluster had C. dilatatus, C. lativentris, C. fuscipennis and C. marginatus with slight modification in different phylograms, revealing monophyly as observed by Liu et al. in Coranus species of China, Baskar et al. and Ambrose et al. in Rhynocoris species of India [5,11,29].

Cyt c. The five phylograms (Figure 11,12,13,14 and 15) of Cyt c gene of three undetermined species of Coranus from Australia, Brunei and Nigeria C. callosus from western Australia, probably from two localities, i.e., two ecotypes revealed affinity between Coranus sp.₁ of Brunei with Coranus sp.₂ of Australia. Coranus sp.₂ of Australia thus instead of clustering with C. callosus of Australia aligns with that of Coranus sp.₁ from Brunei. Similarly two ecotypes of Coranus callosus of Western Australia instead of

aligning together, one aligns with Coranus sp.₃ of Nigeria while another with Coranus sp. of Brunei and Coranus sp.₂ of Australia. Thus, monophyly phylogenetic affinity is pronounced despite geographical isolation. However, we admit that it is premature to suggest the role of geographical isolation without knowing the molecular characteristics such as number of segregating sites, nucleotide diversity and haplotype diversity and the geographical genetic structure. Moreover, the quantity of sampling is too small. Baskar (2010) and Baskar et al. (2012, a, b, c) reported genetic diversity among the ecotypes of four Indian Rhynocoris species viz., R. kumarii Ambrose and Livingstone, R. marginatus (Fabricius), R. longifrons (Stål), and R. fuscipes (Fabricius) based on mitochondrial genes and correlated the affinity with the ecological diversity of semiarid, scrub jungle, and tropical rainforest habitats [29,30,31,32]. Ambrose et al. (2014) also reported a similar phenomenon in four ecotypes of R. kumarii [5]. The present results corroborates with the findings of Liu et al. (2009), Baskar (2010), Baskar et al. (2012 a, b, c) and Ambrose et al. (2014) suggesting the existence of genetic diversity, with low level of gene flow in Coranus species [5,11,29,30,31,32]. However, these observations are contrary to those of Giordano et al. (2005) in Triatoma infestans (Klug) [33]. This contradiction might be the result of the non-dispersal haematophagus feeding behaviour of Triatoma in contrast to the dispersal predatory behavior of Coranus. The findings further suggest that the Cyt b fragment is a useful marker to describe the genetic structure of ecotypes of

closely related habitats (Naranjo et al., 2010) [34].

28S rRNA. Five phylograms (Figure16,17,18,19,20) were constructed for twelve species of Coranus, i.e., except Coranus sp.₃. In maximum likelihood method (Figure 16), all the nine Coranus species from China formed a major cluster. Coranus sp.₁ of Brunei diversified as a separate lineage. From this common node a sub cluster formed with two Australian species viz., C. callosus and Coranus sp.₂. An almost similar kind of phylogency is revealed by NJ, ME, UPGMA and MP methods (Figure 17,18,19,20). Thus, the affinity between the nine species of Coranus from China and that of two species from Australian is well pronounced. Although Liu et al. (2009) reported that 28S rRNA is a highly conserved gene and may not be an optimum molecular marker for Coranus, the present analysis contradicts their view and suggests its usefulness in phylogenetics [11].

The results obtained not only have enriched our knowledge on Coranus biosystematics but also supplemented multidisciplinary data of the genus. The results further reveal the utility of mitochondrial gene 16S, Cyt b and Cyt c oxidase subunit I and nuclear gene 28S rRNA sequence in phylogenetic analysis in Coranus. The findings further suggest intraspecific and interspecific phylogenetic affinity of Coranus species from four countries and three continents. Moreover, the genetic diversity observed among probably two ecotypes of C. callosus, from western Australia suggest progression of speciation warranting further studies in this direction that could lead to meaningful revision, regrouping or replacement of species with new revelation through molecular analysis. The analysis further suggests the usefulness of Cyt b fragment as a useful marker to understand the phylogenetics of ecotypes of closely related habitats and that of 28S rRNA as an optimum molecular marker for Coranus. However, our sampling of only 13 species of the genus Coranus which has more than forty species emphasizes further studies with more species.

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). The authors thank Prof. Carl W. Schaefer, University of Connecticut, USA, and Prof. Wanzhi Cai, China Agricultural University, China for their guidance and timely support respectively. The authors also gratefully acknowledge the anonymous reviewers whose review helped them to improve the quality of the manuscript.

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