RAPD STUDIES RAPD STUDIES

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1 RAPD STUDIES RAPD STUDIES The ability of PCR based markers to reveal inter- and intraspecific variations among and between the populations subsequently makes it useful for screening different parameters of genetic diversity like gene mapping, genetic differentiation, linkage analysis etc. The chapter deals with the importance of Random Amplification of Polymorphic DNA (RAPD) markers in order to supplement the taxonomic status of studied species. RAPD analysis of 17 species (Figs ) has been represented with agarose gel band photograph (a), tabulated form of variability matrices in percentages (b) and cladogram (c). It has been theoretically predicted in genetic history that local population sizes and population turnover among the populations of the same species strongly bring about the changes in genetic diversity and genetic differentiation. The possible reasons for the same may be interpreted as the populations of the same species that are geographically isolated (allopatric populations) have to adapt to different climatic conditions. It may result in bottle neck populations, effects of genetic drift causing a change in even single allele of a gene pool (due to fixation or mutation) leading to decrease in gene flow among them, resulting in substantial decrease in genetic diversity and increased genetic differentiation throughout the species range. The results of RAPD profiles in the present study clearly suggest that the genetic variability or dissimilarity comes out to be moderately significant in most of the populations of different species collected from different States of North India i.e. Punjab, Himachal Pradesh, Uttarakhand, Jammu and Kashmir. Some of the regions have extreme types of vegetation, especially the dense forest areas of Himachal Pradesh, differential vegetations in Uttarakhand plains and Hilly areas, as compared to exclusive food crops vegetation of Punjab. Morphologically similar populations of Eysarcoris montivagus (Distant) (Fig a, b, c), collected from Jammu and Kashmir (S 76), Himachal Pradesh (S 77), Uttarakhand (S 78) and Punjab (S 79) with similar external male and female genitalia, show significant different RAPD profiles as suggested by genetic variability index in terms of percentages i.e. Between S 76 - S 77: 62.00%, S 76 - S 78: %, S 76 - S 79: 62.50%, S 77 - S 78: 52.00%, S 77- S 79: 62.00%, S 78 - S 79: %. The cladogram for the same clearly suggests S 76 to be the parent population while S 77 and S 78 tend to form the sister clads revealing their more close relationship as compared with the parent population. The most 225

2 RAPD STUDIES appropriate explanation for the above results may be a recent divergence of various populations from the parent population, or may be due to the increased DNA evolution in a gene pool. The above geographically separated populations, however, are not separated by huge barriers; some are even available on the border lines of two areas, showing genetic variability that needs a critical review. As discussed earlier, founder effect tends to be the closest and appropriate reason for a variable profile, leading to decreased gene flow and increased genetic variability. There is another case where RAPD profiles showed significant variability index for the populations of predatory bug Perillus bioculatus (Fabricius) (Fig a, b, c) that exists in different colour morphs as red, orange and white forms collected from Jammu and Kashmir (S 73), Punjab (S 74) and Himachal Pradesh (S 75). The results suggested significant intraspecific variations; S 73 S 74: %, S 74 S 75: %, S 73 S75: %. This may be due to increased mobility of these predaceous bugs as compared to other non predaceous species like Nezara viridula Linnaeus (Fig. 544 a, b, c), Nezara antennata Scott (Fig a, b, c), Dolycoris indicus Stål (Fig a, b, c), and Dolycoris baccarum (Linnaeus) (Fig a, b, c), that show slight morphological variations with respect to colour and size, tend to show moderate intraspecific variations between the range of 40-60%. Carbula insocia (Walker) (Fig a, b, c) has shown maximum variabilty index of % among populations of Himachal Pradesh (S 82) and Punjab (S 83). Similar results were obtained for other predatory bug Eocantecona furcellata (Wolff) collected from Uttarakhand (S 59), Himachal Pradesh (S 60), Punjab (S 61) and Jammu and Kashmir (S 62) as S 59 S 60: 61.29%, S 59 S 61: %, S 59 S 62: %, S 60 S 61: %, S 60 S62: %, S 61 S 62: %, indicating maximum diversity among populations of Uttarakhand and Himachal Pradesh. RAPD profiles for Erthesina fullo (Thunberg) (Fig a, b, c), clearly suggested that long distances lead to decreased gene flow and increased genetic variability among populations of Himachal Pradesh (S 99), Punjab (S 100) and Uttarakhand (S 101) as S 99 S 100: %, S 99 S 101: %, S 100 S 101: % indicating higher intraspecific variations among populations of Himachal Pradesh-Punjab and Punjab-Uttarakhand as compared to Himachal Pradesh and Uttarakhand. Similar results are obtained in other studied species. 226

3 RAPD STUDIES Therefore, the present results clearly indicate the importance of RAPD that requires no prior knowledge of DNA sequence, to analyse the all important intraspecific variations among geographically separated allopatric populations that usually go unnoticed during taxonomical studies. The technique has the ability to reveal significant genetic differentiation and population structure of a well established species. However, the present results show that significant level of intraspecific variations are seen in morphologically similar populations, but there is no trend of increase or decrease in variation for different species as far as distance between two particular areas is concerned, as some populations tend to show significant variation between Himachal Pradesh and Uttarakhand, and vice versa in others. So, it cannot be concluded that only distance can bring variations. Other factors might be involved in this process that may be clarified by DNA sequencing of the samples. These RAPD profiles suggest that migratory events do occur in natural populations of these agricultural pests, but the rate and mode of dispersion of these still need to be analysed. Since, most of these are serious agricultural pests, it becomes necessary to determine the molecular markers for populations of a species to evaluate their resistance or susceptibility to a particular insecticide, in order to avoid some unnecessary biocontrol methods and insecticide resistant management programmes to increase the overall yield of economically important crops. 227

4 General Conclusions and Scope for Further Research A monumental work on family Pentatomidae has been done by Distant (1902, 1908, 1918) in the series on Fauna of British India. His study dealt with morphological aspects that included general coloration and markings on head, antennae, pronotum, scutellum, abdomen and illustration of metathoracic scent gland. Being serious pests of many economically important agricultural and horticultural food crops, the focus on this group grabbed attention of many workers throughout the World, especially in countries like Australia and United States, but it was very vague in India. Some of the Indian authors who worked towards the taxonomy and some other related aspects of this group included: Pruthi (1925), Mathew (1969, 1980), Pawar (1974), Azim and Shafee (1979, 1981a, 1981b, 1982, 1983, 1986, 1987), Sucheta (1979), Azim (1980, 2011), Agarwal and Baijal (1982, 1984), Pajni and Sidhu (1982), Chopra and Sucheta (1986), Johri and Baijal (1992), Tyagi and Baijal (1992), Schaefer (1997), Mehta (2001), Dhiman et al. (2004), Pandey et al. (2005), Sucheta and Khohkar (2006),; Azim et al. (2008), Aland et al. (2010), Azim and Bhat (2010), Tara and Sharma (2010), Salini (2011), Ghate et al. (2012) and Kaur et al. (2012). As per the present scenario, Rider ( ) is preparing the World Catalogue for this family. The present work included the taxonomic study of this group from North India supplemented with RAPD markers. Extensive and intensive collection-cum-survey tours were made to cover the maximum far flung unexplored localities of Punjab, Haryana, Uttarakhand, Himachal Pradesh, parts of Jammu and Kashmir, Union territory of Chandigarh and NCR region of Delhi. A total of 52 species under 36 genera referable to three subfamilies; Pentatominae, Asopinae and Podopinae, were collected and their identification was confirmed with the help of keys and descriptions given in Distant s Fauna of Brisish India (1902, 1908, 1918) and from the published literature procured from national libraries and internet. The identification was also authenticated by comparison with the reference collection preserved in National Museums of India and from National History Museum, London. Out of the three subfamilies, Pentatominae forms the largest group of phytophagous bugs recognized by scutellum reaching half way to abdomen or passing 228

5 just slightly beyond it, but never reaching its apex. Out of the collected 52 species, 46 belong to this subfamily. One genus and species, Halyabbas unicolor Distant, has been reported as first record from India, while 23 species have been reported for the first time from North India. Some new localities have also been added for few studied species. Subfamily Podopinae is characterized by large U-shaped scutellum, almost reaching the apex of abdomen, covering more or less entire forewings. The present study includes Scotinophara ochracea (Distant), that has been reported for the first time from North India. The third subfamily, Asopinae, is primarily a predaceous family, characterized by elongate crassate labium considerably exceeding forecoxae and free basal labial segment, thickened and not concealed between bucculae. Five species belonging to this subfamily are included in the present work out of which two (Cazira verrucosa (Westwood) and Picromerus obtusus Walker) have been recorded for the first time from North India. Members of this family show a lot of variations with respect to shape of head that include its overall shape from being acuminate and tapering at apex as in Erthesina Spinola and Halys Fabricius, triangular as in Mecidea Dallas, subrounded distinctly broad at apex as in Paranevisanus Distant or truncated at apex as in Dalpada Amyot and Serville and Cahara Ghauri, length of lateral lobes with respect to central lobe, presence or absence of spines before apex and level of sinuosity. Various colour patterns and different types of punctures, specific markings, spots, callosities on pronotum and scutellum may act as reliable taxonomical tools for differentiating closely related genera and species e.g. species under genus Nezara Amyot and Serville have predominently green colour, while those of Eurydema Laporte and Bagrada Stål are distinctly bright colored while species under genera Eysarcoris Hahn and Carbula Stål have the shades of yellowish brown. Some populations within a species tend to show distinct colour polymorphism, e.g. populations of Nezara viridula (Linnaeus) and Nezara antennata Scott show orange or yellow streaks on head and anterior margins of pronotum while Perillus bioculatus (Fabricius) exists in different colour morphs (red, orange yellow, pale whitish) in the same geographical area. Labium usually shows variation with respect to the ratio of its length to that of abdomen at both generic and species levels. It may be short just reaching the middle coxae (Dalpada Amyot and Serville, Cahara Ghauri, Paranevisanus 229

6 Distant) or may extend upto 4 th abdominal segment (Halys Fabricius) or may even reach upto 7 th abdominal segment (Erthesina Spinola). Pronotum shows various reliable characters that are of immense taxonomic significance to differentiate various genera and most importantly closely related species. Anterolateral margins of pronotum may be denticulate (Halys Fabricius, Cahara Ghauri, Dalpada Amyot and Serville) serrated (Erthesina Spinola) or entire in most genera (Nezara Amyot and Serville, Eysarcoris Hahn, Carbula Stål etc.). They may be obliquely straight, moderately or strongly sinuous or even calloused. The humeral angles of pronotum are another character of taxonomic importance. They may be angulated prominently or even nodulated in some. Angulation varies a lot at generic as well as at species level i.e. obtusely angulated, acutely produced, spinose, bifid, subrounded without any prominent angulation. Metathoracic scent gland complex also shows variations at the generic level and slightly at specific level too. The peritreme may be very well developed or strongly reduced (Eurydema Laporte, Bagrada Stål), varying in distance it covers with respect to metapleural margin. The anterior and posterior margins of peritreme vary, but have less importance in terms of variability at species level. Legs have usually no taxonomical significance, except a few genera showing modifications as in Erthesina Spinola in which both anterior and posterior tibiae are dilated, while in genera like Dalpada Amyot and Serville, only anterior tibiae are dilated. As far as morphological variation of abdomen is concerned, it shows sexual dimorphism with respect to ventroanterior and ventroposterior margins of 7 th abdominal segment. In males, the ventroanterior margin is usually narrowly concave, an inverted V-shaped, while in females it is broadly concave, somewhat an inverted U-shaped. The genital segments of abdomen show distinct variation as external male and female genitalia, respectively modified for copulation and oviposition. In females, the 8 th and 9 th abdominal segments get modified to form the ovipositor. The shapes of 1 st gonocoxae, 2 nd gonocoxae, 9 th paratergites and ratio of lengths of 9 th paratergites to 8 th paratergites are important from taxonomical point of view. Similarly, the spermatheca, which shows variable number of processes on spermathecal bulb (one in Chaubattiana rubrovittata, Piezodorus rubrofasciatus and Palutia crossota, two in Nezara viridula, Nezara antennata, Dalpada jugatoria and Halyabaas unicolor); variable shape of bulb (elongated in Bagrada picta, oblong in 230

7 Aeliomorpha lineaticollis and Eocanthecona furcellata, flattened in Glaucias albomaculata, small lobe like in Acrosternum graminea, rounded in Carbula scutellata), the ratios of lengths of distal and proximal spermathecal ducts, ratio of distal spermathecal duct to pump region, presence or absence of proximal sclerotized dilation are some of the reliable taxonomical characters. In males, various parts of pygophore, which is the modified 9 th abdominal segment, shows important reliable differentiating tools, not only at generic level but also between very closely related species. Some characters of morphological variance include the overall shape of pygophore and dorsolateral lobes produced variably (subacutely, roundish, quardrate or even pointed posteriad). Pygophore inside contains a pair of parameres and an aedeagus. The overall shape of parameres (unilobed or bilobed.) provides a diagnostic feature for a species. The shape and size of its blade and presence or absence of any process or hump on outer or inner margins can be a differentiating key character both at generic and specific level. Aedeagus has considerable taxonomical significance consisting of proximal basal plate with appendages, and a distal theca (bilobed as in Andrallus Bergroth, Eocanthecona Bergroth or unilobed in others) containing inside ejaculatory reservoir, vesica and conjunctiva with membranous appendages (Dorsal, ventral and median). These appendages vary in number, being uni- or bilobed and in the level of sclerotization among various taxa. As far as molecular aspect is concerned, the RAPD analysis of 17 species belonging to 2 subfamilies (Pentatominae and Asopinae), collected from different states of North India (Punjab, Himachal Pradesh, Uttarakhand and Jammu and Kashmir) was done. The results show significant level of intraspecific variations between populations from different areas. It can be suggested on the basis of foregoing discussion that there is an urgent need to revise this family by covering maximum unexplored localities of India in order to arrive at a definite conclusion about the exact status of these agricultural pests in India. However, the future studies should also include molecular aspects, particularly DNA sequencing to supplement the taxonomy based on morphological characters only. The intraspecific variations within species that are morphologically similar clearly emphasize the importance and need of molecular techniques to study 231

8 some major and confusing species complexes that cannot be separated merely on the basis of morphotaxonomy and external genitalia. 232