MOLECULAR CHARACTERIZATION AND GENETIC DIVERSITY OF SNAILS IN AGROECOSYSTEM OF FAISALABAD

By

Javaria Altaf 2008-GCUF-5561-226 Thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

IN

ZOOLOGY

DEPARTMENT OF ZOOLOGY GOVERNMENT COLLEGE UNIVERSITY, FAISALABAD.

SEPTEMBER 2014

i DECLARATION

The work reported in this thesis was carried out by me under the supervision of Prof. Dr. Naureen Aziz Qureshi Department of Zoology, GC University, Faisalabad, Pakistan.

I hereby declare that the title of thesis “Molecular Characterization and Genetic Diversity of Snails in Agroecosystem of Faisalabad City” and the contents of thesis are the product of my own research and no part has been copied from any published source (Except the references, standard mathematical or genetic models / equations/ formulae/ protocols etc). I further declare that this work has not been submitted for award of any degree / diploma. The University may take action if the information provided is found inaccurate at any stage.

Signature of the Scholar______

Registration Number: 2008-GCUF-5561-226

ii CERTIFICATE BY THE RESEARCH SUPERVISOR

I certify that the contents and form of thesis submitted by Ms Javaria Altaf, Registration No. 2008-GCUF-5561-226 has been found satisfactory and in accordance with the prescribed format. I recommend it to be processed for the evaluation by the External Examiner for the award of degree.

Signature______

Prof. Dr. Naureen Aziz Qureshi

Supervisor,

Department of Zoology,

Government College University, Faisalabad.

Chairperson

Signature______

Dr. Farhat Jabeen

Department of Zoology

GC University, Faisalabad.

Dean / Academic Coordinator

Signature______

Faculty of Sciences and Technology,

GC University, Faisalabad.

iii SUPERVISORY COMMITTEE

Supervisor

Signature______

Name: Prof. Dr. Naureen Aziz Qureshi

Designation with Stamp______

Member-1

Signature______

Name: Dr. Shabana Naz

Designation with Stamp______

Member-2

Signature______

Name: Dr. Syed Hammad Raza

Designation with Stamp______

iv List of Contents

ACKNOWLEDGEMENTS...... xi ABSTRACT ...... 1 INTRODUCTION ...... 3 1.1 ...... 3 1.2 COLLECTION OF THE SNAILS...... 5 1.3 ECOLOGY ...... 6 1.4 GENETIC DIVERSITY OF SNAILS ...... 9 1.5 PROVIDING CONSERVATION MANAGEMENT UNIT INFORMATION TO THOSE CHARGED WITH CONSERVING ...... 13 1.6 FRESHWATER MALACOFAUNNA OF PAKISTAN ...... 14 2 REVIEW OF LITERATURE ...... 16 2.1 FRESH WATER GASTROPODS AS PRACTICAL CONSERVATION MANAGEMENT UNITS16 2.2 ECOLOGY ...... 18 2.3 QUADRAT SIZE AND SAMPLING...... 23 2.4 SAMPLING PREVIOUS GENERATIONS...... 25 2.5 DEFINING THE THEORETICAL CONSERVATION MANAGEMENT UNIT ...... 26 2.6 ABUNDANCE DISTRIBUTIONS...... 29 2.7 POPULATION AND EVOLUTIONARY GENETICS ...... 33 3 MATERIALS AND METHODS...... 37 3.1 STUDY AREA ...... 37 3.2 COLLECTION OF THE SNAILS...... 39 3.3 PHYSICAL AND CHEMICAL PARAMETERS OF SOILS ...... 39 3.4 SOIL SATURATED PASTE...... 39 3.5 pH OF SATURATED SOIL PASTE (pH) ...... 39 3.6 ELECTRICAL CONDUCTIVITY OF SATURATION EXTRACT (EC) ...... 40 3.7 HEAVY METAL ANALYSIS ...... 40 3.8 IDENTIFICATION OF THE SNAILS...... 40 3.9 MOLECULAR CHARACTERIZATION OF MOLLUSCS ...... 46 3.9.1 COLLECTION OF MOLLUSCS ...... 46 3.9.2 GENOMIC DNA EXTRACTION...... 46 3.9.3 QUANTIFICATION OF GENOMIC DNA ...... 47

v 3.10 POLYMERASE CHAIN REACTION ...... 48 3.10.1 RANDOM AMPLIFIED POLYMORPHIC DNA ...... 48 3.10.2 OPTIMIZATION OF PCR CONDITIONS:...... 49 3.10.3 RAPD-PCR ANALYSIS ...... 49 3.10.4 PCR TEMPERATURE PROFILE...... 50 3.11 DATA ANALYSIS...... 50 3.12 SOFTWARES USED ...... 51 3.12.1 SHANNON-WEINER DIVERSITY INDEX...... 51 3.12.2 INTER SPECIFIC ASSOCIATION ...... 51 3.12.3 TWO WAY ANALYSIS OF VARIANCE...... 52 3.12.4 MULTIPLE REGRESSION ...... 53 3.12.5 CLUSTER ANALYSIS ...... 53 4 RESULTS AND DISCUSSION ...... 55 4.1 TAXONOMIC CHARACTERIZATION OF THE SNAILS ON THE BASIS OF MORPHOMETRICS ...... 55 4.1.1 FAMILY ZONITIDEA (Mörch, 1864)...... 57 4.1.2 (TRYON 1866)...... 74 4.1.3 PHYSIDAE (FITZINGER, 1833)...... 84 4.1.4 SUBULINIDAE (FISCHER AND CROSSE, 1877) ...... 87 4.1.5 FERUSSACIIDAE (BOURGUIGNAT 1883)...... 91 4.1.6 SUCCINEIDAE (BECK, 1837)...... 95 4.2 MOLECULAR CHARACTERIZATION OF SNAIL SPECIES...... 102 4.2.1 OPTIMIZATION OF THE PCR CONDITIONS ...... 102 4.2.2 RAPD (PCR) ANALYSIS...... 102 4.2.3 GENETIC SIMILARITY AMONG SNAIL SPECIES ...... 105 4.2.4 GENETIC RELATIONSHIP AMONG SNAIL SPECIES...... 105 4.2.5 ANALYSIS OF MOLECULAR VARIANCE ...... 110 4.3 DISTRIBUTION AND ABUNDACE OF SNAILS IN FAISALABAD ...... 112 4.4 SPECIES INTERACTION WITH HEAVY METALS AND OTHER SOIL FACTORS ...... 136 4.5 INTERACTION OF SNAIL SPECIES ...... 144 CHAPTER 5...... 157 SUMMARY...... 157 REFERENCES...... 160

vi FIGURES

Figure 3-1: Location of Faisalabad, Punjab, Pakistan (Saleem, 2014)...... 37 Figure 3-2: Map of the study areas...... 38 Figure 3-3: General characteristics of gastropod shell..l ...... 41 Figure 3-4: Shell morphology...... 41 Figure 3-5: General shape...... 42 Figure 3-6: General Terminology...... 42 Figure 3-7: Types of surface sculpture...... 43 Figure 3-8: Shell morphology:...... 43 Figure 3-9: Morphology of the shell shaped periphery and the last round...... 44 Figure 3-10: Shell morphology: ...... 44 Figure 3-11: Apertural variations in gastropod shell...... 45 Figure 3-12: Shell shape of the last round...... 45 Figure 4-1: Ariophanta bistrialis ceylanica Zeitschr, 1837...... 59 Figure 4-2: Ariophanta bistrialis cyix (Beck, 1837)...... 61 Figure 4-3: Ariophanta bristrialis taprobanensis (Dohm, 1859)...... 63 Figure 4-4: Ariophanta bristrialis Beck, 1837...... 66 Figure 4-5: Ariophanta solata Godwin- Austen, 1898...... 68 Figure 4-6: Ariophanta belangeri bombayana Desh, 1847 ...... 70 Figure 4-7: Oxychilus draparnaudi (Beck, 1837) ...... 73 Figure 4-8: catiana (Montagu 1803)...... 76 Figure 4-9: Cernuella virgata (Da Costa, 1778)...... 79 Figure 4-10: Pupoides albilabris (C. B. Adams, 1841)...... 83 Figure 4-11: Physa fontinalis (Linnaeus, 1758)...... 86 Figure 4-12: Zooctecus insularis (Ehrenberg, 1831) ...... 88 Figure 4-13: Juvenile Zooctecus insularis (Ehrenberg, 1831)...... 90 Figure 4-14: Cecilioides acicula O. F. Müller 1774...... 94 Figure 4-15: Oxyloma elegans (Risso, 1826)...... 97 Figure 4-16: RAPD (PCR) of 15 snail species with prmer GLL-7. M is the 1 Kb ladder...... 104 Figure 4-17: RAPD (PCR) of 15 snail species with prmer GLK-19. M is the 1 Kb ladder...... 104 Figure 4-18: RAPD (PCR) of 15 snail species with prmer GLK-16. M is the 1 Kb ladder...... 104 Figure 4-19: Dendrogram showing relationship among 15 snail species...... 108 Figure 4-20: Principal component analysis of 15 snail species based on molecular data...... 109 Figure 4-21: Percentage of Molecular Variance...... 110 Figure 4-22: Abundance of Gastropod Species in Various Villages & Different Agro- Regions of Faisalabad during 2011...... 112 Figure 4-23: Species Diversity Indices for Snails in Different Villages of Faisalabad...... 116 Figure 4-24: Species Diversity Indices for Snails in Different Villages of Faisalabad...... 116

vii Figure 4-25: Species Diversity Indices for Snails in Villages through Different Irrigation Canal of Faisalabad...... 118 Figure 4-26: Evenness for Snails in Villages through Different Irrigation Canal of Faisalabad...... 118 Figure 4-27: Regression Analysis of Species Diversity and Evenness of Snails in Different Irrigation Canals in Faisalabad ...... 119 Figure 4-28: Relative Abundance of Snails in different Habitats of the Agroecosystem of Faisalabad....122 Figure 4-29: Species Diversity Indices for Snails in Different Crops of Faisalabad...... 124 Figure 4-30: Evenness for Snails for Snails in Different Crops of Faisalabad...... 124 Figure 4-31: Regression Analysis of Species Diversity and Evenness of Snails in different crops in Faisalabad ...... 125 Figure 4-32: Relative Abundance of snails in different Months in Faisalabad...... 128 Figure 4-33: Species Diversity Indices for Snails in Different Months in Faisalabad...... 131 Figure 4-34: Evenness for Snails in Different Months in Faisalabad...... 131 Figure 4-35: Regression Analysis of Species Diversity and Evenness of Snails in different months in Faisalabad...... 132 Figure 4-36: Effect of Maximum Temperature on the Number of Snails...... 134 Figure 4-37: Effect of Minimum Temperature on the Number of Snails...... 134 Figure 4-38: Effect of Average Humidity on the Number of Snails...... 135 Figure 4-39: Effect of Average Rainfall on the Number of Snails...... 135 Figure 4-40: Effect of Average Sunshine on the Number of Snails...... 135 Figure 4-41: Comparison of Soil Cadmium concentration with National Environmental Quality Standards (Pakistan)...... 138 Figure 4-42: Comparison of Soil Lead Concentration with National Environmental Quality Standards (Pakistan)...... 138 Figure 4-43: Comparison of Soil pH with National Environmental Quality Standards (Pakistan)...... 138 Figure 4-44: Comparison of Soil Electrical Conductivity with National Environmental Quality Standards (Pakistan)...... 139 Figure 4-45: Effect of Average Cd concentration on the number of snails ...... 139 Figure 4-46: Effect of Average lead concentration on the number of snails...... 140 Figure 4-47: Effect of Average Electrical conductivity on the Number of Snails...... 140 Figure 4-48: Effect of Average pH on the Number of Snails...... 140 Figure 4-49: Cluster analysis of Variables:Villages...... 152 Figure 4-50: Cluster Analysis of the Variables: Wheat, Sugarcane, Fodder, Vegetables, Ditches...... 153 Figure 4-51: Cluster Analysis of the Variables: March, April, May, June, July, August...... 154

viii List of Tables

Table 3-1: RAPD primers along with their sequences...... 48 Table 4-1: Morphometrics of the Ariophanta bistrialis ceylanica Zeitschr, 1837...... 59 Table 4-2: Morphometrics of the Ariophanta bistrialis cyix (Beck, 1837)...... 61 Table 4-3:Morphometrics of the Ariophanta bistrialis taprobanensis(Dohm, 1859)...... 63 Table 4-4: Morphometrics of the Ariophanta bistrialis Beck, 1837...... 66 Table 4-5: Morphometrics of the Ariophanta solata Godwin- Austen, 1898 ...... 68 Table 4-6: Morphometrics of the Ariophanta belangeri bombayana Desh, 1847...... 70 Table 4-7: Morphometrics of the Oxychilus draparnaudi (Beck, 1837)...... 73 Table 4-8: Morphometrics of the Monacha catiana (Montagu 1803)...... 76 Table 4-9: Morphometrics of the Cernuella virgata (Da Costa, 1778)...... 79 Table 4-10: Morphometrics of the Pupoides albilabris (C. B. Adams, 1841)...... 83 Table 4-11: Morphometrics of the Physa fontinalis (Linnaeus, 1758)...... 86 Table 4-12:Morphometrics of the Zooctecus insularis (Ehrenberg, 1831)...... 88 Table 4-13: Morphometrics of the Juvenile Zooctecus insularis (Ehrenberg, 1831) ...... 90 Table 4-14: Morphometrics of the Cecilioides acicula (O. F. Müller 1774)...... 94 Table 4-15: Morphometrics of the Oxyloma elegans (Risso, 1826)...... 97 Table 4-16: Characterization of the Snails on the Basis of Morphometery...... 99 Table 4-17: RAPD primers with sequence, number of polymorphic bands (NBP), and mean band frequency (MBF)...... 103 Table 4-18: Genetic similarity matrix of 15 snail species based on Nei's genetic identity...... 107 Table 4-19: Summary AMOVA Table...... 110 Table 4-20: Accession number and Order of the species...... 112 Table 4-21: Species Diversity Indices for Snails in Different Villages of Faisalabad...... 115 Table 4-22: Species Diversity Indices for Snails in Different Villages of Faisalabad...... 118 Table 4-23: Distribution of Snails in Different Habitats of Agroecosystem...... 121 Table 4-24: Pecent (%) Abundance and Distribution of Different Species in different Habitats of Faisalabad...... 123 Table 4-25: Species Diversity Indices for Snails in Different Crops of Faisalabad...... 123 Table 4-26: Distribution of snails in different months...... 127 Table 4-27: Pecent (%) Abundance and Distribution of Different Species in different Habitats of Faisalabad ...... 129 Table 4-28: Mean Envoirnmental Variables in Different Months...... 130 Table 4-29: Species Diversity Indices for Snails in Different Months in Faisalabad...... 130 Table 4-30: Regression Analysis of Envoirnmental Parameters and Numbers of Snails...... 134 Table 4-31: Comparison of Soil parameters with National Environmental Quality Standards (Pakistan)...... 137 ix Table 4-32: Regression Analysis of Soil Parameters and Number of snails...... 139 Table 4-33: Analysis of Variance for Number of Specimen, using Adjusted SS for Test...... 144 Table 4-34: Interspecific association indices and test stastistics between fifteen snail species in different months...... 145 Table 4-35: Interspecific association indices and test stastistics between fifteen snail species in different habitats of agroecosystem...... 149 Table 4-36: Cluster Analysis of the Variables: Wheat, Sugarcane, Fodder, Vegetables, Ditches...... 153 Table 4-37: Cluster Analysis of the Variables: March, April, May, June, July, August...... 154

x ACKNOWLEDGEMENTS

I kneal in front of Almighty Allah, who gave me courage and resources to complete this work. I also pay respect and heartiest homage to His Prophet (PBUH) for whom this Universe was created.

First of all, I would like to pay thanks to my worthy supervisor, Prof. Dr. Naureen Aziz Qureshi, Dean Faculty of Science and Technology, for her continued guidance and support during the course of my Ph. D. studies despite her busy schedules. I would never be able to complete this uphill task without her continuous encouragement.

I would like to acknowledge the laboratory facilities that were extended by Dr. Faisal Saeed Awan, Assitant Professor, Centre of Agricultural Biochemistry and Biotechnology, UAF. I pay a very special thanks to Dr. Javaid Iqbal Siddiqui, Associate Professor Department of Zoology, Government Science College, Samanabad Faisalabad, and Dr. Tahira Riasat, Assistant Professor Department of Zoology Government College Univeristy Faisalabad for their continuous help guidance and support during statistical analysis. I am thankful to Dr. Shabana Naz, Assistant Professor Department of Zoology, Dr. Hammad Raza, Assistant Professor Department of Botany. I am extremely grateful to the faculty members and collegues of Department of Zoology, Wildlife and fisheries and Dr. Mushtaq-ul-Hassan Associate Professor (Retired) Government College Univeristy Faisalabad for their time to time support. I owe extreme thanks to students, Mr. Rana Muhammad Saif Ur Rahman student GCUF, and Inayat-ullah, Department of Agriculture, Government of Pakistan, who supported me in sampling throughout Faisalabad.

My deepest gratitude goes to my family for their unflagging love and support throughout my life; this dissertation is simply impossible without them. I am indebted to my father who infused confidence in me to bear all thicks and thins of life. I am also indebted to my mother without whose prayers I could not have any achievement in my life. I am extremely supported by my mother-in-law and father- in- law Dr. Muhammed Afsar Awan, Ex-Head Mutation Breeding Division NIAB for their encouragement and understanding without which this work would have been impossible.

I pay special thanks to my sisters and brother without whom I could not struggle to this extent. The encouragement of my brothers in law was great enough. I feel great care from my dear and sweet daughters who sacrificed a lot during my busy life and were always there for me.

xi ABSTRACT This is a first attempt towards a detailed understanding of malacofaunna of Faisalabad Pakistan. The present study has been carried out on the morphometric and molecular characterization and genetic diversity of snails in the agroecosystem of Faisalabad City from March 2011 through August 2011. During this period 19290 snails were randomly collected from four agroecosystems (sugarcane, wheat, fodder, vegetables fields) and ditches from villages linked with Rakh branch, Jhang branch and Ghogera branch. The snails were isolated from the soil samples through sifting through screen of mesh size less than 1 mm. The snail specimens were studied under the microscope. The snail species were identified by using recent identification keys i.e., Blandford and Godwin (1908), Bouchet and Rocroi (2005), Sturm et al., (2005), Anderson, (2008), Watson and Dallwitz (2005) and diagrammatic description provided in them. The identification of the specimens was made on the basis of number of whorls, coiling of the shell, umbilicus, shape, colour, shape of the aperture, presence or absence of operculum, height (mm), diameter (mm), and the diameter of the aperture (mm) using vernier caliper. The snails were found belonging to two suborders, seven families, nine genera, fifteen species out of which six species have been reported first time in this region. Molecular characterization of snails has been done with the help of randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique for understanding the biodiversity in this region. Genetic characterization of 15 snail species was done by using 23 RAPD primers and out of which 15 RAPD primers produced polymorphic amplification. On the basis of Analysis of Molecular Variance there was found 11% variation among populations of the five habitats and 89% variation within populations in species population found in all the habitats. Genetic similarity among snail species was estimated by Nei’s genetic similarity indices showing a range of 0.5 to 0.74. Maximum genetic similarity was found between Ariophanta belangeri bombayana and Ariophanta bristrialis taprobanensis as well as Ariophanta belangeri bombayana and Ariophanta solata. Minimum genetic similarity based on Nei’s genetic indices was observed among Cernuella virgata and Ariophanta bistrialis cyix. Considering the Zooctecus insularis and Juvenile Zooctecus insularis they are quite distant from each other in the cluster due to which it is expected that might belong to some new species, which need further investigations. The data of distribution and abundance was subject to different statistical tools i.e., shannon and wiener

1 diversity index, index of overall association, two way analysis of variance, multiple regression, cluster analysis shows that as we progress towards south, the diversity of the snail species in Faisalabad is highly reduced. However the species diversity in all the villages linked to R.B., G.B. and J.B. was highly significant with a strong positive relationship between species diversity and species evenness (97.03%). The diversity indices in all the habitats of the agroecosystem were highly significant while in ditches the results were non-significant with a strong negative correlation between species diversity and evenness in the crops that clearly shows that when diversity is low the evenness was high and viceversa in the distribution of snails in the different crops. There is 95.3% relationship between species diversity and species evenness in different crops of Faisalabad. The species diversity is highly significant in all the months except in April.Regression analysis between species diversity and species evenness shows that there is 57.36% relationship between them in different months in agroecosystem of Faisalabad. There is a significant effect of maximum and minimum temperatature on the number of snails while other abiotic factors i.e., humidity, rainfall, sunshine, soil cadmium concentration, soil lead concentration, soil pH, soil electrical conductivity has a non-significant effect on them. The soil parameters have been found much less than National envoirnmental quality standards Pakistan.

However the association between the species and months and between species and agroecosystem has been found highly significant. Interspecific association indices suggests an overall positive association however no association has been found during different months while a strong association has been found with different habitats.This study has given us a baseline data of the malacofaunna in Faisalabad which will help us to identify the indicator species, develop ecological models, and conservation strategies by the policy makers.

2 CHAPTER 1

INTRODUCTION

1.1 TAXONOMY Phylum are the largest of the marine phyla and snails comprise about 23 % of the marine organisms. There are a number of forms of gastropods in terrestrial and freshwater envoirnment, while most gastropods are marine in their habit.The historical background of snails reveals its history of nearly 500 million years, while the number of molluscs has been estimated to vary from 80,000 species to 135000 (Boss, 1973). There are few groups which differ on the basis of the presence of either reduced or internal shell or no shell at all. Snails are generally shelled forms while slugs are the forms which are without shell. There are various marine forms of the snails, which are without shells, and are not closely related to terrestrial slugs. biodiversity, is defined as the number of native species per unit of land area, (Holland and Cowie, 2009). Next to the insects the second large and most successful inverteberate are snails (Abbot, 1989 and Hapman, 2009). Considering the number of the species that have been described, there are non marine molluscs belonging to phylum Mollusca becomes the second most diverse Phylum in Kingdom Animalia. Although the freshwater and terrestrial, the molluscs are found to be one of the most diverse group, yet there are only a few scientists who are aware of their importance and are working on these creatures. One of the most important and effective approach to the biodiversity conservation and management is the compilation and publication of the Red Data Book (Bouchet, 1997). According to Baillie et al. (2004) there are total 708 freshwater and 1222 terrestrial molluscs, out of which 42% of the 693 extinctions from the species are molluscs, comprising 260 gastropods and 31 bivalves as reported in Red List of Threatened Species ((Baillie et al. 2004). Inverteberates are not generally noticed by the biologists and conservation agencies as most of the work is being done on terrestrial vertebrate regarding their extinctions and has been well documented (Lydeard et al., 2004). Mostly the animal taxa which are being focused are verteberates with an apparent maturity of the taxonomy of birds and mammals due to their prevalence. This also shows a global interest in certain attractive classes. These classes are important due to public awareness and aesthetic significance. Although even common people are aware of the importance of Giant

3 panda or White Rhinoceros yet the inverteberate species are extremely neglected subject in the Red data (Gorbatovsky, 2003). Although the diversity of inverteberate species is much higher than verteberates yet they are being ignored. The major reason for this negligence is the ignorance about these creatures. The continental molluscs have been less represented in the regional Red Data Books of Siberia and Urals which led to the conclusion that there are far few professional malacologists who can cover the malacological surveys especially due to the remoteness and absence of communication (Grebennikov and Vinarski, 2010). Even in a country like United States of America the conservation strategies planned for gastropods are very less. In USA, the species which have been presumed to be extinct in freshwater gastropods are nearly 60 in number. There are 20 threatened or endangered species while the remaining 290 species are of extreme conservation concern. So there are 48% of the fresh water gastropods which are important conservation targets while 9% of freshwater gastropods are extinct in USA. The destruction at this rate is more than that of any other major group exceeding the freshwater mussels also, in which the species which are conservation targets are 42% (Johnson, 2003). The development of any conservation plan and frame work of scientific investigations requires considerable taxonomic efforts with extremely careful and thorough systematic approach (Wheeler, 2004). The gastropods belong to the 99% of the organisms like the inverteberates on the surface of the earth (Ponder and Lunney, 1999); but the basic understanding about their biology and taxonomy is wanting. The populations and species, which need to be protected due to genetic diversity, which helps them to adapt according to their envoirnment, and unique life history are called conservation management units. The proper identification of these populations and species, referred as conservation management units, is very important. Not only this, there is a requirement of a stable system to communicate those units, which explains the evolutionary history, which is of immense importance in devising the conservation strategies. This has been a matter of intense research on fresh water gastropods in the recent years (Hershler et al., 2007a, Minton and Savarese, 2005, Perez et al., 2005, Miller et al., 2006, Liu et al., 2003, Strong and Glaubrecht, 2003, Michel, 2004). The field of systematics has been in the middle of its evolution especially in terms of its procedures and philosophies due to the presence of the data sets that are independent and testing of strategies so that the system of classification using multiple lines of evidence can be used

4 together (Wiens and Penkrot, 2002; Hershler et al., 2007b; Wheeler,2004; Godfray, 2002 Strong and Frest, 2007). Systematics is defined as the process of construction of phylogenies, and understanding the patterns of evolutionary relationship among organisms. Classification is defined as the process of translation of phylogenies with its application into systems with nested biological organization. The conservation efforts, which are done having meaningless classifications, worsen the situation and are extremely harmful for the protection of biodiversity (Daugherty et al., 1990, Lang et al., 2006). The field of taxonomy and systematics play extremely important for the conservation of organisms (Vane-Wright et al., 1991; Soltis and Gitzendanner, 1999; Moritz and Faith, 1998) and freshwater gastropod faunna (Cranston, 1990). This small creature is constantly being ignored due to megafaunal biasness (Platnick, 1991) against invertebrates. On the other hand, it may be due to a casual attitude toward taxonomy where naming is given less importance when compared to the concern shown to protect a taxon. Due to examples of bad taxonomy, there is a great hinderance in conservation efforts (Daugherty et al., 1990; Karl and Bowen, 1999;Mayden and Kuhajda, 1996; Bowen and Karl, 1999; Engstrom et al., 2002; Berry et al., 2002; Funk and Fa, 2006; Flanagan et al., 2006). It is generally taken for granted by the scientific community, of the recognition that field of taxonomy is fundamental and is integrally linked to all spheres of the biological studies. This can be clearly understood that the role of taxonomy in the biological science is not being appreciated as reflected by the continuous decline in its support. There is a great difficulty in gathering sufficient information about the species biology and the interaction among those species of freshwater gastropod taxa, so that an effective conservation action can be carried out (Lydeard et al., 2004). This review highlights the significance of taxonomy and systematics of freshwater gastropods for conservation actions which can be carried out in Pakistan. In the light of the forecoming review, a few areas of research can be pointed out which puts light on the applied scientific approach, and recommends for incorporation of taxonomic and systematics information into future conservation plans gastropod. A good sampling methodology is of an immense importance in this regard.

1.2 COLLECTION OF THE SNAILS A descriptive or analytical study about the species community always demands a controlled sampling method. A statistical design reffered as nested sampling, mostly consists of

5 three replicate units, which is generally called quadrat is generally used by the students of tropical forest snail communities at a larger site which can be grouped into areas, later, and can be analyzed (Cameron and Pokryszko, 2005). Generally a surface area from a range of 8 m2 microscale (Liew et al., 2008; Clements et al., 2008) to macroscale 400 m2 (Emberton et al.,1996; Emberton, 1997) have quadrats, which comprise more or less homogeneous macrohabitats, as explained by many scientists in Africa (Oke et al., 2008; Oke and Alohan, 2006; De Winter and Gittenberger, 1998) and Asia (Liew et al., 2010; Schilthuizen and Rutjes, 2001). In a random sampling method a number of quadrats samples are taken from a whole site either randomly (Fontaine et al., 2007a, b) or systematically along the transects (Raheem et al., 2009; Clements et al., 2008; Schilthuizen et al., 2002; Emberton, 1997; Emberton et al., 1999). According to Cameron and Pokryszko (2005) the sites which are less than 1 km2 were considered to be small. In most of the studies the quadrat samples are taken only once, some of the workers do repeated sampling continue sampling in the consecutive seasons (Bloch et al., 2007; De Winter and Gittenberger, 1998) or even decades (Bloch et al., 2007; Vermeulen, 2003). In the patch of oil palm in Nigeria, the diversity of land snails was studied by direct search and sieving of litter by Oke and Alohan (2008) who studied the diversity and found 22 species in 833 specimens from 6 molluscan families. The diversity in the agroecosystem was low i.e. 25 species, when compared with the diversities of Okomu National Park (50) species, Cameroo (97), Malaysian Borneo (61) and Tanzania (84) species, however the number of individuals in this agro ecosystem were higher than the other rainforest.

1.3 ECOLOGY Biodiversity refers to the variety or richness of life i.e. plant, animal, and other life in a given area, from the plant or tiniest snail to the largest predator. Biodiversity covers not only the species themselves, but also the complex interactions among species, their natural communities and ecosystems (Gruver and Finley, 2006)

Molluscs are widely diverse in their size, anatomical structure, behavior and are distributed in a range of habitats from sea water i.e. littoral, pelagic and at great depths while some are more adapted to fresh water while others to brackish water. In the terrestrial ecosystem

6 there are many ecological factors e.g. temperature extrimities, acidity, dryness and mineral content of the soil, which have impact on their distribution. There are almost 7 families of Order 20 families of Order Prosobranchia of the freshwater gastropods on the earth. There are major physiological differences in the mode of nutrition between Prosobranchia and Pulmonata. The pulmonates obtain their food by scraping their food with the help of their radula. However most of the members of the Prosobranchia obtain their food by traping food particles, suspended in the water, in mucous which is ingested by the snails. Most of the freshwater snails depend on the organic debris and algae from the leaves, stones, and substrates (SWCSMH, 2006). The terrestrial slugs and snails generally live on a variety of decaying plant material and living plants parts. They produce irregular holes by chewing and cutting leaves, flowers, succulent plant parts, fruit and young barks. They are designated as serious pests of ripening fruits i.e tomatoes and strawberries as they are grown near the ground. However, they also depend on foliage and fruit of some trees like citrus (Flint, 2003). They are so widespread, that they are even found at high altitudes and in some of the Polar Regions. Land snails are generally considered as typical herbivores, fungivores and detrivores (Burch and Pearce, 1990) that show intraspecific competition at weak levels (Cain, 1983, Barker and May hill, 1999). Annual litter input of about 0.5% per year can be consumed by land snail communities (Mason, 1970). To prevent themselves from dessication snails have developed a number of behavioral i.e. high aggregation characteristics and morphological adaptations i.e door-like clausilium, internal lamellae thick white shell,thick epiphragm, strengthened and reflected apertural lips, which help the snails in their survival during temperature extremes and desiccation (Storey, 2002; Cook, 2001). Giokas et al., (2005) investigated the biochemical composition and physiology of the land snail, considering seasonal pattern with reference to its morphology, ecology and climatic data. The annual cycles of humidity, photoperiod, reproduction, temperature, water availability and have an impact on land snail biochemical composition and physiology (Storey, 2002; Cook, 2001). Snail meat is rich in protein with a protein content of 88.37%, which is more than pork (82.42%) and less than beef (92.75%) (Imevbore and Ademosun, 1988; Ademolu et al., 2004), low in fats and a source of iron (Orisawuyi, 1989). It has an important position in the food webs of different ecosystems as snails are mostly consumed by fish, water fowl, crayfish, leeches, and

7 sciomyzid flies (SWCSMH, 2006). This has been supported by Johnson (2003), that fresh water snails are also an important source of food for many fish, turtles and other species of wild life including pathogens, ground beetles, toads, snakes,turtles, and birds, but most are rarely effective enough to provide satisfactory control in the garden (Flint, 2003).

Snails are found in all types of habitat i.e., large water bodies or small pools of water, in pastures, in woodlands, in mosses, under rocks, on cliffs , underground, in trees, on the of other bodies. They have adapted themselves to all forms of habitat except aerial locomotion (Hickman et al., 2001). Different ecological factors affect the distribution of the snail. The structure of macroinverteberate community is generally determined by a variety of physical, chemical and biological factors in the streams (Malmqvist, 2002). Not only this, the abundance and diversity of mollusca differs significantly between different macroalgea assemblages (Chemello and Milazzo, 2002), which is also confirmed by Takahiro (2006) that temperature and algal abundance is directly proportional to shell morphology within same population in Okinawa Islands. Temperature along with relative humidity is possibly the most driving factor (Suominen, 1999) and distribution of snails and soil moisture are directly proportional (Prior, 1985) and especially the juveniles of many gastropod species are extremely sensitive to dessication (Asami, 1993).

The sunlight and the soil moisture also influence land snail community composition in dry and sunny habitats (Nekola, 2002b) as hot and dry sunny days kill many snails due to the change in soil composition and properties (Chen et. al., 1993), while on the other hand the fire management may lead to major reductions in gastropod species abundance and richness (Nekola, 2002b). There is a declines in the snail fauna after fire (Karlin, 1961) and clearcutting (Walden, 1998) in temperate and boreal forests.

Soil inverteberates are badly affected by soil compaction and the impact is more severe on animals than plants living in the shared communities (Duffey, 1975). Heavy grazing has a negative impact on the grassland snails (Cameron and Morgan, 1975); as snail diversity and abundance is significantly reduced by homogenous grazing of the herb layer. On the other hand suitable habitats for the rare snail species are expanded due to sheep grazing, and with an increase in the heterogeneity of vegetation leading to the intricate cover of the soil surface

8 enables more species to co-exist (Labaune, 2002), however, nearly 90% of the snails generally live in top 5 cm of soil surface (Hawkins et al., 1997). While in the summer the snails climb on the plant stalks and expose themselves to the stalks to escape the summer heat which increase the crop losses near harvest time (Desbiolles et al., 2003).

Flowing waters predict the presence of greatest number of species (Johnson, 2003). Schiltuizen (2005a) reported that abundance of the snails is positively and strongly correlated with calcium carbonate and in turn with pH. The pulmonate snails were found significantly abundant at disturbed localities when compared with the prosobranch snails, however, abundance for both orders was similar at the sites which were undisturbed. There is a strong correlation of their abundance pH and Calcium carbonate concentration. In addition habitat, humidity, slope aspect, plant species, number and herb cover are very important predictors of the snail abundance at the sites (Anna, 2005).

Snails are extremely sensitive to certain chemicals such as petroleum, certain metals and agricultural pesticides and fertilizers, in very small amounts; many species are excellent water quality indicators. The most important heavy metals water pollutants are Cu, Zn, Pb, Hg, Cd, Ni, and Cr. Some of these metals i.e. Cu, Cr, Ni, and Zn are essential trace metals to living organisms, while some cause toxicity at higher concentrations. The decline of freshwater snails began in early 20th century when dam construction and other channel modification siltation and industrial and agricultural pollution have all degraded the river habitat on which most species depend (Johnson, 2003). The rivers have played a very important role in the dispersal of snails throughout Punjab, through their tributaries (Arshad et al., 2011). In Faisalabad there is a network of irrigation canals which empty into small distributries and form a mosaic network, which may support snail to reach the agricultural fields through these water bodies due to moist habitat in land snails, the dispersal values are extreme despite their low mobility (Backeljau, Baur and Baur, 2001) which best suits these soft bodied animals.

1.4 GENETIC DIVERSITY OF SNAILS In marine inverteberates, the molluscs are considered to be the best phylum in marine inverteberates that have been studied genetically.They are the best models to study the reproductive barriers, leading to evolution, due to gamete recognition proteins (Swanson and

9 Vacquier, 1998; Hellberg and Vacquier, 1999; Metz et al., 1998b). There is a great potential for combining genetic and paleontological approaches as, molluscs have an excellent fossil record (Reid et al., 1996; Collins et al., 1996). Species of gastropods have been extensively investigated include, Littorina (Kyle and Boulding, 1998; Reid et al., 1996; Tatarenkov and Johannesson, 1998). Other genera include Nucella (Kirby et al., 1997) Hydrobia (Ponder and Clark, 1988), Nassarius (Sanjuan et al., 1997) Patella (Ridgway et al., 1998; Côrte-Real et al., 1996a,b) Stramonita (Liu et al., 1991), Alviniconcha (Denis et al., 1993), Trochus (Borsa and Benzie, 1993), Tectus (Borsa and Benzie, 1993), Austrocochlea (Parsons and Ward, 1994), Drupella (Johnson and Cumming, 1995), Columbella (Oliverio, 1995), , and Dendronotus (Thollesson, 1998). As the shells can be readily collected and studied so the alpha taxonomy of gastropods has attracted the attention of professional and amateurs for a long time. There are many descriptions of the shells which are available in the past literature; however, mostly the phenotypic plasticity results in differences in shell morphology. As a result in molluscs there is development of synonymization of species, or same name of different species due to the increase in the number of genetic studies, when compared with the other inverteberate phyla. The differences in the shell morphology (or other characters), which are of taxonomic importance can be understood with the help of genetic data (Johnson and Cumming, 1995; Borsa and Benzie, 1993; Parsons and Ward, 1994; Mokady et al., 1994; Oliverio, 1995; Izuka et al., 1996; Thollesson, 1998; Sanjuan et al., 1997). It is not common to have an unexpected discovery of highly divergent sympatric taxa in the groups which are morphologically uniform; however, it is possible in case of deep sea (Peek et al., 1997; Craddock et al., 1995) and in cephalopods (Yeatman and Benzie, 1994). While in the case of allopatric species there is confusion, in most of the cases, that the shell variability may be due to the genetic and environmental differences. Besides this large amount of genetic differentiation or very low amount of genetic difference can be observed in the comparison of same geographic domain and sometimes this difference can be found even in same genus (Côrte-Real et al., 1996 a, b ;Adamkewicz and Harasewych, 1996). The mechanism of species boundaries is evident along continuous coastlines where there is the existence of narrow zones of overlap due to which there is low hybridization (Ridgway et al., 1998; Liu et al., 1991), however sometimes there are quite intricate patterns of hybridization (e.g. Mytilus) while no zone of overlap exists in other cases. Many animal genera have been studied extensively, with the help of various approaches, and the resultant data sets is

10 straightforward. A variety of patterns of hybridization have been revealed in the studies of Mytilus, which depend on the geographical locale and taxa along with a different attractive and fantastic sex-linked mitochondrial transmission system (Gardner, 1996; Beynon and Skibinksi, 1996; Comesaña et al., 1999; Quesada et al., 1998). There are examples in Littorina, that organisms in different habitat closely related pairs of taxa have shown reproductive barriers due to environmental variations within a site, however on the other hand there are specimens that are from different environments within sites which are genetically more similar than are those specimens which are geographically isolated but are similar in terms of environment (Kyle and Boulding, 1998; Tatarenkov and Johannesson, 1998). The immunochemical methods were used (Andrews 1964, Burch and Lindsey 1968) and, are still valueable today alongwith the chromosomal studies, (Choudhury and Pandit, 1997; Natarajan et al., 1966, Garbar and Korniushin 2003) and DNA sequence fragments (Davis et al., 1999, Remigio et al., 2001) supported by studies on the allozymes (DeVries et al., 2003; Bandoni et al., 1995; Viyanant et al., 1988; Dillon and Lydeard, 1998). There is the augmentation of the previous methods with the help of the additional data that has been developed by the genetic markers like Amplified Fragment Length Polymorphisms (AFLP) and micro- and minisatellite markers (Emery et al., 2003, de Boer et al., 2004, Miller et al., 2006). The taxonomy with the inclusion of molecular data has guided scientists to the identification on the basis of one- molecule identification, e.g DNA barcoding and DNA taxonomy (Tautz et al., 2003; Hebert et al., 2003; Blaxter, 2004; Rubinoff, 2006; Scotland et al., 2003; Lipscomb et al., 2003; Hebert et al., 2004). According to DeSalle (2006) a total evidence approach must be supported where conservation management units are defined by all the available information. The species that have been redescribed and defined with circumscription and the new species, which have been discovered have an inherent characteristic of testable scientific hypotheses (Sites and Marshall, 2004; Goldstein et al., 2000, Dunn, 2003; Hey et al., 2003; Lipscomb et al., 2003; Seberg et al., 2003). This approach will lead to an increase in the effort required for the identification of the species and conservation management units. As a result of this effort the classifications would be more clear and accurate as compared to the situations where these approaches have not been used and these efforts have been used in classification of other taxa of inverteberates (Bond and Sierwald, 2003; Giribet and Wheeler, 2002; Guralnick, 2005; Hershler et al., 2006; 2007b; Roe and Hartfield, 2005;Williams et al., 2003). It is extremely important to carry out taxonomic work

11 with extreme precision and accuracy using precautionary principles and sound science especially when handling very narrow range taxa or the taxa, which are of great concern independent of the procedures which are being adopted for species delineation practical identification of species of interest (McGarvey, 2007). A likely consequence of something of taxonomic oversplitting is protection of non distinguishable units having negative political, financial and regulatory results. There are also positive aspects, the permanent loss of the evolutionary lineages and processes can be protected and balanced this way. On the other hand, there is taxonomic undersplitting which is a Type II error in the field of taxonomy and have implications with extreme potential as in this way when a species is unrecognized, endangered unprotected or unlisted and consequently (Buhay et al., 2002). Uptill now, no scientific debate has been rigorously carried out to determine the level and direction of error in species delineation and identification methods. There are two approaches with which the taxonomic work must be proceeded for the species delineation, one of which is the rigorous hypothesis (Wheeler and Platnick, 2000; Nixon and Wheeler, 1992) and the second is the development of framework on the basis of equivalence-testing (McGarvey, 2007) that are important and lays emphasis on the precautionary measures and reduces the negative effects, which are potentially permanent of the statistical errors to the minimum. Taxonomic undersplitting has been well depicted by a famous example on the Scaphirhyncus suttkusi i.e. Alabama sturgeon (Williams and Clemmer, 1991). The Alabama sturgeon remained in recognition due to a single-gene data set, and along with this it came in the conservation limbo, which led to its protection. However the whole process cost a large amount of money and finally there was an ultimate decline of species. A situation similar to the already mentioned example in case of freshwater mussel with reference to genus Epioblasma, where there is limited genetic sampling (Buhay et al., 2002) didn’t show exactly the evolutionary processes in the group. To make future conservation strategies, there is a need of necessary data for the recognition of diversity in the taxa for which there should be a detailed treatment of natural history traits, morphological and genetic (Jones et al., 2006). There is an example of extremely thorough and careful approach in the taxonomy of the freshwater gastropods Pecos assiminea. in which lineage based approach was used in which there was also a comparison of parsimony, neighbor joining likelihood in the multiple data sets considering shell measurements and cytochrome oxidase subunit I sequences. Divergences along with the geological events and biogeographic hypotheses relate to the delineated groups. A new species of Assiminea cienegensis has been

12 thoroughly described with special emphasis on the shell morphology and anatomical features. These kinds of efforts are required for designation of taxonomic unit which helps all the workers to solve the problems which are caused due to limited data sets and sampling efforts (Hershler et al., 2007a).

1.5 PROVIDING CONSERVATION MANAGEMENT UNIT INFORMATION TO THOSE CHARGED WITH CONSERVING The species description, redescription and classification are extremely important for the discovery, identification of the conservation management units, which helps in devising the conservation strategies. The information regarding species description atleast fixes the scientific name, its natural history, locality, its external (i.e morphological, behavioural features) as well as its internal characters i.e molecular data, and the most important of all its status among remaining biodiversity (Dubois and Neme´sio, 2007). In conservation plans these names are generally used and are also used as a reference when there is the question about the basic knowledge of an organism. There are set guidelines for the description and redescription of the species in ICZN i.e.International Code of Zoological Nomenclature (ICZN, 1999). To obtain a minimum level of information about any taxon it is extremely important for the taxonomic workers, scientists and agency workers to follow the ICZN. On the other hand, if the information and the description of species provided by the scientists is incomplete and does not fulfill the requirements or the information provided fails to support the taxonomic consumer to incorporate the information given at all levels (combinations of new name,new species, etc.), the problems will arise for the taxonomic workers. In USA and Canada there are almost 10 % of the total species that have been just named while they have not been described, which are both native and exotic (Nature Serve, 2007). These taxa belong to hydrobiids, and other which belong to them have been referred in the gray literature species as letter (A, B, C) or numbers (1, 2, 3) (Frest and Johannes, 2000). The scientific names with this type of generic and species combinations are mostly not unique and cause ambiguity due to which they cannot be used in relational taxonomic databases (Froese, 1999). With a reduction in the ability to correctly identify the species and to have a reference collection subsequent conservation efforts become out of space for a unit of diversity. It is extremely important to study populations with reference to the taxonomic work, with the appropriate names and descriptions of the species. The formal taxonomic work gives

13 effective information for making conservation strategies. The conservation efforts are greatly affected due to the inconsistent use of names as it becomes greatly difficult to find the related information. Assignment of correct generic and species help to achieve conservation goals,and can be tested as a scientific hypothesis that should be can be used by the conservationists. A list of names has been provided by Turgeon et al., (1998) which is generally accepted in this field and this can be used a reference point for starting the modern nomenclature, hoping that the future work will possibly change. Dealing with the snails the taxonomy has become a major issue (Bogan and Hoeh, 2000) as the taxonomy of this organism is based on characters of little importance due to which there is a dire need for the reconstruction of the families and genera.

1.6 FRESHWATER MALACOFAUNNA OF PAKISTAN

The importance of this little creature has always been underestimated. Most species of the snails play a dominant role in the fresh waters by providing food for other organisms like fish and improve water quality by consuming large quantities of detritus and algae (Johnson, 2003). While in some parts of the world, they have been given the status of pest threatening the agricultural yield and being themselves targeted by the chemical and pesticides. The use of mollucan pesticides for the control of molluscs, which are serious pests, are still being considered highly effective control measure. On the other hand, they impose a deleterious effect on the non target species and damage the overall envoirnment (Awad, 1995) and leads to ecological diversity imbalance. In the forest ecosystem a decline in the reproduction in the passerine birds due to decrease in the snail abundance on acidic soils have been reported by Graveland et al., (1994). The freshwater malacofaunna of Pakistan is least known in Asia (Begum and Nazneen, 1991, 1992a, b). The first study known about the snail taxonomy in Pakistan is in the time of British rule by Hutton (1849) which were collected at the western terminus of the Bolan Pass, Balochistan. Later in 1881, Theobald and many other workers explained the geographical limit of the Himalayan snail fauna in Murree, Pakistan. Blandford and Austen (1908) and Gude (1914) have made a major contribution in the field of malacology and conchology in South Asia which was later carried out by many others. Recently researches have been conducted on the biodiversity of soil macroinverteberate in the low and high input fields of Wheat and Sugarcane

14 in District Faisalabad (Rana, 2012) with the major focus on impact of chemical on the diversity of different macroinverteberates as molluscs are very important for monitoring the toxins in aquatic ecosystem (Salanki et al., 2003).Very less work has been done with reference to the taxonomy and ecology of snails in the agroecosystem of Faisalabad. The information generated previously by Ali (2005) and Rehman (2010) is an attempt which has augmented the information of the malacofaunna in Faisalabad. The contribution of Auffenberg (2009) in studying the distribution of land snails in South Asia, including Pakistan, is recent and quite important. We hypothesize that due to use of pesticides and other chemical in the agriculture land the diversity of the species is impacted. To check our hypothesis the following are the objectives of our study.

. To determine the diversity of the snails in the agro-ecosystem of Faisalabad. . The relative occurrence of snails in the villages of the Faisalabad. . Genetic diversity of species of snails in the agro-ecosystem of Faisalabad.

Here we give the following review about the analysis of species boundaries which highlights the most recent work of the multiple studies.

15 CHAPTER 2

2 REVIEW OF LITERATURE 2.1 FRESH WATER GASTROPODS AS PRACTICAL CONSERVATION MANAGEMENT UNITS The identification and classification of the freshwater gastropods is generally done on the basis of morphological as well as shell characteristics for the past two centuries. The qualitative and quantitative data is rarely provided by authors at comparable levels and the original descriptions that are provided with descriptive terms mostly have different interpretations, which depends on the prior experience of the taxonomist (i.e. ‘‘broadly conic’’ vs‘‘tapering’’). Usually, description of specimens on the basis of juvenile shells, single shells, or sometimes even partial shells and the level of natural variation at the level of philosophy of taxonomy is not considered. The identification keys for the classification of many groups are available (Thompson, 1999; Wu et al., 1997; Burch, 1982) however, they are difficult to understand for amateur taxonomist as there is an inconsistency in differential diagonostic and descriptive approach among them. The shells have phenotypic plasticity and variation due to which relying completely on the morphology of the shell becomes strongly confusing to systematics. In fresh water the phenotypic plasticity is well documented (Adams, 1900; Holomuzki and Biggs, 2006; Heller et al., 2005) and has been generally observed due to envoirnmental pressures (Prezant et al., 2006; DeWitt, 1998; Krist, 2002). According to Schilthuzen et al. (2006), the biotic factors play major role in the allopatric speciation. Due to genetic divergence and geographical variation the shell morphology also varies strikingly, as the geographical differentiation evolves the shell shape and behavior. The level of the shell variation that is related to the local adaptation or adaptation due to envoirnmental pressures as compared to the characters valid species is not completely understood. As noted by Burch (1982) there are many generic groups that have been classified on the basis of shell characteristics and the characters, which have been used historically, have many interveneing forms at one or the other point. The areas which are considered to be extremely important for the identification of species or subspecies, are the intervening areas as they are the regions where there is intergradation, especially in case of the allopatric populations

16 (Mayr, 1991). The hypothesis that the mouth of the Gulf of California is a barrier for the migration of fauna into and out of the Gulf has been rejected with the help of sliding window technique, and it was confirmed that the fauna in the Gulf of California has not been structured by this isolated water body (Fredensborg, 2006). In the unrelated taxa there may be the presence of similar shapes and sculptures of shell (Minton and Lydeard, 2003; Minton et al., 2003; Chambers, 1980). There are many phenotypes within a same species or population of species of fresh water molluscs which can be distinguished and their presence pose severe problems in the process of identification (Dillon, 1984). This also gives convincing evidence that shell structure and scultures cannot be used only for the exact identification and accurate species delineation. There are other morphological characters which adds more information about the gastropods, that help in the confirmation of the correct taxonomic unit. The characters like radula morphology and soft-part anatomical characters have been proved as very important, along with the shell characters, in classification of the freshwater gastropods as they are strongly non plastic (Schander and Sundberg, 2001; Falniowski and Szarwoska, 1995 Brown and Berthold, 1990) however, it varies in others (Minton, 2002; Dazo, 1965). The radula taxonomically is a very useful character in many groups of gastropod molluscs (Alan et al., 1999). It is extremely important phenotypic character as it has a basic relationship with diet and envoirnment (Andrade and Solferini, 2006). The combination of an array of morphological features, as well as its microanatomy in Acochlidian gastropod Hedylopsis ballantinei, plays a very important role in the construction of Acochlidian phylogeny (Sommerfeldt, 2005). The characters, which can be strongly informative but have not been completely explored for their utility during identification of freshwater gastropods are mucous compositon, body color, gamete recognition, and genetic loci code associated with reproduction or for traits under selection. According to Malaquis (2006) the major taxonomic features in the snails are external morphology, shell, animal coloration, jaws, radula, gizzard plates, seminal duct, prostrate gland, egg masses and penis. In almost all species with European origin except Haminoea orbignyana, the anatomical data is considered to be the most important for identification, considering male reproductive system as the most important. The genital-anatomical characters can be equally mistrusted like conchological characters, when used at the tribe level, especially when they are in conflict with the distributional and conchological patterns (Weerd, 2004).

17 The snail species, which have opposite chirality have genitalia on the wrong side due to which they are not able to mate and are reproductively isolated. The chirality in the snails originates during the early stages of the development and can be observed in the adults later. Single genetic locus generally determines the coiling of the shell which is on the basis of delayed inheritance. 90% of the snails have dextral coiling, while the sinistral coiling is due to the mutation in the genera and families, which are entirely dextral. It is generally considered there should be the strong frequency dependent selection against the establishment of the specific chirality as dextral species are in minority and find it difficult to have a mating partner (Schilthuizen and Davison, 2005). Xeropicta derbentina, shows plasticity and can switch from annual cycle to biennial cycle depending on the population density or climatic conditions (Kiss et al., 2005). The biochemical and molecular features are potential characters that help in understanding of freshwater gastropod diversity, in addition to the morphological characters (Mangenelli et al., 2001; Raahauge and Kristensen, 2000; Hershler and Liu, 2004; Minton and Lydeard, 2003). Still there are gaps need to be filled as there are many traits that are plastic and have been used in description of many species in taxonomic reviews (Cuezzo, 2003; Hovingh, 2004; Haase, 2003) and therefore are not very useful in determination of systematic relationships (Wullschleger and Jokela, 2002; Dillon et al., 2002; Backeljau et al., 2001;Parmakelis et al., 2003).

2.2 ECOLOGY The lands molluscs generally live on a varied range dead and decayed material or living herbaceous plants, fungus, algae, rotting wood and bark. They feed on empty snail shells live snails, nematodes, animal wastes and carcasses, and even rasp limestone rock or cement (Nekola, 2012). The gastropods are found under large stones, old logs, lying in wood, under the decaying bark of trees on the wet lichens-clad barks, on the damp moss near waterfalls, on walls, on the leaves of the shrubs, the plantain and bamboos, under decaying leaves, beneath the surface of ground in worm burrows, in the roots of plants and in the exuviae left by floods on river banks and many shells were found on land washed by the flood (Blanford and Godwin (1908).

18 The faunistic composition of the land snails varies with vegetation types and habitat (Burch, 1956; Nekola, 2002; Ports, 1996; Stamol, 1993; Stamol, 1991; Theler, 1997;Van Es and Boag, 1981; Wäreborn, 1970; Young and Evans, 1991). The contrast between forest faunas and open-ground is not limited to terrestrial snails, other soil invertebrate members also show this pattern including fungus-feeding microarthropods (Branquart et al., 1995). According to Nekola (2003) conservation of snails require protection of soil surface architecture. The structure of the community in molluscs is best expressed by soil moisture content (Machintosh 2002) and the protection of the architecture of soil is required for their conservation (Nekola, 2003). Almost 90% of snails live in top 5 cm of the soil surface (Hawkins et al., 1997). The architecture of organic litter (Alvarez and Willig, 1993), and the soil which forms the underlying layer (Hermida et al., 2000), may also put a strong influence on the structure of the terrestrial gastropod community. They have possibly some kind of competition, for the inorganic nutrients, with the plant roots (Lavelle et al., 1995). The other important feature of terrestrial gastropod communities is the thickness of organic litter as the abundance of snails (Berry, 1973), community composition (Barker and Mayhill, 1999) diversity (Locasciulli and Boag, 1987) of terrestrial gastropods often has a positive correlation with litter depth. Machintosh et al., (2002) studied the macro fauna in the Ranong mangrove in southern Thailand and found low molluscan diversity in the area of tin mining (pollutants), whereas in the areas of Rhizophora plantation (higher organic matter) there was the highest diversity. The anthropogenic disturbance may impact the duff soil more severely as compared to the turf soil; however, the grassland snails face a negative impact due to heavy grazing (Cameron and Morgan-Huws, 1975). The differences in the faunal composition may be due to disturbance resulting in changes in the architecture soil surface. The organic litter layers are less complex, structurally, and are thinner in undisturbed turf soils which goes less to compaction when compared to the duff sites (Nekola, 2003), whereas sheep grazing helps the rare gastropod species to survive by providing them habitats that are suitable for them. The heterogeneity of the vegetation and a complex cover of top soil help co-existance of more species, whereas there is a significant reduction of the gastropod abundance and diversity due to the homogenous grazing (Labaune and Magnin 2002) and fire-management (Nekola, 2002b). The Monadenia fidelis and Deroceras reticulatum were identified as indicator species, in unburned wetland prairie. On the

19 other hand, Vertigo modesta and Catinellar hederi were found to be the indicator species for burned habitat. The terrestrial gastropod species richness was found to be low in the burned area, however, there is an increase in the abundance of molluscs after the first post burn year, in which it was low. The source population of the molluscs and other related animals should be provided an opportunity to colonize them by conducting the burns as prescribed according to the refuges, without prior information about their response to fire, since it appears that fire helps in the decrease of molluscs’ abundance and diversity in wetland prairies (Severens, 2005). The species survival is closely correlated with the ground moisture and the presence of bryophyte cover that remains intact due to which snail remains unaffected after clear cutting. In the buffer strips, species number remains unaffected while there is a decrease in the individuals’ number. This indicates that in the riparian forests buffer-strip retention is a successful practice for protection of land snails. The moist or wet floor of the forest generally serve as refuge even when studied at very small scales i.e. hollow and shallow crevices, which help in the survival of the gastropod fauna for long term. Clear cutting decreased the number of individuals from each site along with decrease in the mean number of species (Hylander et al., 2004). Initial declines of snail fauna in temperate and boreal forests after fire have been reported Karlin (1961), which was reconfirmed by Waldén (1998) and Strayler et al. (1986) with similar effect of clearcutting. Shrubs or trees which are broad leaved generally help in species density and richness after disturbance (Hawkins et al., 1997). Poor snail communities are found in the coniferous plantations that are when compared to forests that are old grown (Shikov, 1984). A very short term disturbance effects are very long lasting for the snail survival as the land snails are generally poor dispersers (Baur and Baur, 1988), which may lead to local extinction for a long period of time (Hylander, 2004). Two things are extremely important, for species that cannot survive any disturbance, firstly the dispersal capacity of species for the recolonization in those areas where they are no longer observed (Green and Johnson, 2000); and secondly, the spatial structure of patches in which its source populations are generally found (Kafka et al., 2001). The juveniles member of most of the species are very sensitive to dessication (Asami, 1993) and a positive correlation exist between the moisture of the soil and gastropod distribution (Prior, 1985). The number and abundance depend on the presence of bryophyte layers, and is more intact in the wet areas as compared to the drier areas. This protects the litter layers

20 underlying these bryophytes, on which the snails survive, and the moisture content of the soil. Generally, during sunny days there are dry and hot conditions, which modify the characteristics of the top soil litter of the clearcuts (Chen et al., 1993) and as a result many snails are killed. Hawkins et al. (1997) reported that some of the snails have developed their survival strategies from severe conditions. They quickly move to the deep soil layers where the site is unaffected. The wet or moist soil served as a useful refuge as and even this moist ground predicts the survival of the snails (Prior, 1985). The areas are generally cleared and prepared by sweeping up the well drained, steep lime stone slopes. This results in the vegetation cover loss, greater impact of sun radiations, loss of topsoil, resulting in desiccation, greater risk of fire, and in the reduction of the number of drought-intolerant snail species (Vermeulen and Whitten, 1999). The lime stone hills may take years to recover (Kiew, 2001) once damaged by fire are often eventually overgrown by secondary vegetation of grasses, invasive climbers, and pioneer tree species (Vermeulen and Whitten, 1999). The limestone outcrops in the Southeast Asia are rich in terrestrial gastropods with 74 species in 16000 sampled individuals. Prosobranhia species are extremely endemic to very short ranges and are restricted to the limestone regions and are less abundant in the disturbed sites as compared to Pulmonata snails. The gastropod diversity are not found to be different between undisturbed and disturbed sites in most sites. This leads to the result that Prosobranchia may lead to extinctions if continuously exposed to disturbances. The organisms which have high calcium carbonate requirement and high pH along with low tolerace for acidity need high limestone content regions for their growth (Schiltuizen, 2005a). Snails generally have high calcium requirement for their reproduction and shells (Graveland et al., 1994) and their abundance has a positive correlation with both pH and Calcium carbonate (CaCO3) (Schilthuizen et al., 2003; Waldén, 1995). There are a few groups, which are extremely endemic to these limestone hills, showing the presence of 106 species which are obligately calcicolous snails on 28 karst hills. Out of these, 70 species were restricted to a single hill, as they have extremely high requirement of calcium due to which they are found exclusively on limestone and show great diversity (Tweedie,1961). Vermeulen and Whitten (1999) found that in the state of Sarawak there are nearly 50 species of the terrestrial gastropods which are endemic to only one large outcrop of karst. In Pulmonata, there is less pronounced impact of inter relationship of calcicoly and endemism as compared to the Prosobranchia. Not only this,

21 while comparing Pulmonata with Prosobranchia, the dessicating effects of terrestrial life are more pronounced on Prosobranchia due to its open mantle cavity (Solem, 1984; 1974 ). Nearly 50 percent of the species belonging to terrestrial gastropod in Malaysia belong to the order Prosobranchia, but their distribution is more patchy when compared to the order Pulmonata (Schilthuizen et al., 2002), which shows their tolerance ranges are narrow for humidity and various abiotic factors. Due to human intervention, in Southeast Asia the limestone is exposed to deterioration and degradation as excavation for obtaining marble, cement, for construction of roads is leading to the extinction of various site endemic species (Vermeulen, 1994). In Turkey, limestone meadows are in danger to be affected by human activities and are important site for snail conservation. They have 24 species out of which, 21 are native and 3 are located in Istanbul (Orstan, 2005). There are other ecological factors which are important to assess the diversity and distribution of the snail species such as bottom sediments, flow of water, diversity of macrophyte. The number of species depends on the number of bottom sediments, an increase in the flow velocity decreased the number of the species density and snail number while snail species richness is not effected by the vegetation density except Elodea canadensis and a few other species (Strzelec and Królczyk, 2004). The adverse climatic conditions do greatly effect pulmonate species i.e., caerrulea, which survives in adverse conditions as they can prevent themselves from desiccation by showing a series of morphological and behavioural characteristics that can support their survival during adverse climatic conditions as there is no correlation between the climatic conditions and biochemical variables (Giokas, 2005). The shell size is directly proportional with some ecological factors as temperature and algal abundance while the callus thickness and shell size decreases with an increase in density of individuals (Takahiro, 2006). Elkarmi and Ismail (2007) studied the effect of the temperature of the water on the morphometrics, age and growth of two morphologically different species Melanoides tuberculata and found that the size and life of the snails in the hot water springs is almost twice greater than the snails of the pools having ambient temperature. While according to the Bergmann’s rule there is a positive relationship between latitude and body size of the species which could not be confirmed for Northwest Europeon snails (Hausdorf, 2003).

22 Chokor and Oke (2009) reported that the snail diversity is decreasing in Nigeria due to deforestation as they found that the number of species, rather than species composition, was greatly reduced in the sunny places as there was a highly significant difference in species number between shady places and in the sun plot. Sunlight and moisture content of the soil seems to a major impact on the composition of terrestrial gastropod community in dry and sunny habitats. However, the other important possible driving factors are relative humidity and temperature but not sunlight (Suominen, 1999). It was concluded that fire, plant species, plant number, herb cover, slope aspect, and habitat humidity are the important ecological factors that predict the snail abundance at the sites and also be used as biological indicator of ecological changes (Anna, 2005).

2.3 QUADRAT SIZE AND SAMPLING Manual searching is done while sampling within a quadrat during night and /or day of suitable microhabitats and is generally standardized with the help of searching effort and is written in the unit of person hours or per meter square (m2). For these studies the bulk method is generally followed i.e beating of the specific number of branches over an inverted umbrella. The sampling may be carried out by random collection sieving and sorting of a specific amount of leaf litter or soil (Emberton et al., 1996) or following the method of Cameron and Pokryszko (2005). The sampling may be carried out with a variety of ways. The living slugs and snails can be sampled at night for two person hour in circle with a radius of 3-m without disturbing the litter and returning the animals after identification (Bloch et al., 2007). The time for sampling and sampling site can vary as De Winter and Gittenberger (1998) sampled by spending two hours by two person in quadrats measuring 20 × 20 m quadrats in Cameroon. This can also be done by 0.5 h tree trunks searching, beating the vegetation and collection of the well defined amount leaf litter, which can be later dried, sieved and manual searching in the laboratory. A quadrat sampling measuring 2 × 4 m at the base of limestone cliffs in west Malaysian karst forests were sampled with the help of collection of litter and topsoil which was later enriched with the help of coarse sieving, floatation, drying and further sieving (Clements et al. 2008). Most of the scientists have adopted nearly same sampling methods for comparable results (De Winter and Gittenberger, 1998; Schilthuizen and Rutjes, 2001; Cameron et al., 2003; De Chavez and De Lara, 2011), while others are of the view that sampling methods can be best optimized

23 for completeness under the prevailing conditions (Cameron and Pokryszko, 2005). Incidence- based completeness estimators (ICE) (Chao and Lee, 1992; Chazdon et al., 1998) were computed by Liew et al. (2008) for hexaplets of quadrats in limestone and non-limestone forests across Malaysia and it was found that there was a 90% completeness irrespective of the quadrat sampling intensity, whereas the one which were away from limestone were quite incomplete. For most community-ecological purposes, sampling regimes are best optimized for the expected local species diversity and abundance (since completeness is generally a prerequisite for any kind of analysis), rather than for comparison with other studies. As Cameron and Pokryszko (2005) imply, an inventory of a quadrat is complete if all species actually present are found. However, in species with such limited active dispersal capacities as land snails, species ‘presence’ is an elusive concept. Even in medium and large-bodied (sub) tropical land snails, active dispersal rates are often in the order of just 1-5 m, leading to extremely narrow demes of tens of metres across or less (Schilthuizen and Lombaerts, 1994; Parmakelis and Mylonas, 2004; Giokas and Mylonas, 2004; Schilthuizen et al., 2005b). In the small-bodied microsnails in complex three-dimensional microhabitats that often dominate tropical snail communities, the scale of well-mixed population units is likely to be much smaller, and substantial heterogeneity may already be present below the scale of standard quadrats. In other words, a 20 × 20 m quadrat may contain 35 species, but only subsets of these may actually be coexisting in the sense that they share the same resources and potentially engage in ecological interactions with one another (MacArthur, 1965). It is important to keep in mind that such clumping in land snails is fundamentally different in character from similar patterns of micro-scale clumping in organisms with mobile individuals, gametes, or seeds; in the latter cases, micro-scale clumping of adults affects demographic and genetic population structure much less than in land snails, and some of the unevenness seen in adjacent quadrats along transects (Schilthuizen et al., 2002) may thus be real rather than sampling artefacts. It also means that, in order to decide on the quadrat size adequate for capturing true alpha diversity in a certain habitat (Rosenzweig, 1995). The largest quadrat size that has an S not significantly different from q could be taken as the preferred quadrat size (Schilthuizen 2011) when fitted in Arrhenius equation S = q + cAz (Rosenzweig, 1995).

24 2.4 SAMPLING PREVIOUS GENERATIONS A very important issue which comes under consideration is the inclusion of the empty shells, when appear in a sample, atleast in the studies on the non-acidic soils. Most of the scientists include empty shells while sampling necessarily as in the tropical forest it becomes really difficult to get live snails and slugs while some authors have limited themselves to the contemporary populations by sampling living snails only (Alvarez and Willig, 1993; Bloch et al., 2007). Clements et al. (2008) and Schilthuizen et al. (2002), explained of sampling empty shells only while in other studies living snails along with empty shells were sampled. According to Schilthuizen et al. (2005a), the majority of the specimens in the Pristine and Bornean karst forest were the empty shells containing less than 1% living animals. There are advantages and disadvantages of sampling empty shells, depending on the study objectives. Considering the advantages of the above mentioned sampling strategy is that almost the entire malacofauna can be obtained in an unbiased fashion because otherwise there will be a biasness towards the larger and thicker shelled species to survive weathering for much longer. These sampling methods have greatly supported to answer questions related to species- area relationships (Clements et al., 2008). In the forest strata, generally all shelled snails and semi-slugs, end up as empty shells on the forest floor unless they live in enclosed spaces like phytotelmata (Kitching, 2000), nest ferns (Ellwood and Foster, 2004), or social insect nests (Eguchi et al., 2005). Sampling shells from the forest floor is thus comparable in efficiency to insecticide fogging for canopy insects (Coddington et al., 1991). The changes in the species composition can be revealed, making comparisons between the present day species with the previous diversities and can understand the level of changes in the species composition. Empty shells are considered to be the representatives of the contemporary malacofaunna, which is only possible if the shells are younger than an average snail generation (Cernohorksy et al., 2010). Basically the rate of degradation of shells on the forest floor is still unknown. According to Cameron and Pokryszko (2005), the empty shells which are present in the calcarious areas may be actually a part of ancient or subfossils, while Solem (1984) states that the rate of shell decay is less than ten days when the forest floor litter is wet and occur in a period of three weeks during dryer periods. The places where land use has been majorly changed, the presence of old specimens in the sample is particularly problematic (Schilthuizen et al., 2005a), which is quite important for biogeographical related problems specific to species- area relationships (Clements

25 et al., 2008). The effect of logging, quarying and forest to plantation on the snail community has been studied with the help of the inclusion of empty shells in the samples (Oke et al., 2008; Schilthuizen et al., 2005a; Tattersfield et al., 2001b). The impacts of these disturbances can be underestimated if the samples include the remains of the fauna from predisturbed time. The same is in the case of verteberates as their bones are left as remanants of predisturbed times (Terry, 2010a; 2010b). The empty shell species can be compared with contemporary species to calculate diversity in an area and if there is no difference and other procedural problems then the samples can be pooled (Rundell and Cowie, 2004; Cameron and Pokryszko, 2005) and can be considered the present-day populations. In the field of conservation biology the roles of systematics are well understood and accepted for many organisms. The role of systematics and taxonomy for the species protection and management of freshwater gastropods has not been reviewed. A thorough review of literature in systematics and taxonomy of freshwater gastropods, with recent examples in the International scenario, to identify the key roles of these fields in species delineation, taxonomy and final designation of conservation management units is discussed (Perez and Minton, 2008).

2.5 DEFINING THE THEORETICAL CONSERVATION MANAGEMENT UNIT The major pillar for the increase in the success of conservation measures is the identification of the appropriate conservation management unit. The populations and species which need to be protected, as they have unique life history and are genetically diversified that helps them to adapt according to their envoirnment and other historical events, are called conservation management unit (Perez and Minton, 2008). The taxonomist does not have to undertake routine identifications of materials collected during archeological or ecological collections. However it is the duty of an ecologist to carry out such expeditions, who wants the material to be identified. Primarily the taxonomists have to draw clear boundaries (taxonomic entities) across vague outlines. The major responsibility of the taxonomists is to provide scientific names and data so that there can be the development of conservation guidelines by the lay men, agency workers, decision makers and legislators. Another responsibility of the taxonomists is to convey these scientific changes to the end users as these change are for good reasons, these changes in name is a sign of better understanding of a group of organisms or

26 single organisms. These changes resulting in instability brought about due to additional data, must be understood and willingly accommodated by the managers. To grant protection status and proper recognition of managements units, different organization working at the national level frequently conduct reviews of species, group taxonomy and natural history of these species (Nicholopoulos, 1999, COSEWIC, 2006). It is a major responsibility of a taxonomist to identify synonym species and summarize the already available accumulated knowledge. Unfortunately, a several scientific species give umbrella to single species which makes the inventory difficult due to which scientific names change for several reasons. There are few reasons due to which name changes become a necessity. Firstly a species may have been described by different scientists and resultantly even named more than once as they belong to different sexes, geographical regions or due to lack of knowledge of earlier descriptions. The conservation plans are implemented prior to the appropriate definition of management units, which can give very negative results for the conservation of species (Avise and Nelson, 1989; Greig, 1979). The conservation plan was established prior to the research on freshwater snail Leptoxis crassa anthonyi (Minton and Savarese, 2005), which clearly indicates that the units of the conservation managemnt (USFWS, 1997), which were inconsistent with the evolutionary history of population while they had been established on the basis of the assumption that the genetic and geographical distribution were correlated (Minton and Savarese, 2005). Field survey resulted in establishment of a number of new records during Pennsylvania Land Snail Atlas Project. These new records (104 specimens) were not introduced species and were likely having been simply overlooked. The conservation is mainly focussed on the forest species and species like Gastrocopta procera which are open field species are not included or are undersampled. These new records indicate the dire need for surveys so that the gaps in knowledge about land snail distributions can be filled (Pearce, 2010). The freshwater snail literature includes examples working plans for conservation of fresh water snails with detailed information about the taxonomic work. The recent studies on Hydrobiidae (Ponder et al., 1989; 1993; 1994; 1995; 2000; Hershler, 1994; Hershler et al., 2007 a,b; ; Hershler and Liu, 2004; Hershler and Ponder, 1998) has helped understanding of the taxonomy and systematics of these species. For example, the two separate management units were distinguished i.e. Assiminea cienegensis from Assiminea pecos, for conservation with the help of new molecular data as well as the morphological and conchological studies, when studied

27 with the help of electron microscope (Hershler et al., 2007a) a rare as well as disjunct populations in the Cuatro Cie´negas basin, Coahuila (Mexico). The most of the taxonomic studies on the snails are done on the basis of shell measurements characters, while further work on gastropods has revealed two problems about species delineation with this approach. Plasticity in the shell character in the gastropods makes the demarcation of the taxonomic unit difficult. Secondly, there is a great chance of the exclusion of morphologically cryptic, valid species from the list. Therefore for the accurate identification of the species there is the need of methods which reveal the taxonomic status in more detail, in addition to shell morphology (Mayden, 1997; Adams, 2001). The precise description and demarcation of species on the basis of its distinguishing characters, helps to understand its systematic relationship of the group under study. The taxonomists are relying on the biological species concept, abbreviated as (BSC), for many years, which supports identification of species on the basis of reproductive isolation (Dillon et al., 2002, de Queiroz, 2005a). Later faults and failure in the biological species concept to meet the standards were suggested (Wheeler and Meier, 2000) and species concept based on lineage such as unified (de Queiroz, 1999; 2005b) and the phylogenetic (Cracraft, 1983) species concepts became popular. The freshwater gastropods literature also includes examples of thorough systematic work that has extremely supported and has showed fruitful results in conservation plans of freshwater gastropods (Holznagel and Lydeard, 2000; Mulvey et al., 1997; Lydeard et al., 2000; Minton and Lydeard, 2003; Roe et al., 2001; Roe and Lydeard, 1998). The species cannot be identified on the basis of morphological characters only while species identification on the basis of lineage-based concepts is extremely valueable (Wilke and Falniowski, 2000). The identification on the basis of the lineage based method considers species as independent evolving units without considering the criteria on the basis of which the species has been described and delineated. The description of species on the basis of lineage gives more understanding and recognition about the management units corresponding with the subspecies level approach (IUCN, 2001). According to the United States Endangered Species Act (USFWS, 2003) protection should be provided at the subspecies level rather than the distinct populations. In case of the countries like, Canada (COSEWIC, 2006) and Australia (Australian Government Attorney-General’s Department 1999) the protection must be extended to smaller, groups at the subspecific level. When addressing taxa at the subspecific levels groups that are of evolutionarily significance (Ryder, 1986) are of prime

28 attraction; to be considered as it relies more on the populations that are genetically and morphologically distinct populations rather than on a specific concept. There are a few questions, which arise regarding level of distinctness, in the context of evolution (Pennock and Dimmick, 1997). There is the development of very low level of agreement as the protection at and below species level has been recognized by most of the conservationists. The best approach applicable to delineate the units for conservation management allows the flexibility with reference to the organism; however, specific exceptions are there and existence of few changes is to do systematic studies in combination with the population genetic data regardless of the taxonomic level. This helps to recognize more valid species (Pfenninger and Magnin, 2001; Ponder et al., 1994) and as well in policy making for conservation management units. The major benefit of modern systematics is the increased objectivity and recognition of biodiversity (Isaac et al., 2004; Wheeler and Cracraft, 1996). The goal of defining and identifying species can be accomplished scientifically with the help of lineage-based approach to species delineation which helps to reduce the negative effects of statistical errors (McGarvey, 2007).

2.6 SPECIES ABUNDANCE DISTRIBUTIONS The researchers of the tropical land snail expressed and demonstrated the patterns of intraspecific rarity and commonness that have been found (Oke and Alohan, 2006; De Winter and Gittenberger, 1998; Schilthuizen et al., 2002; 2005a; Schilthuizen and Rutjes, 2001). Species abundance distribution has been a point of interest for the scientists and researchers due to many reasons. This is because in this way the community can be undersood in a more better way rather than by just counting the species, as in this way heterogeneity and abundance can be incorporated, which is the basis for the calculations (Magurran, 2004). Secondly in understanding species abundance distribution, the rare-species tail can also provide information regarding the estimation species number missed giving true scenario of species richness. Chao (1984) estimated the number of singletons and doubletons in the sample with the help of a simple non parametric estimator. Various scientists found high percentages of singletons i.e., (23%,12%, and 11%) (Fontaine et al., 2007b; De Winter and Gittenberger, 1998; Liew et al., 2010) with high estimations of richness. Thirdly, the species abundance distribution helps us to understand changes in species dominance induced due to the season (De Winter and Gittenberger, 1998) disturbance on the basis of changes in physical factors (Schilthuizen et al.,

29 2005a) and to deduce the ecological processes developing the community structure. The log normal distribution seen in the natural communities including gastropods can be approached by a model (Sugihara et al., 2003; Sugihara, 1980) for the subdivision of niche space in sequence. A sigmoid shaped curve is formed in a Whittaker plot when a log normal distribution was calculated. Whenever there is the over representation of the rare species a distinct right-skew is often observed (Tokeshi, 1999). This niche free model can predict the skew in a better way (Hubbell, 2001) as it fills the slots randomly in a community. The predictions of particular models was tested by the goodness of fit to understand the potential ecological processes that structures the communities for some of the non-tropical and tropical land snail (Cameron and Pokryszko, 2005). However species abundance distribution studies available in the literature largely suffer from biased sampling methodology. There are many patterns which are quite similar in properties with that of SAD (species abundance distribution) which are present in nature which can lead to complex dynamical properties. This limits to understand the ecological structuring processes from species abundance distribution alone (Nekola and Brown, 2007). These models are mostly applicable to only one guild in a same community, which in case of the gastropods needs a little more elaboration. If the spatial scale for the determination of species abundance diversity is quite large then the chances of obtaining patterns due to ecological reasons rather than statistical reasons declines rapidly (Sizling et al., 2009). A community of species which are performing ecological function that are similar (Rosenzweig, 1995); and they compete for almost the similar resource (Hubbell, 2001) is called a guild. It would be probably wrong to consider tropical terrestrial gastropods communities as guilds. Although meager knowledge about tropical terrestrial gastropod’ autecology is available and there are many predators in the African faunas (Wronski and Hausdorf, 2008; De Winter and Gittenberger, 1998), which are in high numbers including fungivores, foliovores and detrivores, even considering the non-molluscivores, yet there is another factor to be considered and measured, is its body size. The snail’s body mass varies in several magnitude throughout its life, and there are stronger ecological interactions in the individuals of the similar size so animals of different sizes should be considered as separate communities as individuals of the same size have stronger ecological interactions so different size classes should be really considered as different communities. This gives us an idea that there is a difference in the community of the juveniles and adults. This is not necessary that this community is composed of land snails only, rather this

30 community may include other inverteberates with low mobility e.g. isopoda, diplopoda and certain coleopteran, and these snails will have a stronger competition than it would have with other snails, as occurs in the rocky intertidal community in marine inverteberates (Connell, 1961; Wootton, 1994). The shells of the molluscs can be easily collected and studied due to which it has been a source of extreme attraction by professional and amateurs for the molluscan alpha taxonomy. However, phenotypic plasticity mostly results in differences in shell morphology due to which past descriptions were often unjustified. The plasticity in the phenotype due to ecosystem variation poses a difficulty in morphospecies identification (van Damme and Pickford, 2003) due to which adult shell shape and ornamentation become unreliable characters. The same difficulty arises when identifying on the basis of radula morphology (Michel, 1994), due to this methods are needed which give evidence of the separate species besides morphological methods. A system based on shell morphology and colour was devised by Samadi et al. (1999), which were later supported molecular investigations and this system of morphological identification system was used by Facon et al. (2003) and Genner et al. (2004). The understanding of importance of molecular methods taxonomy was done by Facon et al. (2003). They studied the invasion of Melanoides of the NewWorld using molecular methods and suggested that these morphs were paraphyletic and the two morphs camouflaged in Lake Malawi (Genner et al. 2004) and according to the authors they originated from Southeast Asia. This has been supported by development of molecular DNA markers to find the genetic relationships among taxa at a higher and more advanced level to get information about their phylogeny and biodiversity and they can be further used to solve general biological problems (Kupriyanova, 2000). The cases of synonymization of species in molluscs are probably higher as compared to other marine inverteberate phyla as a result of number of genetic studies. There are examples of the Nautilus (Wray et al., 1995) and Donax as studied by Adamkewicz and Harasewych, 1996 that clearly show the fact that there may be cases where genetic data does not support drastic differences between taxa that are sympatric on the basis of shell morphology. While on the other hand, very less morphological differences in shell or other various characters have been confirmed by the genetic data to be important taxonomically (Izuka et al., 1996; Borsa and Benzie, 1993; Johnson and Cumming, 1995; Oliverio, 1995; Mokady et al., 1994; Parsons and Ward, 1994; Thollesson, 1998; Sanjuan et al., 1997). In molluscs, there are very less chances of the discovery of

31 morphological similar groups which are extremely divergent, however it may occur; for example, in cephalopods (Yeatman and Benzie, 1994) in the deep sea (Craddock et al., 1995; Peek et al., 1997). In allopatric studies, the genetic and envoirnmental basis for the differences are mostly confused and the uncertainty in the taxonomy on the basis of shell variability is worsened. In some taxa there is a very less or no genetic differentiation when comparing in same geographic entity while on the other hand a large amount of genetic differentiation is found taxa, at generic level (Adamkewicz and Harasewych, 1996; Côrte-Real et al., 1996a,b). There are zones of overlap along continuous coastlines, which are narrow may have a little evidence of hybridization providing definite evidence for species boundaries (Liu et al., 1991; Ridgway et al., 1998), while sometimes quite complex patterns of hybridization are observed. Using a variety of approaches in studying several molluscan genera have helped to reduce the complexity of the results making them more understandable. Depending on the taxa and geographical locale, a variety of patterns of hybridization have been revealed in studies of Mytilus (Beynon and Skibinksi, 1996; Quesada et al., 1998; Gardner, 1996; Comesaña et al., 1999). The importance and actuality of the molecular markers in the field of taxonomy can be understood from its application. On the other hand using different molecular markers may lead to a conflict, due to different reasons, within the views of the zoologists about the morphological taxonomy. The complexity of the molecular data analysis is explained by modeling of the evolutionary processes, on the basis of the various assumptions, which proceed at unequal and nonuniform rates in different taxa. However some scientists think that with the advent of the sequencing of human genome the significance of molecular marker studies has been diminished, while others consider it to be a huge task when comparing with the identification with the help of molecular markers reducing the comparison of genomes to that of analysis of genome parts (Lavrentieva, 1999). The molecular biologists working on the evolutionary perspectives of the species need to learn about the cladistic approach in systematics (Pesenko, 1989). The traditional taxonomists accomplishing the task with the help of cladistics develop phylogenetic relationships among elementary evolutionary units, and are unable to suggest a better method. The satisfaction is achieved when the conclusions drawn from both approaches lead to a common end. However, if the results donot coincide then the preference is given to those more reasonably integrating the molecular and morphological results.

32 2.7 POPULATION AND EVOLUTIONARY GENETICS The use of molecular markers has proved to be a useful tool for complex taxonomic identification where morphological characteristics are ambiguous or cryptic (Douek et al. 2002, Westheide et al. 2003, Miura et al. 2005, Park et al. 2005). The randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique is a relatively simple and cheap method capable of differentiating taxa without the need to know their genomes (Welsh and McClelland 1990, Williams et al. 1990). RAPDs are dominant markers that result from the use of short (10 bases long) primers (synthetic oligonucleotides) of random sequence that can amplify multiple segments of genomic DNA by PCR. The number of segments depends on the number of sites of the genome recognized by a particular primer. The main reason for the success of RAPD analysis is the gain of a large number of genetic markers that require small amounts of DNA without the need of a molecular characterization of the genome of the taxa under study. Species in which such markers were used include oyster genera Crassostrea, Saccostrea, and Striostrea (Klinbunga et al., 2000), amphipods (Gammarus: Costa et al., 2004) and the tropical abalones Haliotis asinina, Haliotis ovina and Haliotis varia (Klinbunga et al., 2004). The RAPD technique has received a great deal of attention from population geneticists (Hedrick, 1992) because of its simplicity and rapidity in revealing DNA-level genetic variation. The utility of RAPD markers in the phylogeny of cichlid fishes (Sultmann et al., 1995) and the genus Xiphophorus (Borowsky, et al., 1995) gave support to classical hypotheses of their phylogenetic relationships. RAPD markers have been successfully used to detect genetic variability in Gliricidia (Chalmers, et al., 1992) mosquito species and populations (Kambhampati et al., 1992) closely related species of black Aspergilli (Megnegneau, et al., 1993), cocoa (Russel et al., 1993.), medfly (Baruffi, et al., 1995) and parasitic protozoa (Tibayrenc et al., 1993). Species-specific markers were developed in species and strains of microorganism (Welsh and

McClelland, 1990; Skibinski, 1994; Fani et al., 1993). Clone-specific markers have been identified in hydroids (Hadrys et al., 1992) and in fungal mycelia (Smith et al., 1992). The technique has also been used to study genetic variation in several fish species. Bardakci and

Skibinski, (1994) and Naish et al., (1995) used RAPD markers to discriminate between commercially important tilapia species, subspecies and strains of tilapia. RAPD markers were also generated for several tropical fish species representing 7 families (Dinesh et al., 1993). Furthermore, RAPD analysis revealed high levels of genetic variation among individuals from

33 the same broodstock of sea bass (Dicentrarchus labrax) (Allegrucci et al., 1995). Finally, 721 strain-specific RAPD markers were identified in 2 laboratory strains of zebrafish (Johnson et al.,

1994). RAPD markers unique to individuals from one species within a genus will be species- specific (inter-specific). Similarly, genus-specific markers can be generated if the fragment is a unique polymorphism to individuals belonging to a certain genus. Species-specific markers can be used in inter-specific gene flow and hybrid identification. Similarly, population-specific markers will be useful in identification of hybrid populations (Hadrys et al., 1992). Inter-specific gene flow was shown between two Iris species, Iris hexagona and I. vulva using species-specific RAPD markers (Arnold et al., 1991) F1 hybrids from different inbred lines of maize were identified using AP-PCR (Welsh, et al., 1991). RAPD polymorphism detected among individuals within a given species has been used to determine paternity and kinship relationships in large progenies of dragonfly (Hadrys et al., 1992). In this study, Anax parthenope males guard ovipositing females. It was suggested that the male might guard a female in order to assure a subsequent mating rather than immediate fertilisation. RAPD analysis of several unrelated males, the guarding male, the guarded female and offspring clutches demonstrated that the guarding male was the father. A specific RAPD marker was found in the guarding male and in the offspring but was rare in the population as a whole Parentage analyses with RAPD markers are based on the presence of diagnostic markers (present in only 1 of the putative parents) in the offspring. A high frequency of non-parental RAPD bands has been reported in primate pedigrees

(Riedy et al., 1992.). However, Scott et al., (1992) found much lower frequencies of non-parental RAPD bands in beetles (Nicrophorus tomentosus) and strawberries (Fragaria vesca). The presence of species-specific RAPD characters makes it possible to use these markers in studying hybridization in the context of speciation. The data on individual variation of parental populations are required for experiments on interspecific hybridization upon investigating, e.g., reticulate evolution. The probability of speciation through cross evolution is particularly high for plants (Grechko, 2002). The genital differences in nitidus and Zonitoides excavatus, are not clearly found but there are clear differences in the shell morphology. A great variation has been revealed by using (RAPD) markers in Zonitoides excavatus. Sperm transfer has not been observed to be completely absent. The species status of both taxa is supported by the strong genetic differentiation suggested by allozyme and RAPD data (Jordaens et al., 2003). Due to this it has

34 become extremely important to use molecular techniques so that they can be identified with more accuracy. Histone gene primers have been developed for land snails and it has been observed that land snails and bivalves seem to differ in their histone gene organization. Nucleotide polymorphism is of significant importance to understand phylogenetics and systematics of closely related species and genera. The coding regions exhibit no amino acid substitution among land snail species (Armbruster, 2005). Apparently, one can expect to find any locus among RAPD fragments depending largely on the primer specificity. Thus, if adequately used and interpreted, the RAPD method is an informative modern tool for studying genetic diversity of species in nature. Most of the studies on the land snails have been carried out approximately a century before in the Indian subcontinent. Hutton (1842) collected and studied the land snails of neighborhood of Bolan pass, from Suliman range and the hills of south laying west to Indus. According to the Blanford and Godwin (1908), in the areas of Sawat, Dir or Chitral no terrestrial snails have been observed except Petraeus snails, while in the Kuram valley only very few species were found. They worked chiefly on the conchological side of the families Testacellidae and Zonintidae of Indian subcontinent. Recently 329 specimens of snails were collected by Ali (2006) from sugarcane fields of village Gatti Faisalabad pertaining to 6 families and 9 genera belonging to order Pulmonata. Pokryszko et al, (2009) collected 3500 dry shells and described 22 species out of which 12 were new species from 77 localities. The specimens were presereved in alcohol. The shell variations in most species were described and nine species were illustrated with figures of detailed reproductive system. In the Northern area of Pakistan there is highest diversity of Pupilliods due to wetter climate and wider altitudinal range. Ten out of twenty two species were considered to be endemic to Pakistan, while the distribution of the rest of species was extended to other regions i.e. Asia, Europe and Holarctic. Pupilloid fauna showed great diversity of Palaearctic/Holarctic influence on general. In the light of above meagre scientific studies we can say that the studies related to the faunal distribution of snails have been ignored in Pakistan and there is a scanty and scattered knowledge about the snails in this area of the world.

35 The goal of the present study was to obtain species-specific RAPD molecular markers that could distinguish between fifteen species of snails along with their diversity and distribution in different fields of Faisalabad.

36 CHAPTER 3

3 MATERIALS AND METHODS

3.1 STUDY AREA The third largest city of Pakistan, Faisalabad, is situated in the central Punjab covered an area of 122 km2 at spherical coordinates of 31025/ N and 73009/ E located at an altitude of 300 m above mean sea level (Kahlown, 2006) (Figure 3.1). Faisalabad City has total land area of 52130 acres of which 19805 acres is cultivated land areas (Final Mouza List, 2008). 24 villages were sampled in agricultural areas of Faisalabad city from March 2011 through August 2011 (Figure 3.2).

Figure 3-1: Location of Faisalabad, Punjab, Pakistan (Saleem, 2014)

37 Figure 3-2: Map of the study areas.

38 3.2 COLLECTION OF THE SNAILS The snails were collected from different sites in the agroecosystem of Faisalabad from March 2011 to August 2011 (Figure 3.1-3.2). Collection of molluscs was carried out by random sampling from the open edges, under tree, and inside field on 25 acres of each selected village following Afshan et al. (2013). The samples were taken from open edges of the field (without shadow) using iron quadrat (30 cm2) whereas the core samplers were used to collect the samples under tree (with shadow) and inside field. Snails were also sampled with the help of handpicking while doing an extensive search from 24 villages of Faisalabad along the Rakh branch, Jinnah branch and Ghogera branch. The number of samples taken from each village varied from 50-125 samples due to diffence in the agricultural land area. Soil samples were packed in the bags and were assigned a number, labeled with the site, collector’s number and date of collection. All samples were taken to the Laboratoy for molluscan separation with hand sorting and for smaller specimen different sieves were used (0.2 mm, 2.0 mm, 4.75 mm). The dry shells were packed in air tight plastic bags, while the living snails collected were preserved in 70% ethanol (Thompson, 2004).

3.3 PHYSICAL AND CHEMICAL PARAMETERS OF SOILS The physical and chemical parameters of the soil samples were determined i.e. pH , electrical conductivity and heavy metals ( Pb and Cd). Breif description of the protocols has been given as under.

3.4 SOIL SATURATED PASTE The soil saturated paste was made following USSLS, (1954). For preparation of soil paste known weight (200 g) of soil was soaked with distilled water and allowed to stand overnight. The saturated paste was made which glistened, did not accumulate water if depression is made in the surface and fell freely from spatula.

3.5 pH OF SATURATED SOIL PASTE (pH) With the help of pH meter Jenway Model -671 P, pH was recorded after standardizing it with buffer solution of pH 4.02 and 9.20 (Method 21a).

39 3.6 ELECTRICAL CONDUCTIVITY OF SATURATION EXTRACT (EC) Electrical Conductivity of a saturation extract was determined by following Method 4b as prescribed by USSLS, (1954) with the help of conductivity meter Jenway Model 4070, EC was noted after standarding it with 0.01N KCl solution.

3.7 HEAVY METAL ANALYSIS Soil sampling after extracting reducible fraction were extracted with 2 mL of 0.02 M 0 HNO3 and 5 mL of 30% H2O2. The mixture was heated to 85 C for 2 h with occasional agitation. Another 3 mL of 30% H2O2 (buffered to pH 2 using concentrated HNO3) was then again heated to 850C for 3 h with intermittent agitation. After cooling 5 mL of 3.2 M

CH3COONH4 in 20% HNO3 (v/v) was added and the samples were diluted to 20 mL and then agitated it continuously for 30 minutes. Metals in the filtrate were measured by using Hitachi Polarized Zeeman 8200 Atomic Absoption.

3.8 IDENTIFICATION OF THE SNAILS The identification of the specimens was carried out on the basis of following characteristics such as number of whorls, coiling of the shell, umbilicus, shape, colour of shell, shape of the aperture, presence or absence of operculum, height and diameter of the snails. The diameter of the aperture was measured with the help of vernier caliper. The snail’s samples were studied under the microscope and are identified by using the keys and diagram provided by the Blandford and Godwin (1908), Bouchet and Rocroi (2005), Sturm et al. (2005), Anderson (2008), Watson and Dallwitz (2005) .

40 Figure 3-3: General characteristics of gastropod shell.. Source: http://en.wikipedia.org/wiki/Gastropod_shell

Figure 3-4: Shell morphology.. Source: http://snailsandslugs.wordpress.com/2010/09/28/andalucia/

41 Figure 3-5: General shape. Source: http://snailsandslugs.wordpress.com/2010/09/28/andalucia/

Figure 3-6: General Terminology. Source: http://snailsandslugs.wordpress.com/2010/09/28/andalucia/

42 Figure 3-7: Types of surface sculpture. Source: http://snailsandslugs.wordpress.com/2010/09/28/andalucia/

Figure 3-8: Shell morphology: Way to measure height and width of the shell and way to count number of counts. . http://snailsandslugs.wordpress.com/2010/09/28/andalucia/

43 Figure 3-9: Morphology of the shell shaped periphery and the last round.

Figure 3-10: Shell morphology: Naval and peristome. http://snailsandslugs.wordpress.com/2010/09/28/andalucia/

44 Figure 3-11: Apertural variations in gastropod shell. http://snailsandslugs.wordpress.com/2010/09/28/andalucia/

Figure 3-12: Shell shape of the last round. http://snailsandslugs.wordpress.com/2010/09/28/andalucia/

45 3.9 MOLECULAR CHARACTERIZATION OF MOLLUSCS

3.9.1 COLLECTION OF MOLLUSCS Fifteen species of molluscs (Table 17) were used for molecular analysis. The samples were collected from different agricultural fields of Faisalabad region and tagged and immediately stored in 70% ethanol for further analysis.

3.9.2 GENOMIC DNA EXTRACTION Genomic DNA was extracted from all the samples of snails by CTAB method (Doyle and Doyle, 1990). The snail samples of 0.3-0.5 g were ground to a fine and homogeneous paste by using 2 ml of 2XCTAB and transferred into 50 ml falcon tubes. After that, 15 ml of hot (65 °C) 2xCTAB was added to the sample tube and mix gently by inverting the tube for several times and incubated at 65 °C for half an hour. 15 ml of chloroform isoamylalcohol at the ratio of 24:1 were added and mixed by inverting for 30 sec followed by centrifugation at 10,000 g for 10 min at room temperature. The aqueous phase was transferred to another tube and repeated this step by adding 15 ml chloroform: isoamylalcohol (24:1) in eppendorf tube. To the aqueous phase, 0.6 volume of isopropanol were added, precipitated the genomic DNA and centrifuged at 1000 rpm for 5 minutes and discarded the supernatant solution. Genomic DNA was then washed three times with 70% ethanol, air dried and resuspended in 0.5 ml 0.1XTE buffer. The supernatant was treated with 5 µl of RNase and incubated for one hour at 37 0C followed by addition of equal volume of chloroform: isoamylalcohol (24:1). Thereafter the genomic DNA was centrifuged at 13000 rpm for 10 minutes and transferred aqueous phase in a new eppendorf tube followed by precipitation by adding of 1/10th of 3 M sodium chloride and 2 volume of ethanol and collected by centrifugation at 13,000 rpm for 10 minutes at room temperature. The pallet was air dried and resuspended in 0.1X TE buffer and measured the concentration of the DNA by loading in a 0.8% agarose gel with DNA Quant 200 fluorometer. The DNA quality evaluation was verified on 1% agarose gels stained with ethidium bromide and visualized under ultraviolet light.

46 3.9.3 QUANTIFICATION OF GENOMIC DNA Genomic DNA concentration of samples of snails was quantified with a IMPLEN NanoPhotometerTM. The optical density (OD) of each sample was calculated and the working dilutions of all the DNA samples were made by diluting each DNA sample to a uniform concentration of 15 ng ml-1 with double distilled deionized water and used for RAPD (PCR) reactions.

IMPLEN NANOPHOTOMETERTM PROTOCOL

Started up the apparatus and selected the measurement option as Nucleic acid for DNA and RNA samples.

1. Cleaned pedestals with tissue and MilliQ water.

2. For a test measurement with MilliQ water, added 1 µl of MilliQ water to front pedestal.

3. Added 1 µl Nuclease Free water to pedestal and selected “Blank”.

4. Added 1 µl of sample and selected “Measure” option and noted down the readings.

5. Values for DNA or RNA conc. were recorded in ng µl-1 at 260 nm wavelengths.

6. Cleaned pedestals with tissue before measuring each new sample.

7. Turned off the apparatus by clicking “Exit” twice.

8. Retrieved data by clicking on shortcut to “nanodrop data”.

47 3.10 POLYMERASE CHAIN REACTION

3.10.1RANDOM AMPLIFIED POLYMORPHIC DNA Oligonucleotide RAPD primers of different series were custom synthesized from Genelink Co. USA were used in this study. Total of 15 RAPD decamers were selected, which were polymorphic in amplification profile (Table 3.1).

Table 3-1: RAPD primers along with their sequences. Sr. No. Primer Code Sequence 1 GL DecamerL-07 CAGGCCCTTC 2 GL DecamerL-08 AGTCAGCCAC 3 GL DecamerK-08 AGGGGTCTTG 4 GL DecamerK-12 GAAACGGGTG 5 GL DecamerK-19 TTCCGAACCC 6 GL DecamerK-20 GGTGACGCAG 7 GL DecamerL-12 CTGCTGGGAC 8 GL DecamerI-02 GTGAGGCGTC 9 GL DecamerK-07 GGGGGTCTTT 10 GL DecamerL-04 AAAGCTGCGG 11 GL DecamerL-01 GACGGATCAG 12 GL DecamerK-16 GTTGCCAGCC 13 GL DecamerB-07 ACTTCGCCAC 14 GL DecamerB-15 AGCGCCATTG 15 GL DecamerL-15 CAGAAGCCCA

48 3.10.2 OPTIMIZATION OF PCR CONDITIONS: The PCR thermal cycler (Eppendorf AG No. 533300839, Germany) was used for the RAPD-PCR amplification and with different concentration of template DNA, 10X PCR buffer,

MgCl2, dNTPs, primer and Taq DNA polymerase. PCR conditions were optimized for concentration of template DNA, 10X PCR buffer, MgCl2, dNTPs, primer and Taq DNA polymerase.

3.10.3 RAPD-PCR ANALYSIS Total reaction mixture of RAPD (PCR) to amplify the genomic DNA of fifteen snail species is as follows with the total volume of 25 µl.

Reagents Concentrations

DNA template 3.5 µl

10x Buffer 2.5 µl

Gelatin 2.5 µl

MgCl2 3.5 µl

dNTPs 4.0 µl

Primers 2.0 µl

Taq DNA polymerase 0.2 µl (MBI, Fermentas, Vinius, Lithuania)

d3H2O 6.8 µl

Total 25 µl

49 3.10.4 PCR TEMPERATURE PROFILE Follwing reaction temperature profile was used for RAPD (PCR) reactions.

Step 1 Denaturation for 5 min at 94 °C

Step 2 Denaturation for 1 min at 94 °C

Annealing for 1 min at 36 °C 40 cycles

Extension for 2 min at 72 °C

Step 3 Extensions for 10 min at 72 °C

Step 4 Hold at 4 °C

3.11 DATA ANALYSIS The RAPD (PCR) products were run on 1.2 % agarose gel and stained with ethidium bromide. The robust and visible RAPD fragments were scored as present (1) or absent (0) except those fragments with low amplification and that could not be clearly distinguished. The fingerprints were examined under ultra violet Transilluminator and photographed using SyneGene Gel Documentation System. The data generated from the detection of polymorphic fragments were analyzed using popgen 32 software (Ver. 1.44) (Yeh et al., 2000) whereas calculation of principal compoment analysis and analysis of molecular variance (AMOVA) was done using the PAST software.

The Polymorphic Information Content (PIC) value of each RAPD primer was calculated using the equation developed by Anderson et al., (1993).

PICi = 1 - ∑P2ij (pij is the frequency of the jth allele for locus i)

Mean band frequency was computed by the following equation

MBF = n/N

Where n = number of organisms carrying particular band

N = total number of organisms

50 The genetic similarity between 15 snail species was estimated according to the method developed by Nei (1973). Based on similarity data, an un-weighted pair group method of arithmetic averages (UPGMA) cluster analysis was used to assess genetic diversity among the snail species.

3.12 SOFTWARES USED

3.12.1 SHANNON-WEINER DIVERSITY INDEX Shannon’s index accounts for both abundance and evenness of the species present. The proportion of species I relative to the total number of species (pi) was calculated, and then multiplied by the natural logarithm of this proportion (in Pi). The resulting product was summed across species, and multiplied by -1.

H is a more reliable measure as sampling size increases.

The addition of the calculation of evenness (J) or equitability (EH) was also applied. Shannon’s equitability (EH) was also applied. Shannon’s equitability (EH) was calculated by dividing H by Hmax (here Hmax = In S).

The evenness index measures how evenly species are distributed in a sample. When all species in a sample are equally abundant an evenness index will be at its maximum, decreasing towards zero as the relative abundance of the species diverges away from evenness (Sebastian et al., 2005). It means evenness assumes a value between 0 and 1 with 1 being complete evenness i.e., a situation in which all species are equally abundant.

3.12.2 INTER SPECIFIC ASSOCIATION Usually, the association of more than a single pair of species is of interest; we may be interested in from 5 to perhaps 50 or more species. The number of all possible pairwise species association or combinations that may be computed increases rapidly according to the equation S(S-1)/2, where S is the number of species.

51 σ 2T =∑ pi(1-pi)

Where pi =ni/N. Next we estimate the variance in total species number as

S 2T= 1/N∑(Tj-t)2

Where t is the mean number of species per sample

The variance ratio,

2 2 VR=S T/ σ T serves as an index of overall species association. The expected value under the null hypothesis of independence is, VR˃1 suggests that overall the species exhibit a positive association. If VR ˂1, then a negative net association is suggested.

A statistic, W, is computed that may be used to test whether deviations from 1 are significant. For example, if the species are not associated then there is 90% probability that W lies between limits given by the chi-square distribution.

2 2 Χ 0.5,N˃W˂ Χ 0.95,N where

W=(N)(VR).

3.12.3 TWO WAY ANALYSIS OF VARIANCE The means of the two populations are often compared by two sample t-test however it is sometimes required to compare the means of populations simultaneously. For this we cannot apply two sample t-test for all possible pair wise comparisons of means. There are two disadvanges when we run multiple two sample tests when we compare their means. First of all the procedure is quite time consuming and tedious. Secondly the level of significance greatly increases as the number of t- test increases. Thus, a series of two sample t-tests is not an appropriate procedure to test the equality of several means simultaneously.

Evidently we require a procedure for carrying out a test on several means simultaneously. When each observation is classified, according to two criteria (or variables) of classification simultaneously, we use the two way analysis of variance technique.

52 There are two basic forms of two way analysis of variance depending upon whether the two variables of classification are independent or whether they interact. Two variables of classification are said to interact when they together have an added effect that they donot have individually (Chaudhry and Kamal, 1996). Two way analysis of variance was calculated by using Minitab version 16.

3.12.4 MULTIPLE REGRESSION The technique of simple regression which involves one dependent variable and one independent variable is often inadequate in most real world situations where a variable depends upon two or more independent variables or regressors. A regression which involves two or more independent variables is called a multiple regression. Thus, in case of multiple linear regression where k independent variables influence the dependent variable Y, the general format of a model is

Yi=α+β1X1i+ β2X2i+……….+βk Xki+εi (i=1,2,…….n)

Where εi’s are the random errors,

Α and βi’s are the unknown population parameters, α is the intercept and β1, β2,……… βk are the regression coefficients for variables X1, X2,………..Xk. respectively.

X1i, X2i,………..Xki are fixed vales of K independent variables, the first of the two subscripts attached to each regressor denotes the variable and the second refers to the observation number (Chaudhry and Kamal, 1996).

The multiple regression was calculated with the help of excel software.

3.12.5 CLUSTER ANALYSIS The term cluster analysis (first used by Tryon, 1939) encompasses a number of different algorithms and methods for grouping objects of similar kind into respective categories. A general question facing researchers in many areas of inquiry is how to organize observed data into meaningful structures, that is, to develop taxonomies. In other words cluster analysis is an exploratory data analysis tool which aims at sorting different objects into groups in a way that the degree of association between two objects is maximal if they belong to the same group and minimal otherwise. Given above, cluster analysis can be used to discover structures in data without providing an explanation/interpretation. In other words, cluster analysis simply discovers

53 structures in data without explaining why they exist. Cluster analysis was used to investigate the degree of association or resemblance of sampling sites. It is a useful data reduction technique that is helpful in identifying patterns and groupings of objects. The Minitab version 16 program were used for cluster analysis using flexible strategy and chord distance, a measure of dissimilarity by following Ward’s method (Ward, 1963; Lance and William, 1967; Faith, 1991 Sebastian et al., 2005).

54 CHAPTER 4

4 RESULTS AND DISCUSSION

The snails were collected from different sites in the agroecosystem of Faisalabad from March to August.The species were identified on the basis of morphometrics description e.g. height, width, aperture height, height/width ratio, coiling of the shell, no. of whorls, shape of the shell (depressed, globose, ovate and conical), and presence or the absence of the operculum and umbilicus, shape of the outer lip/ peristome and the shape of the periphery i.e. shouldered, convex or keeled, color of the specimens and the ecological and habitat information..

The results and discussion of this study are presented as under:

4.1 Taxonomic Characterization of the snails on the basis of morphometrics

4.2 Molecular characterization of snail species.

4.3 Diversity and distribution of snail species

4.4 Species interaction with abiotic factors

4.5 Interspecific interaction of snail species

4.1 TAXONOMIC CHARACTERIZATION OF THE SNAILS ON THE BASIS OF MORPHOMETRICS Freshwater and land snails belong to order Pulmonata of class (Blandford and Godwin Austen, 1908). However the order Pulmonata now is considered to be an informal group according to Bouchet and Rocroi (2005). The suborder Stylomatophora was identified on the basis of the presence of two pair of tentacles and is in conformity with Dayrat and Tillier (2002) illustrating two major characters i.e. a long pedal gland placed beneath a membrane and two pairs of retractile tentacles that are shared by the members of this suborder. However the suborder Bassomatophora bears only on pair of tentacles.

55 Suborder Families Genus Species

Stylomatophora Zonitidae Ariophanta Ariophanta bistrialis ceylanica

Ariophanta bistrialis cyix

Ariophanta bistrialis taprobanensis

Ariophanta bistrialis

Ariophanta solata

Ariophanta belangeri bombayana

Oxychilus Oxychilus draparnaudi

Hygromiidae Monacha Monacha catiana

Cernuella Cernuella virgata

Succineidae Oxyloma Oxyloma elegans

Subulinidae Zootecus Zootecus insularis

Juvenile Zootecus insularis

Pupillidae Pupoides Pupoides albilabris

Ferussaciidae Cecilioides Cecilioides acicula

Bassomatophora Physidae Physa Physa fontinalis

56 4.1.1 FAMILY ZONITIDEA (MÖRCH, 1864) The Family Zonitidea commonly referred as glass snails belongs to the sub order Stylomatophora. Specimens belonging to the Family Zonitidea having small to large sized fragile nearly flat to low conical shells which are generally termed as sub-globosely depressed. The shell is horny brown with 5-5.5 whorls, which are very weakly convex, and the width of the last whorl is more than 2 times of penultimate whorl. The umbilicus is moderately wide. The dimensions of the shell are 6-7 x 12-14 mm. The shell diameter is usually larger than 12 mm having a range of (13 mm at 5.3 whorls, 11 mm at 5 whorls, 8-8.5 mm at 4 whorls, 5 mm at 3 whorls).

They live under dead leaves, rotten logs open and sub-shadow edges of the fields and other similar places (Blandford and Austen, 1908) and have been referred as glass snails by Bouchet and Rocroi (2005).

According to Blandford and Austen (1908), the members of the genus Ariophanta posses shell with 4.5- 5.5 whorls with the last whorl being large, smooth with low spire umbilicus of moderate size and with uniform color. Ariophanta shell is smooth, with low spire 4.5-5.5 whorls the last whorl being large. The shells spirally banded with color more or less distinctly, are decussated but not grooved can be classifies as A. ligulata, A. basilessa, and A. gardener, and A. bistrialis. This genus is endemic to India (http://en.wikipedia.org/wiki/Ariophanta#Distribution) and due to this less literature is available for its citation.

57 4.1.1.1 ARIOPHANTA BISTRIALIS CEYLANICA Zeitschr, 1837 The specimens belonging to Ariophanta bistrialis ceylanica have an average height of 5.80 mm, diameter of 9.20 mm, mean aperture height 4.6 mm and height/diameter of 0.63 mm. The number of whorls range from 4.5-5, the shell has dextral coiling having pale straw colour having a dark line around the periphery and in suture with a subglobose shape, with a thin and simple peristome and the umbilicus present (Table 4.1 and Figure 4.1)

Colour: The unpreserved specimens were pale straw in color with a dark line around the periphery and in sutures.

Habitat: This species has been found in terrestrial ecosystems, under logs, vegetation and ground litter, stone, etc., in diverse habitat. During present study the specimens were collected from the fields of Barseem, Sugarcane, and wheat and also from the bark of the trees.

Distribution: This speices has been reported from Madras Presidency, Ceylon, Shevroy Hills and in cropfields, Pakistan.

Locality: This species was found in following villages. 204 R.B., 07 J.B., 119 J.B., 214 G.B., 121 J.B., 123 J.B., 124 J.B., 124 J.B., 217 J.B., 202 R.B., 208 R.B., 103 J.B., 214 R.B., 221 R.B., 219 R.B., 222 R.B., 223 R.B., 225 R.B., 235 R.B., 295 R.B., 71 J.B., 105 G.B., 65 G.B., 56 G.B., 73 G.B.

58 Table 4-1: Morphometrics of the Ariophanta bistrialis ceylanica Zeitschr, 1837. Species Number 1 Ariophanta bistrialis ceylanica Mean Height 5.70 Mean Diameter 9.10 Height/Diameter 0.63 Aperture Height 4.5 No. Of Whorls 4.5-5.5 Shell coiling Dextral Peristome Thin/simple Umbilicus Present Shape Sub-globose

Figure 4-1: Ariophanta bistrialis ceylanica Zeitschr, 1837.

59 4.1.1.2 ARIOPHANTA BISTRIALIS CYIX (Beck, 1837) The specimens belonging to Ariophanta bistrialis cyix have an average height of 6.60 mm, diameter of 9.50 mm, mean aperture height 4.70 mm and height/diameter of 0.70 mm. The number of whorls ranges from 4.5-5, the shell has dextral coiling having pale straw colour light black/ reddish brown line around the periphery and in suture with a subglobose shape, with a thin and simple peristome and the umbilicus present. (Table 4.2 and Figure 4.2)

Colour: they have pale straw color with light black/reddish brown line around the periphery and in sutures.

Habitat : They are terrestrial and are found in vegetation, fodder, sugarcane, wheat fields and at the bark of the trees in the field.

Distribution: They are distributed in Madras Presidency, Ceylon, Gavadavri at Dumagudian, shevroy Hills and Pakistan.

Locality: 204 R.B,07J.B, 119J.B, 214 G.B,121J.B, 123J.B, 124 J.B, 124J.B, 217J.B. 202R.B. 208R.B. 103J.B., 214R.B., 221 R.B., 219 R.B., 222R.B., 223R.B., 225R.B., 235 R.B., 295R.B. 71 J.B. 105 G.B., 65G.B., 56 G.B. 73G.B.

60 Table 4-2: Morphometrics of the Ariophanta bistrialis cyix (Beck, 1837). Species Number 2 Ariophanta bistrialis cyix Mean Height 6.50 Mean Diameter 9.40 Height/Diameter 0.69 Aperture Height 4.72 No. Of Whorls 4.5-5.5 Shell coiling Dextral Peristome Thin/Simple Umbilicus Present Shape Sub-globose

Figure 4-2: Ariophanta bistrialis cyix (Beck, 1837).

61 4.1.1.3 ARIOPHANTA BISTRIALIS TAPROBANENSIS (Dohm, 1859) The specimens belonging to Ariophanta bistrialis taprobanensis have an average height of 6.70 mm, diameter of 9.80 mm, mean aperture height 4.90 mm and height/diameter of 0.68 mm. The number of whorls ranges from 4.5-5, the shell has dextral coiling having pale straw colour white shade, line around the periphery and in suture with a subglobose shape, with a thin and simple peristome and the umbilicus present (Table 4.3 and Figure 4.3)

Colour: They have pale straw color with white shade/line around the periphery and in sutures.

Habitat: They are terrestrial in habit and are found in vegetation.

Distribution: Madras Presidency, Ceylon, Gavadavri at Dumagudian, Shevroy Hills and Pakistan.

Locality: 204 R.B.,07J.B., 119J.B., 121J.B., 123J.B., 124 J.B., 124J.B., 217J.B., 202R.B., 208R.B., 103J.B., 214R.B., 221 R.B., 219 R.B., 222R.B., 223R.B., 225R.B., 235 R.B., 295R.B., 71 J.B., 105 G.B., 65G.B., 56 G.B., 73G.B.

62 Table 4-3:Morphometrics of the Ariophanta bistrialis taprobanensis(Dohm, 1859). Species Number 3 Ariophanta bristrialis taprobanensis Mean Height 6.60 Mean Diameter 9.70 Height/Diameter 0.68 Aperture Height 4.91 No. Of Whorls 4.5-5.5 Shell coiling Dextral Peristome Thin/Simple Umbilicus Present Shape Sub-globose

Figure 4-3: Ariophanta bristrialis taprobanensis (Dohm, 1859).

63 4.1.1.4 ARIOPHANTA BISTRIALIS Beck, 1837 Ariophanta bistrialis Beck (Nanina), Ind. Mol. I, p, 2 (1837, descr.); Pfr. in Chemn. ed. 2, 1846, Helix, no. 61, pl. 11, figs. 10, 11; id, t. c. vii, 1876, p.122; H. & T. (Helix) C. I. 1876, pl. 29, fig.1; Godwin_Austen (Nilgiria), Mol. Ind. ii, 1898, p. 80, pl. 81, fig. 4 (genitalia), pl. 82, fig. 5 (radula).

Helix ceylanica, Pfr. Zeitschr, Mal, 1850, p. 67; id. Mon. Hel. iii, 1853, p.71; id. t. c. vii, 1876, p. 122; H &T. C. I. 1876, pl. 29, fig. 3; Godwin-Austen (Nilgiria), Mol. Ind. ii, 1898, p. 126 [(description of animal, genitalia and radula)]

Helix taprobanensis, Dohrn, Mal. Bl. vii, 1859, p.206; Pfr. Mon. Hel. v, 1868, p. 116; H&T. C. I. 1876, pl 29, fig. 2.

Helix cyix, Bs. A. M. N. H. (3) v, 1860, p.382; Pfr. Mon. Hel. v, 1868, p.236; H. &T. C. I. 1876, pl. 29, fig. 4.

The specimens belonging to Ariophanta bistrialis have an average height of 6.30 mm, diameter of 8.80 mm, mean aperture height 5.8 mm and height/diameter of 0.72 mm. The number of whorls range from 4.5-5, the shell has dextral coiling having pale straw colour having a dark line around the periphery and in sutures alongwith dark lines around the umbilicus on the lower side of the shell with a subglobose shape, with a thin and simple peristome and the umbilicus present. (Table 4.4 and Figure 4.4)

Colour: Pale straw color, dark line around the periphery and in sutures and dark lines around the umbilicus on the lower side of the shell.

Habitat: They are terrestrial. They are found in vegetation, ground litter, under logs and stone etc., in diverse habitat e.g fodder, sugarcane, wheat fields and from the bark of the trees.

Distribution: They are present in Madras Presidency, Ceylon, and Gavadavri at Dumagudian, Shevroy Hills and Pakistan.

Locality:204 R.B.,07J.B., 119J.B., 214 G.B.,121J.B.,123J.B., 124 J.B., 124J.B., 217J.B., 202R.B., 208R.B., 214R.B., 221 R.B.,219 R.B., 222R.B., 223R.B., 225R.B., 235 R.B., 295R.B., 71 J.B., 105 G.B., 65G.B., 56 G.B., 73G.B.

64 The snail specimens have perforated shell with sub-globosely depressed shape with thin, finely striations and is decussated on the dorsal surface. On the ventral side there are spiral lines impressed with polished surface. They are pale horny in color, encircled by two rufous lines with a whitish band between them, the upper line continued insides the suture. The shells are spirally low to nearly flat or 4.5 convex whorls with rapidly increasing last whorl. The aperture is large with lunately ovate shape. The peristome is thin with the columellar margin slightly reflected is identified as Ariophanta bistrialis.However the shell of the form present in the south India is pale in its colour with two rufous lines.The form of the Ceylon is darker in colour with a single line is classified as Ariophanta bistrialis ceylanica (Zeitschr, 1837); however both varieties occur in each area. There is another variety from Ceylon classified as Ariophanta bistrialis taprobanensis with shell having no band and is a large rather thick variety. The species classified as Ariophanta bistrialis cyix (Beck, 1837) is a dwarf form rather globose, generally with the color- line faint.

65 Table 4-4: Morphometrics of the Ariophanta bistrialis Beck, 1837. Species Number 4 Ariophanta bristrialis Mean Height 6.20 Mean Diameter 8.90 Height/Diameter 0.70 Aperture Height 5.79 No. of Whorls 4.5-5.5 Shell coiling Dextral Peristome Thin/Simple Umbilicus Present Shape Sub-globose

Figure 4-4: Ariophanta bristrialis Beck, 1837.

66 4.1.1.5 ARIOPHANTA SOLATA Godwin- Austen, 1898 Ariophanta solata Bs. (Helix) A. M. N. H. (2) ii, 1848, p. 159; Pfr. (Helix) Mon. Hel, iii, 1853, p. 67; id. t. c. iv, 1859, p. 170; id. t. c. vii, 1876, p.274; H. & T. (Helix) C. I. 1876, pl. 28, fig. 6; Godwin-Austen (type of Nilgiria), Mol. Ind, ii 1898, p.77, pl. 80, figs, 1-4 (anatomy), pl. 82, fig. 2 radula.

The specimens belonging to Ariophanta solata have an average height of 6.0 mm, diameter of 8.20 mm, mean aperture height 4.1mm and height/diameter of 0.75 mm. The number of whorls ranges from 4.5-5, the shell has dextral coiling having pale straw colour having a dark line around the periphery and in sutures alongwith bluish tinges on the spire with a subglobose shape, having a thin and simple peristome and the umbilicus present. (Table 4.5 and Figure 4.5)

Colour: The shells are pale straw in color with a dark line around the periphery and in sutures and bluish tinges on the spire.

Habitat: They are terrestrial from vegetation and ground litter, under logs and stone etc, in diverse habitat.

Distribution: They are distributed in Nilgiris

Locality: 204 R.B., 07J.B., 119J.B, 214 G.B.,121J.B., 124 J.B., 124J.B., 217J.B., 202R.B., 208R.B., 103J.B., 214R.B., 221 R.B., 219 R.B., 222R.B., 223R.B., 225R.B., 235R.B., 295R.B., 71 J.B., 105 G.B., 65G.B., 56 G.B., 73G.B.

The specimens have sub-globosely depressed perforate shell, which is thicker with smooth striations. The colour is white in mostly specimens with bluish tinge, being washed with the brownish shade on the last whorl with narrow, spiral rufous band inside the sutures.there are numerous small brownish spots that are distributed irregularly along with traces of other bands. The spire is low or flatly convex above.The last whorl is slightly swollen from the ventral surface at periphery. The aperture is oblique with lunately ovate shape, generally reddish brown from within. The peristome is simple and thin. The columellar margin is slightly reflected are identified as Ariophanta solata.

67 Table 4-5: Morphometrics of the Ariophanta solata Godwin- Austen, 1898 Species Number 5 Ariophanta solata Mean Height 6.10 Mean Diameter 8.10 Height/Diameter 0.75 Aperture Height 4.12 No. of Whorls 4.5-5.5 Shell coiling Dextral Peristome Thin/Simple Umbilicus Present Shape Sub-globose

Figure 4-5: Ariophanta solata Godwin- Austen, 1898.

68 4.1.1.6 ARIOPHANTA BELENGERI BOMBAYANA Desh, 1847

Ariophanta belangeri, Desh, (Helix) in Belanger, Voy. Zool. p. 43, pl. 1, figs, 1-3 (1834); Pfr. (Helix) Mon. Hel, i, 1847, p. 69; id. t. c. vii, 1876, p.172; H. & T. (Helix) C. I. 1876, pl. 29, fig.6. Helix bombayana, Grat. Act. Soc. Lin. Bord. xi, 1841, p. 406, pl. 1, fig. 1; Pfr. Mon. Hel, i, 1847, p. 41; id. t, c. iii, 1853, p.76; id. t. c. vii, 1876, p. 125; H & T (Helix) C. I. 1876, pl. 29, fig. 5; E. A. Smith (Xestina), Faun, Geog. Mald. Lac. Is 1902 p.142.

Helix vitellina, Pfr. P. Z. S. 1848, p. 109; id. Mon. Hel. iii, 1853, P. 72; id t. c. vii, 1876, p. 122; H. & T. (Helix) C. I. 1876, pl. 59, figs. 1,2.

The specimens belonging to Ariophanta belengeri bombayana have an average height of 6.30 mm, diameter of 9.0 mm, mean aperture height 4.4 mm and height/diameter of 0.66 mm. The number of whorls ranges from 4.5-5, the shell has dextral coiling having pale straw colour having a dark line around the periphery and in sutures having a subglobose shape, with a thin and simple peristome and the umbilicus present (Table 4.6 and Figure 4.6).

Colour: They are having shells with pale straw color having a very faint line around the periphery and in sutures Habitat: They are terrestrial and live in diverse habitat i.e. vegetation, under logs and stone and ground litter. Distribution: They are distributed in Madura, South Arcot, and Malabar, and probably all the Southern most part of the peninsula. Auaimalai Hills ; N. Mahlor Atoll, Maldives. Locality: 204 R.B.,07J.B., 119J.B., 214 G.B.,121J.B., 124J.B., 217J.B., 202R.B., 214R.B., 221 R.B., 219 R.B., 222R.B., 223R.B., 225 R.B., 235 R.B., 295R.B., 71 J.B., 105 G.B., 65G.B., 56 G.B., 73G.B. The species identified as A. belengeri bombayana have 5 whorls. They are pale tawny to whitish in color with low spire. The shell is convex with impressed suture. The shell is openly perforate having a depressed globose shape. The specimens have obliquely striated shells with more or less decussated spiral lines impressed on the dorsal surface while the ventral surface is smoother. The aperture is roundly lunate. The peristome is thin and columellar margin slightly reflected (Blandford and Godwin Austen, 1908).

69 Table 4-6: Morphometrics of the Ariophanta belangeri bombayana Desh, 1847. Species Number 6 Ariophanta belangeri bombayana Mean Height 6.40 Mean Diameter 9.10 Height/Diameter 0.70 Aperture Height 4.42 No. of Whorls 4.5-5.5 Shell coiling Dextral Peristome Simple/Thin Umbilicus Present Shape Sub-globose

Figure 4-6: Ariophanta belangeri bombayana Desh, 1847

70 4.1.1.7 OXYCHILUS DRAPARNAUDI (Beck, 1837) Helix (Helicella) Draparnaldi Beck, 1837 Index molluscorum praesentis aevi musei principis augustissimi Christani Frederici –pp. 1-24. Hafniae

The specimens belonging to Oxychilus draparnaudi have an average height of 8.5 mm, diameter of 14.20 mm, mean aperture height 8.6 mm and height/diameter of 0.60 mm. The number of whorls range from 4.5-5, the shell has dextral coiling, horny coloured with translucent and thin shell with a subglobose shape, having a thin and deflected peristome and the umbilicus present (Table 4.7 and Figure 4.7) .

Colour: The shell is translucent, thin with horny colour. Habitat: They are found in or on the edges of the cultivated fields. They inhabit damp and humid places, under the logs and leaves on the trees.

Distribution: They are distributed in Europe and British

Locality: 204 R.B., 07 J.B., 214 G.B.,123J.B., 124 J.B., 202 R.B., 208 R.B., 103 J.B., 214 R.B., 222 R.B., 223 R.B., 225 R.B., 71 J.B., 105 G.B.

The specimens have narrow to wide umbilicus of the shell, however only a few species are without umbilicus. The members of this genus have spiral or reticulate patterns with smooth gloss is classified as Oxychilus (Welter-Schultes, 2010). The Oxychilus shell is generally brown or amber in colour and the size of the shell range from 6- 16 mm. The shell is nearly transparent. the lip of shell is thin. Oxychilus draparnaudi is generally is a large zonitid glass snail. They are reffered as glass snails snail with the maximum diameter of 14 mm of shell. The shell is translucent and glossy.The shell is glossy and the colour is translucent yellowish-brown on the dorsal side and somewhat whiter on the underside. The diameter of the shell range from 12 to 16 mm diameter when fully grown. There are 5 to 5.5 whorls and the last whorl expands more rapidly due to which the width of last shell whorl about 2.1 times width of penultimate whorl; (Watson and Dallwitz, 2005)

71 HABITAT AND DISTRIBUTION The members of this family were terrestrial and were found in the vegetation and ground litter specially under logs and stones. They were found in diverse habitats as in the fields of Sugarcane, Wheat and Fodder along with the bark of the trees as has been supported by Bouchet and Rocroi,(2005) that the glass snails live in the damp places like under stones and similar places, and are nocturnal.

72 Table 4-7: Morphometrics of the Oxychilus draparnaudi (Beck, 1837) Species Number 7 Oxychilus draparnaudi Mean Height 8.60 Mean Diameter 14.22 Height/Diameter 0.60 Aperture Height 8.61 No. of Whorls 4.5-5.5 Shell coiling Dexral Peristome Thin deflected Umbilicus Present Shape Sub-globose

Figure 4-7: Oxychilus draparnaudi (Beck, 1837)

73 4.1.2 HYGROMIIDAE (TRYON 1866) The members of family Hygromiidae (Tryon 1866) of the terrestrial snails () are reffered as the leaf snails. However, according to some older authors these were considered as the members of subfamily (Helicidae). The leaf snails range in size from small to medium size, but generally members of family Hygromiidae are smaller than helicid snails. While classifying the leaf snails into subfamily and genus there is a great confusion and there is a lack in consent (Steinke et al., 2004).

4.1.2.1 MONACHA CANTIANA (MONTAGU 1803) Helix cantiana Montagu, 1803. Testacea Britannica or natural history of British shells, marine, land and freshwater including the most minute: systematically and arranged and embellished with figures .-pp. i- xxxvii [=1-37], [1-2], 1-606, [1-4], pl.1-16. London. (White).

The specimens belonging to Monacha cantiana have an average height of 6.90 mm, diameter of 9.90 mm, mean aperture height 4.5mm and height/diameter of 0.7 mm. The number of whorls ranges from 4.5-5, the shell is white to dusty white in colour having dextral coiling, and the umbilicus is present. The shape of the shell is subglobose, with a thin and simple peristome and the umbilicus present. (Table 4.8 and Figure 4.8)

Colour: The snail shell white to dusty white in colour

Habitat: They are terrestrial in habit and are mostly found in dry, sunny, open moist sheltered places

Distribution: British Isles, Atlantic European coast from Netherlands to Spain, In India, Pakistan and South Asian Countries

Locality: 204 R.B., 07 J.B., 119 J.B., 214 G.B., 121 J.B., 123 J.B., 202 R.B., 208 R.B., 103 J.B., 221 R.B., 219 R.B., 222 R.B., 223 R.B., 225 R.B., 235 R.B., 71 J.B.

The shell colour in the members of Monacha catiana species ranges from creamy white to brownish red, while juvenile specimens have hairs on dorsal surface of the shell. The colour of shell is white to transparent with pinkish tinge near mouth. The shells are slightly convex with a spire which ranges from flattened to almost conical. The aperture colour of this species ranges from whitish to reddish lip. This colour is less distinct in other species of Monacha. The

74 umbilicus is slightly wider than in Monacha cartusiana. The dimensions of shell width range from 16 mm – 20 mm and a shell height of 11mm - 14 mm having 5 – 6 whorls. The body colour of the snail is reddish from body's front and the tentacles are greyish brown (Hlavac and Peltanova, 2010).

HABITAT AND DISTRIBUTION: These snails were found terrestrial in nature. These areas are generally characterized as dry, sunny and moist places near herbaceous plants as supported by (Watson and Dallwitz, 2005). According to the Fechter and Falkner (1990) Monacha cantiana is mostly inhabited in the herbal layers in the dry soils. They are also found near road sides in the plantations or railway areas with vegetation (Hlavac and Peltanova, 2010). These snails are distributed throughout out the world in Italy and Southern France to Northern France, Belgium, the Netherlands and North Germany, reaching in the east of Austria near Vienna. However, the recent appearance of Monacha cantiana as an introduced species is in the Czech Republic. However this species was first identified in the Great Britian in 1803, but was never detected in Ireland.The snail is considered to be critically endangered in Germany according to the IUCN threat categories but in a country like Pakistan, no update of the records is available after 1970 in the Zoological Survey of Pakistan by Khan and Dastagir 1970 (Afsar, Siddiqui and Ayub, 2012).

75 Table 4-8: Morphometrics of the Monacha catiana (Montagu 1803) Species Number 8 Monacha catiana Mean Height 6.80 Mean Diameter 9.91 Height/Diameter 0.69 Aperture Height 4.51 No. of Whorls 4.5-5.5 Shell coiling Dexral Peristome Simple/Thin Umbilicus Present Shape Sub-globose

Figure 4-8: Monacha catiana (Montagu 1803).

76 4.1.2.2 CERNUELLA VIRGATA (DA COSTA, 1778) Cochlea virgata Da Costa, 1778. Mandes da Costa, E. 1778. Historia naturalis testaceorum Brittanniae, or, the British conchology; containing the descriptions and other particulars of natural history of the shells of Great Britian and Ireland: illustrated with figures. In English and French –Historia naturalis testaceorum Britinniae, ou, la conchologie Britannique; contenant les descriptions & autres particularities d/ histoire naturelle des coquilles de la Grande Bretagne & de I/ Irlande: avec figures en taille douce. En anglois & francois.- pp. i –xii [ = 1-12], 1-254, i-vii [=1-7], [1], pl. I- XVII [=1-17]. London. (Millan, White, Emsleys & Robson).

The specimens belonging Cernuella virgata to have an average height of 7.0 mm, diameter of 9.70 mm, mean aperture height 5.0 mm and height/diameter of 0.72 mm. The number of whorls range from 4.5-5, the shell is white to dusty white in colour with a dark line around the periphery having dextral coiling, the umbilicus is present. The shape of the shell is subglobose, with a thin peristome and the umbilicus present (Table 4.9 and Figure 4.9).

Colour: They have white to dusty white shell with dark line around the periphery.

Habitat: These snails live in dry habitat, in sandy soil on the under herbal plants. They are attached high on the tall plants in hot weather and are found in harvested fields.

Distribution: They are distributed in British Isles, Atlantic European coast from Netherlands to Spain, India, Pakistan and South Asian Countries.

Locality: 204 R.B., 07 J.B., 119 J.B., 214 G.B., 121 J.B., 123 J.B., 124 J.B., 217 J.B., 202 R.B., 208 R.B., 103 J.B., 214 R.B., 221 R.B., 222 R.B., 223 R.B., 225 R.B., 235 R.B., 71 J.B., 105 G.B.

The shell shape of Cernuella virgata is more globular than that of Monacha species. The surface of the spire is irregularly ribbed with a height of nearly 3/4th of width, with slight curve. The shape of the shell aperture is circular with a narrow umbilicus. The umbilicus is covered by the apertural lip. The shell colour and form ranges from whitish to reddish yellow with dark brown bands which may be besring discontinuous or dissolve spots. There is the presence of variable stripes on the shell or can be absent. The shell width ranges from 8 - 25 mm and a height of 6 - 19 mm with 5 – 7 whorls in number (Fischer and Duda, 2004).

77 HABITAT AND DISTRIBUTION.

These snail species were found in the dry habitats. They are found under herb cover or on herbal plants. They are found clinging to tall plants or harvested fields in summers and are supported by the findings of Fischer and Duda (2004), who has also discussed their presence in the railway area and industrial land.

They are found in the South Asian countries like India and Pakistan but are also found in Mediterranean region except Levantine coasts, the Atlantic coasts up to the Netherlands, including the British Isles, Breitenlee near Vienna, Germany (Fischer and Duda 2004).

78 Table 4-9: Morphometrics of the Cernuella virgata (Da Costa, 1778). Species Number 9 Cernuella virgata Mean Height 7.10 Mean Diameter 9.72 Height/Diameter 0.73 Aperture Height 5.10 No. of Whorls 4.5-5.5 Shell coiling Dextral Peristome Thin Umbilicus Present Shape Sub-globose

Figure 4-9: Cernuella virgata (Da Costa, 1778).

79 PUPILLIDAE (TURTON 1831) The members of this family are reffered as chrysalis snails due their resemblance with a insect's chrysalis. The same term is being used for the members of the super family Pupilloidea (Anderson, 2008).

This family contains minute snails with a more or less cylindrical or conical in shape with, blunt multispiral shells.Those shells that are cylindrical belong to genus pupilla while the elongated shell belongs to the Pupoides genus. There are 5-9 whorls in pupilla while in Pupoides there are 6-7 whorls.

These snails have dextral symmetry. They have inoperculate shells. They have rising-spiral with 6–7 whorls. The maximum height of the shell ranges from 1.2 mm to 5 mm. with a width of 1.7– 1.9 mm. It is estimated that the height is nearly 1.74–2.1 times the width. However the spire height is about 0.48–0.62 X that of shell. The spire shape is obtuse. The shell shape is ovoid- symmetric with rather shallow suture to deeply suture. The shape of the body whorl and spire whorls is moderately convex. The whorls are neither keeled nor shouldered. The shell has striations that may be conspicuous, regular across the whorls or these may be striatedwith very dim lines. There may be the presence or complete absence of teeth/calluses. There is the presence of umbilicus with folded columella. The shell lip is having a deflected mouth edge or simple, having thin or thick edges which may be translucent to opaque. The colour ranges from brown to horn-coloured with glossy, dull or plain appearance. In some genera the shell colour is white. There apertural lamellae is a very important taxonomic character with special focus on their number and arrangement for species identification in snails. The number of apertural lamellae ranges from 0-9 mm (Watson and Dallwitz, 2005).

The apertural lamellae range from 0-9 or more, as the number and arrangement of the apertural lamellae being important for species-level taxonomy (Perez et al., 2008).

80 4.1.2.3 PUPOIDES ALBILABRIS (C. B. ADAMS, 1841) Cyclostoma marginata Say,1821, Jour. Acad. Nat. Sci. Phila., 2:172 (Upper Missouri). Not Cyclostoma marginatum, G. Fisher, 1807, Mus, Demidoff., 3:219.

Pupoides marginatus (Say), Pilsbry &Vanatta, Proc. Acad. Nat, Sci. Phila., 1900, p.586- Pilsbry, Man. Conch., 26:111, pl. 12, figs, 1-7- Hanna, Proc. Cal. Acad, Sci, (4 ser.), 12:514, - Henderson, 1924, Univ. of Colo. Studies, 13:132; 1926, 23:102 (Colorado)- Woodbury, 1929, Nautilus, 43:57 (Zion Nat. Park, Utah).- Franzen, 1947, Trans. Kansas Acad. Sci., 49:418 (N. W. Kansas).

Pupa fallaz “Say”, Gould, Bost, Jour. Nat. Hist., 4:357.- W. G. Binney, Terr. Moll., 5:203 pl. 52. fig. 1, pl. v, fig. T(teeth), and of most authors prior to 1900. Not Pupa fallax Say,

P[upa] albilabris “Ward’s letter”. C. B. Adams, 1841, Amer. Jour. Sci., 40:271 (New name for Cyclostoma marginata Say; no other description)

Pupa (Modicella) arizonensis Gabb, 1866, Amer. Jour. Conch., 2:331, pl. 21, fig. 6.

The specimens belonging to pupoides have an average height of 9.0 mm, diameter of 3.0 mm, mean aperture height 2.8 mm and height/diameter of 3 mm. The number of whorls range through 8, the shell is white in colour having dextral coiling, and the umbilicus is minute. The shape of the shell is fusiform and tapering towards apex with a peristome that is thin and simple (Table 4.10 and Figure 4.10)

Colour: The snail shells are white in colour

Habitat: they are terrestrial in habit with moist to dry diverse locations.

Distribution: They are distributed in Cuba and Indian Ocean

Locality: 204 R.B., 07 J.B., 119 J.B., 214 G.B., 123 J.B., 202 R.B., 208 R.B., 103 J.B., 223 R.B., 225 R.B., 295 R.B., 71 J.B., 105 G.B.

This is highly distinguishable species when compared with other species as it height is greater than 4 mm. The shells are conical with brown cover however the apertural margin is strongly whitened. The apertural lamellae are absent.

81 HABITAT AND DISTRIBUTION:

According to our results these snails are terrestrial in nature and prefer to live in moist damp places which are supported by Nekola and Coles (2010) and Watson and Dallwitz (2005). They are found in all types of habitats ranging from arctic tundra and semi-tropical grasslands, forests, and peatlands. The microhabitats of these individuals tend to be most common in leaf litter accumulations, wet turf, vertical rock outcrops or mossy tree trunks. These animals are generally detritivores that feed on a variety of either fungal hyphae or algal mats.

Pupillidae are very widely distributed and are found on all continents, except Antarctica however their distribution in Europe their distribution in the north stretches beyond the Arctic circle, in the Alps beyond 2000 m MSL (Anderson, 2008). This family is distributed in Canada, United States, with an unknown number of additional taxa residing in Mexico Pilsbry (1948) (Perez et al., 2008) .The members of this family belonging to Pupoides have also been reported from the Thar Desert and foothills of Himalayas in Pakistan (Auffenberg, 1997) and from sugarcane fields of Faisalabad Pakistan (Ali, 2005).

82 Table 4-10: Morphometrics of the Pupoides albilabris (C. B. Adams, 1841). Species Number 10 Pupoides albilabris Mean Height 9.20 Mean Diameter 3.10 Height/Diameter 2.97 Aperture Height 2.82 No. of Whorls 7.5-8 Shell coiling Dextral Peristome Simple/Thin Umbilicus Small Shape Fusiform tapering towards apex

Figure 4-10: Pupoides albilabris (C. B. Adams, 1841).

83 4.1.3 PHYSIDAE (FITZINGER, 1833) The members of this family have highly distinctive taxonomic characters. They are small in size with sinistral symmetry, in which the spire is pointing upwards with aperture towards the observer having aperture on left side. They have large and long aperture in which operculum is absent. The shells are corneous and thin, rather transparent (Bouchet and Rocroi, 2005).The shells of the members of sub family Physinae are either fusiform or ovately fusiform. The members of the genus Physa are sinistral with fluviatile, spiral, thin, horny, and generally ovate acuminated. The outer lip is simple and sharp and the inner lip which is continuous with the columella is expanded. The columella is tortuous and is single-plaited (Blandford, and Godwin,1908).

4.1.3.1 PHYSA FONTINALIS (Linnaeus, 1758) Physa, Draparnaud, Tabl. Moll. France, 1801, p. 52, & Hist. Nat. Moll. Terr. Fluv. France, p. 54. Bulla fontinalis Linnaeus, 1758 Limnea (Physa) fontinalis Sowerby, 1821 Physa dalmatina Küster, 1844 Planorbis bulla Müller, 1774 Rivicola fontinalis Fitzinger, 1833 Turbo adversus da Costa, 1778 The specimens belonging Physa fontinalis to have an average height of 9.20 mm, diameter of 5.6 mm, mean aperture height 6.4 mm and height/diameter of 1.63 mm. The number of whorls reach 4, the shell is pale horn colour to dark brown colour having sinistral coiling, the umbilicus is absent. The shape of the shell is succiniformes, with a peristome that is thin and reflected (Table 4.11 and Figure 4.11) Colour: They are pale horn to dark brown in colour

Habitat: They are found in all kinds of fresh water aquatic envoirnment i.e. fresh, running and stagnant, hard and soft, polluted and unpolluted water.

Distribution: they are distributed in Holartic region, Europe except South Europe

Locality: 204R.B., 119J.B., 214G.B., 217 J.B., 202 R.B., 103 J.B., 219 R.B., 222 R.B., 71J.B., 105 G.B.

84 The shell of Physa fontinalis is inoperculate with rising-spire. The shells are 4–7 whorled. They have sinistral symmetry. The shell is higher than wide. In Physa fontinalis the shells are 8–12 mm high or it may even range from 10–18 mm high as found in some introduced specimens. The height of the shell is about 1.5–1.7 times the width. The body whorl is predominating having a small and short spire small as in Physa. The height of the spire is about 0.1–0.2 times the height of shell.The shell is much less elongate having a shape of inverted- pyriform. The whorls of the shell are neither keeled nor shouldered. The aperture is with neither calluses nor teeth. The shell is ranges from thick-lip to thin-lip which may be thin and glossy with translucence. The colour of the shell ranges from pale horn to dark brown or blackish or plain. The colour of the animal is grey to nearly black (Watson and Dallwitz, 2005).

HABITAT AND DISTRIBUTION According to our research these snail species wer found in the fresh water, that may be running or still, industrial or non industrial effluent which is in accordance with the findings of Watson and Dallwitz (2005). The members of Physidae are generally reffered as sinistral pond snails (Bouchet and Rocroi, 2005) According to Sturm et al, (2005), the main source of food of these snails is algae or fish food and they can survive in all conditions due to which they are called as the cockroaches of malacology.

85 Table 4-11: Morphometrics of the Physa fontinalis (Linnaeus, 1758). Species Number 11 Physa fontinalis Mean Height 9.20 Mean Diameter 5.70 Height/Diameter 1.61 Aperture Height 6.41 No. of Whorls 4 Shell coiling Sinistral Peristome Thin reflected from the base

Umbilicus Absent Shape Succiniformes

Figure 4-11: Physa fontinalis (Linnaeus, 1758).

86 4.1.4 SUBULINIDAE (FISCHER AND CROSSE, 1877) The snails belonging to the family Subulinidae range in size from small to medium size shells with a few members with sinistral symmetry. The shell characters of the Genus Zootecus , belonging to family Subulinidae, are described by Gude (1914) as pupiform shell and cylindrical with conical summit or cylindrical tapering spire. The shell is small having pale or white colour. There are 7-10 compactly coiled whorls.

4.1.4.1 ZOOTECUS INSULARIS (EHRENBERG, 1831) Pupa insularis Ehrenberg, C. G. 1831Symbolae physicae. Animalia Everteberata exclusis insect is. Series prima cum tablarum decade prima. – pp. [1-135]. Berolini. (Mitter)

The specimens belonging Zootecus insularis to have an average height of 12.43mm, diameter of 4.7mm, mean aperture height 3.94mm and height/diameter of 2.64mm. The number of whorls ranges from 7-8, the shell is white to dull white in colour having dextral coiling, the umbilicus is absent. The shape of the shell is elongate conic, with a thick peristome (Table 4.12 and Figure 4.12)

Colour: They are mostly white with some of shell having dull white in colour.

Habitat: They are terrestrial and are mostly found in ground litters or are often climbing up tree trunks or wall in wet weather.

Distribution: They are distributed in Saharo-Sindian region

Locality: 204 R.B. 07 J.B. 214 G.B. 217 J.B. 202 R.B. 208 R.B. 103 J.B. 214 R.B. 221 R.B. 219 R.B. 222 R.B. 223 R.B. 225 R.B. 235 R.B. 71 J.B 105 G.B.

87 Table 4-12:Morphometrics of the Zooctecus insularis (Ehrenberg, 1831). Species Number 12 Zooctecus insularis Mean Height 12.45 Mean Diameter 4.87 Height/Diameter 2.56 Aperture Height 3.93 No. of Whorls 7.0-8.0 Shell coiling Dextral

Peristome Thick Umbilicus Small Shape Elongate conic

Figure 4-12: Zooctecus insularis (Ehrenberg, 1831)

88 4.1.4.2 JUVENILE ZOOTECUS INSULARIS (EHRENBERG, 1831) There are snails identified as juvenile snails of Zootecus insularis were also found with 5-6 whorls, the peristome is thin with the last whorl shouldered, white in color, having minute umbilicus and closely dextrally coiled. The height was7.90 mm, with a diameter of 4.40 mm, mean aperture height of 3.1 mm and height/diameter ratio is 170 mm, with a conical oval shape (Table 4.13 and Figure 4.13).

Colour: The shells of the specimens are white.

Habitat: They are terrestrial in their habit and are mostly found in ground litters in wood hedgerow, mainly on base rich soils, often climbing up tree trunks or wall in wet weather.

Distribution: They are distributed in the Saharo-Sindian region

Locality: 204R.B.,07J.B.,202R.B.,208 R.B., 103 J.B., 214 R.B., 219 R.B., 222R.B., 223 R.B., 105 G.B.

The shells of snails belonging to this species are thick and white in colour. The shells are turreted with conical and broad shape of the apical whorls. The teleoconch whorls are nearly cylindrical in shape with very fine axial riblets, arched subsuturally. However irregular wrinkles are present on the protoconch. The axial sculpture may be interrupted by the irregular spiral grooves. The peristome is thick. The shell aperture is subquadrate. The shell has an open umbilicus however due to the reflection of the peristome near columellar rim, it may be covered partially. The size of the specimens ranges from11-15 x 3.5-6 mm approximately. In many specimens there is a connection between the peristomial rims through a thick parietal callosity (Welter Schultes, 2010).

HABITAT AND DISTRIBUTION According to our research these snails are terrestrial and are found in ground litters and sometimes on the tree trunks when there is wet weather supported by the findings of Alkhiyat (2010) but according to the findings of Gude, (1914) they are mainly reported in the arid and barren regions of Pakistan while these snails species have also been found in Thar Desert of Pakistan (Auffenberg, 1997).

89 Table 4-13: Morphometrics of the Juvenile Zooctecus insularis (Ehrenberg, 1831) Species Number 13 Juvenile Zooctecus insularis Mean Height 8.00 Mean Diameter 4.50 Height/Diameter 1.78 Aperture Height 3.12 No. of Whorls 5.5 to 6 Shell coiling Dextral Peristome Thin Umbilicus Small Shape Conical oval

Figure 4-13: Juvenile Zooctecus insularis (Ehrenberg, 1831).

90 4.1.5 FERUSSACIIDAE (BOURGUIGNAT 1883) The animal has two pairs of tentacles but eyes are absent (as Cecilioides acicula is blind and subterranean) and are generally referred as ground snails. The shell is inoperculate with the rising-spiral having 5.5 whorled which is typically dextral. The shell is higher than wide having a height of 4.5–5.5 mm and a width of 1.2 mm. The height is about 3.19 x the width with a high- spire which tapers gradually from the body whorl. The spire height is about 0.45 times to that of the shell diameter. The spire is obtuse. The shell is tear-shaped with shallow sutures. The body whorl only slightly convex to moderately convex while on the other hand the whorls of the spire weakly convex. The whorls are neither shouldered nor keeled. Neither teeth nor calluses are present on the aperture. The columella is folded to twisted. The shell is without an umbilicus. The shell is thin-lipped with the simple and delicate mouth edge which is thin and translucent in life, and become opaque after the death of the animal; the colourof the shell is pale or white, and very glossy and plain. The animal body is colourless (Watson and Dallwitz, 2005).

91 4.1.5.1 CECILIOIDES ACICULA (O. F. MÜLLER 1774) Buccinum acicula Muller, O. F. 1774. Vermivm terrestrium et fluviatilium, seu animalium infusorior um, helminthcorum, et testaceorum non marinorum, succincta historia. Volumen atterum. – pp. 1- XXXVI [=1-36], 1-214, [1-10]. Havniae & Lipsiae (Heineck & Faber)

The specimens belonging to Cecilioides acicula have an average height of 4.2 mm, diameter of 1.3 mm, mean aperture height 1.7 mm and height/diameter of 3.2 mm. The number of whorls ranges through 5.5-6. The shell is white in colour having dextral coiling, the umbilicus is minute. The shape of the shell is fusiform and tapering towards apex with a peristome that is thin and simple (Table 4.14 and Figure 4.14). Colour: The shells are white in colour.

Habitat: They are terrestrial in nature with a subterranean life style (in woodland calcareous habitats, sometimes occurring in cervices, rootlet holes, or in cavities).

Distribution: South Europe to Turkey, E Europe and S Sweden, probably introduced to the British Isles (not North Ireland and Scotland)

Locality: 204R.B., 202R.B., 208R.B., 219R.B.,103 J.B., 223 R.B., 225 R.B., 105 G.B.

The general name for the species is known by a variety of common names i.e blind snail, European blind snail, blind awl snail, blind pin snail and blind white snail (Bonham, 2005). Cecilioides acicula which is reffered as common blind snail, or ground snails which are tiny and small snails having a slender turricular shell and a thin wall (Bouchet and Rocroi, 2005) which are in accordance with the other taxonomists stating that which has a very small, thin needle- shaped shell having 5.5-6 whorls with a height of 4-5 mm and a width of 1-1.3 mm. The colour of the shell pale yellow to off-white and is glossy having a sculpture of irregular low radial corrugations. The body whorl is almost around half the shell height and the aperture is elongate, which is around 1.5 mm high by 0.5 mm wide (Watson and Dallwitz, 2005). The common blind snail has a needle shaped fragile and colourless shell with of height 5 mm and a width 1 - 1.4 mm with number of whorls from 6 – 7 which after the snail's death becomes opaque and then milky white. The aperture is about one third of the shell height. The columella of the shell is blunted and curved (Anderson, 2008). These species may have some resemblance with

92 truncatellids in terms of shape and size, but even then they are much less pointed having more rounded apertures and are operculate.

HABITAT AND DISTRIBUTION: They were found in terrestrial areas, in the soil near the roots of the plants which is in accordance with the findings of Watson and Dallwitz, 2005. These snails are found in the loose ground below the stones and near roots and can reach to 1m (Anderson, 2008). According to Kerney and Cameron, 1979 these species inhabit underground, some distance below the surface.

Cecilioides acicula is distributed throughout the Mediterranean, parts of Central, Eastern Europe, Ireland, England and the south of Scandinavia on the Alps up to the height of 1300m (Anderson, 2008). These species are world wide in nature especially in warm and dry climate (Bouchet and Rocroi, 2005). According to Welter-Schultes (2010) it has been reported from South Europe to Turkey, East Europe and Sout Sweden, probably introduced to the British Isles.

93 Table 4-14: Morphometrics of the Cecilioides acicula (O. F. Müller 1774). Species Number 14 Cecilioides acicula Mean Height 8.40 Mean Diameter 14.25 Height/Diameter 0.59 Aperture Height 8.56 No. of Whorls 5.0-6.0 Shell coiling Dextral Peristome Simple/Thin Umbilicus Small Shape Fusiformtapering towards apex

Figure 4-14: Cecilioides acicula O. F. Müller 1774.

94 4.1.6 SUCCINEIDAE (BECK, 1837) These snails of this family are also referred as amber snails due to their characteristically thin and amber-colored shells. The latter is a stronger reason for the name given to this family (Perez, et. al., 2008).

4.1.6.1 OXYLOMA ELEGANS (RISSO, 1826) Succinea elegans Risso, 1826. Histoire naturelle des principles productions de I/ Europe meridionale et particulierement de celles des environs de Nice et des Alpes Maritimes. To me quatrieme – pp. [1-3], j-vij [= 1-7] 1-439, pl. [1-12]. Paris. (Levrault).

The specimens belonging Oxyloma elegans to have an average height of 6.7 mm, diameter of 3.2 mm, mean aperture height 4.0 mm and height/diameter of 2.1 mm. The number of whorls ranges to 3, the shell is amber to greenish having dextral coiling, the umbilicus is absent. The shape of the shell is succiniformes, with a thin and peristome (Table 4.15 and Figure 4.15)

Colour: The shells of these snails are amber coloured with shades of greenish tinges.

Habitat: They are terrestrial and live in a diversity of damp in natural habitats.

Distribution: European countries Great Britain, Ireland, Czech Republic - Poland, Russia - Sverdlovsk oblast, Slovakia and Bulgaria.

Locality: 204 R.B., 119 J.B., 214 G.B., 217 J.B., 202 R.B., 208 R.B., 103 J.B., 219 R.B., 222 R.B., 223 R.B., 225 R.B., 235 R.B. 71 J.B. 105 G.B.

The shell is higher than wide with a dextral symmetry. The height ranges from 5-22 mm. The width of the shell is approximately 4.25–7 mm. The height is 1.6-1.9 times the width of the shell. The aperture is round to ovate and bears no teeth or calluses or lamellae. The shells are inoperculate with rising spire. There are 3number of whorls which are moderately to strongly convex and gradually tapers from the body whorl. The spire is small or short to high spire however the body whorl is predominating. The height of the spire about 0.1–0.23 times that of the shell. The spire of the shell ranges from acute to obtuse. The shape of the shell is succiniform i.e it is either ovoid-asymmetric or inverted-pyriform. The shell is deeply sutured. The whorls are

95 neither keeled nor shouldered. The columella of shell is smooth. The shell umbilicus is absent. The shell has thin-lip. The colour of the shell is greenish or amber. The shell is plain, thin and translucent (Watson and Dallwitz, 2005). The snail specimen belonging Succinea or Oxyloma are usually 12 mm or more in height (Wiese and Richling, 2013). The shell having height greater than 12 mm and colour paler belong to order Succinea, and the shell having no more than 12 mm in height is Oxyloma. ([email protected]). According to Wiese and Richling, (2013) in Oxyloma elegans (Risso 1826) the height of the shell varies from 9 - 17 mm may reach upto 20 mm. The shell width is 6 - 8 mm. The number of whorls is less than 3. The shell has an elongated conical shape with oval aperture. The body of the snail has dark colour wih many dark spots at the back. The shell colour is pale yellow to grayish green with few whorls.

HABITAT AND DISTRIBUTION

According to our research this is found in the damp and natural habitats is supported by the findings of Wiese and Richling, (2013) explaining that these species prefer to live in wet habitats especially bogs or banks of water but cannot live in water and at the times of flooded situations they climb on the vegetation.

96 Table 4-15: Morphometrics of the Oxyloma elegans (Risso, 1826) Species Number 15 Oxyloma elegans Mean Height 6.80 Mean Diameter 3.21 Height/Diameter 2.12 Aperture Height 4.10 No. of Whorls 2.5-3 Shell coiling Dextral Peristome Simple/Thin Umbilicus Absent Shape Succiniformes

Figure 4-15: Oxyloma elegans (Risso, 1826).

97 The mean maximum height was found in Zooctecus insularis reaching upto 12.45 mm. while the least mean height was found of the specimens belonging to species Cecilioides acicula which is 4.25 mm. The mean height of the specimens belonging to genus Ariophanta ranges from 6.10 mm-6.60 mm however, the mean height of Ariophanta bistrialis ceylanica was the least of all species belonging to genus Ariophanta. (Table 4.16)

However, the specimens of Monacha catiana and Oxyloma elegans have nearly similar mean height i.e. 6.88 mm and 6.80 mm. The mean height of the Zooctecus insularis and Oxychilus draparnaudi is 8.00 mm to 8.60 mm respectively. The specimens of Pupoides albilabris and Physa fontinalis is 9.20 mm.

The mean height of the shell becomes a useful parameter when used with mean measurement of the diameter which helps us in the classification of the snails after the calculation of the height over diameter ratio i.e.H/D ratio.

When the diameter of species belonging to the genus Ariophanta was measured it was found to range from 8.10 mm to 9.70 mm. When these measurementswere subject to find the H/D ratio, it was observed that the species having less than 0.65 mm had much flattened spire as compared to the species of the genus Ariophanta with a value of 0.75 mm. This type of shell shape is reffered as sub-globose, which is also observed in Oxychilus draparnaudi, Monacha catiana and Cernuella virgata having sub-globose shell shape. They have been placed under these species due to the variation in peristome, their ecological habitat and their colour, which is pale straw in genus Ariophanta while in Oxychilius draparnaudii the shell are horny coloured, with Monacha catiana having white shells but in Cernuella virgata the white shells have dark line around.

The other important morphological character is the perisome which is thin and simple in all species of Ariophanta and Monacha catiana, and thin but not simple in the case of pupoides species while in the case of Oxychilus draparnaudi it is thin and deflected. All the above species have a dextral symmetry, with number of whorls ranging from 4.5-5.5 whorls.

98 Table 4-16: Characterization of the Snails on the Basis of Morphometery. Mean Mean Mean Sr. H/D No. of Shell Species/Habitats Height Diameter Aperture Peristome Umbilicus Shape Color no. ratio whorls coiling (H) mm (D)mm Height Pale straw color. Dark line Ariophanta 1 5.70 9.10 0.63 4.50 4.5-5.5 Dextral Thin/simple Present Sub-globose around the periphery and in bistrialis ceylanica sutures Pale straw color. Light Ariophanta black/reddish brown line 2 6.50 9.40 0.69 4.72 4.5-5.5 Dextral Thin/simple Present Sub-globose bistrialis cyix around the periphery and in sutures. Ariophanta Pale straw color. White 3 bristrialis 6.60 9.70 0.68 4.91 4.5-5.5 Dextral Thin/simple Present Sub-globose shade/line around the periphery taprobanensis and in sutures Pale straw color, dark line around the periphery and in Ariophanta 4 6.20 8.90 0.70 5.79 4.5-5.5 Dextral Thin/simple Present Sub-globose sutures and dark lines around bristrialis the umbilicus on the lower side of the shell. Pale straw color. Dark line around the periphery and in 5 Ariophanta solata 6.10 8.10 0.75 4.12 4.5-5.5 Dextral Simple/Thin Present Sub-globose sutures and bluish tings on the spire Ariophanta Pale straw color. Very faint 6 belangeri 6.40 9.10 0.70 4.42 4.5-5.5 Dextral Simple/Thin Present Sub-globose line around the periphery and bombayana in sutures Oxychilus Thin Horny coloured, translucent, 7 8.60 14.22 0.60 8.61 4.5-5.5 Dexral Present Sub-globose draparnaudi deflected thin 8 Monacha catiana 6.80 9.91 0.69 4.51 4.5-5.5 Dextral Simple/Thin Present Sub-globose Shell white/ dusty white Shell white/ dusty white, with 9 Cernuella virgata 7.10 9.72 0.73 5.10 4.5-5.5 Dextral Thin Present Sub-globose Dark line around the periphery Fusiform Pupoides 10 9.20 3.10 2.97 2.82 7.5-8 Dextral Simple/Thin Small tapering White albilabris. towards apex Thin reflected 11 Physa fontinalis 9.20 5.70 1.61 6.41 4 Sinistral Absent Succiniformes Pale horn color to dark brown from the base Zooctecus Elongate 12 12.45 4.87 2.56 3.93 7.0-8.0 Dextral Thick Small White, Dull White insularis conic Juvenile Zooctecus 13 8.00 4.50 1.78 3.12 5.5 to 6 Dextral Thin Small Conical oval White insularis Fusiformtaperi Cecilioides 14 4.25 1.35 3.15 8.56 5.0-6.0 Dextral Simple/Thin Small ng White acicula towards apex 15 Oxyloma elegans 6.80 3.21 2.12 4.10 2.5-3 Dextral Simple/Thin Absent Succiniformes Amber, greenish

99 The mean aperture height is another major characteristic in classifying snails in which the three subspecies of the Ariophanta bristrialis ranges from 4.50-4.91 mm. and in Ariophanta solata it is 4.72 mm. and 4.42 mm. in Ariophanta belangeri bombayana. However the mean aperture height is 8.61 mm in Oxychilus draparnaudi and 4.51 mm in Monacha catiana. The significant difference of Monacha catiana from other species is of colour. The mean aperture height of Ariophanta bristrialis and Cernuella virgata is 5.79 mm and 5.1 mm respectively.The diameter of conical shells is much less as compared to the discoidal shell species with maximum mean diameter in Physa fontinalis i.e. 5.70 mm and least in the Ceciliodes acicula it was 1.35 mm. while the diameter of Zooctecus insularis and Juvenile Zooctecus insularis is 4.87 mm and 4.50 mm respectively. The diameter of Pupoides albilabris and Oxyloma elegams is 3.10 mm and 3.21 mm respectively.

According to our findings the H/D ratio is greater than one in the case of conical snail shells and ranges from 1.61 mm. to 3.15 mm. Physa fontinalis has the least H/D ratio and is almost equal to Juvenile Zooctecus insularis. Ceciliodes acicula has the maximum H/D ratio i.e. 3.15 mm. In case of Oxyloma elegans and Zooctecus insularis the H/D ratio is 2.12 mm. and 2.56 mm. respectively which is followed by Pupoides species with a value of 2.97 mm. Almost all the conical species are white in colour having small umbilicus with exception of Physa fontinalis having pale horn colour and Oxyloma elegans having amber greenish colour. In both species umbilicus is absent. There is a great difference in the coiling patterns of both species, however both have succiniformes shell shape, as Physa fontinalis is the only species with sinistral coiling with 4 number of whorls and the latter is the dextrally symmetrical with just 4 whorls. For the comparison of these conical snails the other character for identification is aperture height. The maximum aperture height was found in Ceciliodes acicula i.e 8.56 mm and the minimum aperture height was found in pupoides 2.82 mm with thin and simple peristome in both species. The aperture height is quite similar in Zooctecus insularis and Juvenile Zooctecus insularis i.e 3.93 mm and 3.12 mm respectively but they are different from each other in terms of peristome and number of whorls. In Zooctecus insularis the peristome is thick with 7-8 numbers of whorls while on the other hand Juvenile Zooctecus insularis has thin peristome with 5.5 to 6 whorls due to which we suspect that it may be some new species which previously has not been described or reported in this part of the world.

100 Recently researches have been conducted on the biodiversity of soil macroinverteberate in the low and high input fields of wheat and sugarcane in District Faisalabad (Rana, 2012; Siddiqui, 2005) with the major focus on impact of chemical on the diversity of different macroinverteberates. Rana (2000) studied the ecological distribution of earthworm species along some water bodies in the agroecosystem of Faisalabad Division. Khanum (2010) studied the taxonomy of the plant nematodes of Sugarcane fields. Very less work has been done with reference to the taxonomy and ecology of snails in the agroecosystem. Previously Ali, (2005), Altaf (2006) and Saif (2011) have attempted to study the diversity of snails in agroecosystem of Faisalabad which has augmented the previous information of the malacofaunna in Faisalabad. The work of the Ali (2005) and Altaf, (2006) was just about the sugarcane fields and wheat fields near Gutti village area; however Saif (2011) covered only few villages of Faisalabad focusing only one family of snails. This study has led us to more detailed information of this little creature in 24 villages of Faisalabad with fifteen different species belonging to nine families. The habitat/environment of this little creature has been considered.While according to (Arad et al., 1992) these parameters have an impact on the shell colour leading to significant variation leading to taxonomic errors.

101 4.2 MOLECULAR CHARACTERIZATION OF SNAIL SPECIES.

4.2.1 OPTIMIZATION OF THE PCR CONDITIONS Optimization of the RAPD-PCR conditions was done due to the sensitivity and reproducibility of the RAPD markers. Different concentrations of genomic DNA, 10X PCR buffer, MgCl2, dNTPs, and Taq DNA polymerase were tested for efficient and reliable amplification. Three different concentrations of template DNA of snails (5, 10, 15, nguL-1) were tested and it was observed that 10 ng µL-1 was found optimum for best amplification. Where as it was found that 3 mM concentration of MgCl2 and 1.0 unit Taq polymerase also found optimum for good amplification in a final volume of 25 µl of reaction mixture. It was also found that higher concentration of Taq and MgCl2 gave smear and lower concentrations resulted in light PCR fragments.

4.2.2 RAPD (PCR) ANALYSIS Genetic characterization of 15 snail species were done by using 23 RAPD primers and out of which 15 RAPD primers (Table 4.17) produced polymorphic amplification as shown in figures 4.16-4.18, whereas the remaining 8 primers produced monomorphic banding pattern and thus were excluded from the study. Total of 114 fragments were amplified and 90 were found polymorphic that is 78.94% polymorphism. The range of amplified RAPD fragments was found to be 7 to 9 with an average of 7.6 per primer. Three RAPD primers GLK-08, GLL-12 and GLK- 09 produced maximum number of bands i.e. 9; The Primer GLD-12 produced maximum number of polymorphic bands (8) and the lowest number of polymorphic RAPD fragments was produced by GLL-1 (4). The overall mean band frequency ranged between 0.27 – 0.68 with an average value of 0.41(Table 4.17).

102 Table 4-17: RAPD primers with sequence, number of polymorphic bands (NBP), and mean band frequency (MBF). Sr. No. Primer Code Sequence TNB NPB MBF

1 GL DecamerL-07 CAGGCCCTTC 08 07 0.43

2 GL DecamerL-08 AGTCAGCCAC 08 05 0.51

3 GL DecamerK-08 AGGGGTCTTG 09 07 0.46

4 GL DecamerK-12 GAAACGGGTG 07 07 0.68

5 GL DecamerK-19 TTCCGAACCC 07 05 0.36

6 GL DecamerK-20 GGTGACGCAG 07 06 0.45

7 GL DecamerL-12 CTGCTGGGAC 09 08 0.39

8 GL DecamerI-02 GTGAGGCGTC 08 06 0.40

9 GL DecamerK-07 GGGGGTCTTT 07 05 0.43

10 GL DecamerL-04 AAAGCTGCGG 07 06 0.36

11 GL DecamerL-01 GACGGATCAG 07 04 0.31

12 GL DecamerK-16 GTTGCCAGCC 09 07 0.41

13 GL DecamerB-07 ACTTCGCCAC 07 06 0.33

14 GL DecamerB-15 AGCGCCATTG 07 05 0.27

15 GL DecamerL-15 CAGAAGCCCA 07 06 0.29

103 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M

Figure 4-16: RAPD (PCR) of 15 snail species with prmer GLL-7. M is the 1 Kb ladder.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M

Figure 4-17: RAPD (PCR) of 15 snail species with prmer GLK-19. M is the 1 Kb ladder.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M

Figure 4-18: RAPD (PCR) of 15 snail species with prmer GLK-16. M is the 1 Kb ladder.

104 4.2.3 GENETIC SIMILARITY AMONG SNAIL SPECIES Genetic similarity estimated by Nei’s genetic similarity indices was ranged 0.5 to 0.74. Results depicted that the narrow genetic base was observed among most of the species studies which is more than 50%. Maximum genetic similarity was found between Ariophanta belangeri bombayana and Ariophanta bristrialis taprobanensis as well as Ariophanta belangeri bombayana and Ariophanta solata. Minimum genetic similarity based on Nei’s genetic indices was observed among Cernuella virgata and Ariophanta bistrialis cyix (Table 4.18).

4.2.4 GENETIC RELATIONSHIP AMONG SNAIL SPECIES Genetic relationship among fifteen snail species were estimated using cluster analysis (Figure 4.19) by the unweighted pair group method of arithmetic means (UPGMA) which showed that the two distinct cluster. First cluster includes the species Ariophanta bistrialis cyix, Ariophanta bristrialis taprobanensis, Ariophanta bristrialis, Ariophanta solata, Ariophanta belangeri bombayana, Oxychilus draparnaudi, Monacha catiana. The most closely related species were Ariophanta bristrialis taprobanensis, Ariophanta bistrialis ceylanica, Ariophanta bristrialis and Ariophanta solata, followed by species Ariophanta bistrialis cyix where as the species Ariophanta belangeri bombayana, Oxychilus draparnaudi and Monacha catiana also showed close relationship among the members of this group. The most distinct species were found to be Cernuella virgata and Juvenile Zooctecus insularis which remains unclustered. The other species like Zooctecus insularis, Pupoides albilabris, Physa fontinalis, Oxyloma elegans and Cecilioides acicula were found to cluster in the second group and linked with each other at some distances. Considering the Zooctecus insularis and Juvenile Zooctecus insularis they are quite distant from each other in the cluster, it is expected that might belong to some new species, which need further investigations.

The genetic association among the 15 snail species were also detected by principal component analysis (Figure 4.20) and it was found that close association was observed among the species Ariophanta bistrialis ceylanica, Ariophanta bistrialis cyix, Ariophanta bristrialis taprobanensis, Ariophanta bristrialis, Ariophanta solata, Ariophanta belangeri bombayana, Oxychilus draparnaudi, Monacha catiana and these species closely clustered in one group while

105 the species Zooctecus insularis also present at some distance with rest of the species in this group showing that Juvenile Zooctecus insularis might belong to a separate species. The other rest of the species were found scattered into three other groups.

106 Table 4-18: Genetic similarity matrix of 15 snail species based on Nei's genetic identity. Pop ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 **** 0.6140 0.6842 0.6140 0.6140 0.6842 0.5877 0.6316 0.6140 0.6491 0.5965 0.6491 0.6491 0.6316 0.5614 2 **** 0.6842 0.6316 0.6316 0.6667 0.7281 0.7018 0.5263 0.6140 0.6316 0.6667 0.5789 0.6140 0.5965 3 **** 0.6491 0.6491 0.7368 0.6930 0.7368 0.6316 0.7193 0.6491 0.6842 0.6140 0.6667 0.6140 4 **** 0.6140 0.6491 0.6579 0.6842 0.5439 0.6140 0.6491 0.6667 0.6140 0.6667 0.6140 5 **** 0.7368 0.6053 0.6842 0.5614 0.7193 0.6140 0.6140 0.6316 0.6491 0.5965 6 **** 0.6404 0.6667 0.6140 0.6842 0.6491 0.6667 0.6140 0.6842 0.6491 7 **** 0.7105 0.5877 0.6404 0.6053 0.7281 0.6228 0.6754 0.6228 8 **** 0.5614 0.6842 0.6491 0.7018 0.5965 0.6140 0.6842 9 **** 0.5965 0.6491 0.6140 0.5789 0.5614 0.5965 10 **** 0.6491 0.6667 0.5789 0.6491 0.6667 11 **** 0.6842 0.5965 0.6316 0.6491 12 **** 0.6316 0.6491 0.6667 13 **** 0.5614 0.6316 14 **** 0.5789 15 ****

107 Distance 6.4 5.6 4.8 4.0 3.2 2.4 1.6 0.8 0.0

Cecilioides acicula 14

Oxyloma15 elegans

Physa fontinalis 11

Pupoides albilabris 10

Zooctecus12 insularis

Monacha8 catiana

Oxychilus draparnaudi 7

Ariophanta belangeri bombayana 6

Ariophanta3 bristrialis taprobanensis

Ariophanta1 bistrialis ceylanica

Ariophanta4 bristrialis

Ariophanta5 solata

Ariophanta2 bistrialis cyix

Cernuella9 virgata

Juvenile13 Zooctecus insularis

Figure 4-19: Dendrogram showing relationship among 15 snail species.

108 3

2 15

13 1 2 t

n 12 213658 e 47 n

o -5 -4 -3 -2 -1 1 2 3 4 p

m 11 -1 10

o 9 C -2

-3

-4 14 -5

Component 1

Figure 4-20: Principal component analysis of 15 snail species based on molecular data.

109 4.2.5 ANALYSIS OF MOLECULAR VARIANCE Analysis of molecular variance revealed that the variance component among and within populations was 11% and 89% respectively. Both values are significant at probability less than 0.001 (Table 4.19 Figure 4.21).

Table 4-19: Summary AMOVA Table.

Source Df SS MS Est. Var. %

Among Pops 4 5.300 1.325 0.116 11%

Within Pops 10 9.833 0.983 0.983 89%

Total 14 15.133 1.100 100%

Figure 4-21: Percentage of Molecular Variance.

The fresh water gastropods are of extreme conservation concern for example hydrobiid and pleurocerid freshwater gastropods as they are lacking in dispersing capabilities due to which they are found in isolated populations while in general freshwater pulmonate gastropods typically are excellent dispersers. The major problem which comes in the way of species classification of the gastropods is the correct recognition of the differences between the species at the population level (Edwards and Beerli, 2000; Wiens and Penkrot, 2002; Maddison and Knowles, 2006). The molecular taxonomy is quite effective at this point of time as systematic relationship present in

110 the sister taxa is there even after millions of year and can be studied with the help of the genetic signatures (Thomaz et al., 1996; Arbogast et al., 2002). The processes occurring at the population level must be considered when taxonomies of recently diverged taxa are being studied. The perception of isolation has been supported by the molecular data to some degree. Considering the distributional patterns there have been very few records of pleurocerid or hydrobiid lineages that are found across unconnected water drainages systems (Minton and Lydeard, 2003; Perez et al., 2005; Sides, 2005), and documentation of the level to which there is an exchange in the genes of the upstream and downstream populations. There is an indication of the deep divergence by molecular analyses of intraspecific mitochondrial lineages of freshwater gastropod species (Dillon and Frankis, 2004; Lee et al., 2007). The genetic variation due geographical isolation increases the effective population size (Wright, 1943; 1951; Wakeley, 2000). The long term retention of ancient mitochondrial alleles, 20 million years, is due to an increase in effective population size (Thomaz et al. 1996) much older than the coalescence times expected from mutation rate and population size. In hydrobiid and pleurocerid groups of the gastropods the mutation rates may be elevated up to 8 to 14 base pairs per million years (Thacker and Hadfield, 2000, Holland and Hadfield 2002, Rundell et al., 2004). There may be an effect of the founder events on the genetic diversity of the closely related species events may include local extinction, and random genetic drift, adaptive genetic change (Goodacre et al., 2006). The interpretation of high levels of population subdivision that produce a pattern of reciprocal monophyly as differences at the species level due the fact that sampling is done from restricted localities (Arbogast et al., 2002). These problems are resolved when dense population-level and geographic sampling are combined with use of the appropriate molecular markers do mitigate but taxa whose ranges are reduced by the human actions, the aforesaid problem might be unavoidable. This misleading pattern is particularly likely when data consist solely of mitochondrial DNA sequences because of the smaller effective population size of the mitochondrial genome. A final complication that must be considered is the status of populations that are in the process of speciation, before or after reproductive isolation has evolved. Historical gene flow in recently diverged species is hard to disentangle from recent contact if introgression is possible (Wakeley, 1996; Rosenberg and Feldman, 2002).

111 4.3 DISTRIBUTION AND ABUNDACE OF SNAILS IN FAISALABAD Population density is population size in relation to some unit of space. It is generally assayed and expressed as the number of individuals or population biomass per unit area or volume. Often it is important to know whether a population is increasing or decreasing than to know its size at any one moment. In such cases indices of relative abundance are more important.

Table 4-20: Accession number and Order of the species. Accession Number Species 1 Ariophanta bistrialis ceylanica 2 Ariophanta bistrialis cyix 3 Ariophanta bristrialis taprobanensis 4 Ariophanta bristrialis 5 Ariophanta solata 6 Ariophanta belangeri bombayana 7 Oxychilus draparnaudi 8 Monacha catiana 9 Cernuella virgata 10 Pupoides albilabris 11 Physa fontinalis 12 Zooctecus insularis 13 Juvenile Zooctecus insularis 14 Cecilioides acicula 15 Oxyloma elegans

Figure 4-22: Abundance of Gastropod Species in Various Villages & Different Agro- Regions of Faisalabad during 2011.

112 In the Figure 4.22, the species have been numbered according to the sequence shown in the Table 4.20. Of the total, there were 17% Ariophanta bristalis and it was found maximum in number. The species found with the least number was of Cecilioides acicula (0.3%) followed by juvenile Zooctecus insularis (0.5%). The species Ariophanta bistrialis cyix, Ariophanta bistrialis ceylanica, Ariophanta bristrialis taprobanensis range from 12.4% to 13.3% respectively. The Oxyloma elegans was 9.5% followed by Ariophanta solata, 6.1%. The species which were found in a few hundreds were Physa fontinalis, (5.0%), followed by Cernuella virgata, 4.9%, followed by Zooctecus insularis, 4.5%, followed by Pupoides albilabris and Ariophanta belangeri bombayana, 3.8%. the species Monacha catiana, was 3.3%, Oxychilus draparnaudi, 3.0%.

The diversity indices of the snails, in different villages of Faisalabad, were found to be highly significant i.e. in 204 R.B., 07J.B., 119J.B., 214 G.B., 221J.B., 124 J.B., 202 R.B., 208 R.B., 103 J.B., 222 R.B., 223 R.B., 235 R.B., 71 J.B., 56G.B. and 73 G.B. while the other villages have a significant diversity with a p-value of 0.01 (Table 4.21).

When we compare the diversity indices and the eveness of different villages of Faisalabad it can be clearly observed that the villages having a diversity index more nearly 2.25 are 202 R.B., 103 J.B., 222 R.B., 223R.B. and65 G.B. (Group 1) with highly significant results whereas the evenness (E5) is greater than 0.8 only in 103 J.B. 222 R.B. and 65 G.B. while the other villages have a value of greater than 0.77. These results show that with increasing trend of diversity indices the species have been found to be quiet evenly distributed in the above mentioned group of villages however in the latter group of villages species have been found less evenly distributed.

The diversity indices ranges from 2.0 to 2.2 in the villages, 07 J.B., 119 J.B., 214 G.B. 225 R.B. and 105 G.B. (Group 2) with the the evenness greater than 0.8 in 119 J.B. and 105 G.B. showing a trend that there is an decrease in the diversity index the species have been found to be more evenly distributed. However in the villages of 07 J.B.and 214 G.B. the species are comparatively less evenly distributed with E5 value of 0.77 and 0.78 respectively. The E5 value has greatly reduced in the 225 R.B. reaching 0.70.

113 In the villages, 121 J.B., 123 J.B., 124 J.B., 217 J.B., 214 R.B., 221 R.B., 219R.B., 235 R.B., 295 R.B., 56 G.B., and 73 G.B. (Group 3) the value of diversity index ranges from 1.55- 2.0 showing that these areas are less diverse in terms of snails species. However, the evenness ranges from ranging from 0.83-0.85 in 121 J.B., 123 J.B., 124 J.B., and 295 R.B. showing that although these snails species are more evenly distributed in these areas than the villages of Group 1 yet these villages are less diverse giving significant results. The villages 217 J.B., 214 R.B., 221 R.B., 219 R.B., 235 R.B., 73 G.B., show lesser evenness 0.7-0.79 which is nearly similar to Group 2 villages but the villages of Group 3 are less diverse in terms of snail species. 56 G.B. has the least evenness in all the villages i.e. 0.48 (Figure 23 and Figure 24).

The map of the Faisalabad shows that 123 J.B. is towards the north eastern direction of Faisalabad with least diversity of snail species which increases in 103 J.B. with decrease in diversity again in 121 J.B. 121 J.B. is towards south of the 103 J.B.

Considering the villages linked to the Rakh branch, the 202 R.B. is in Group 1 with highest diversity. To the south of 202 R.B. there is 204 R.B. in same group with similar diversity. However, in 208 R.B. the diversity is least which is at south of 204 R.B. The village 235 R.B. is to the extreme south in the villages linked to Rakh branch in the Group 3 with the least species diversity.In villages linked with Ghogera Branch the diversity indices are least, showing that as we progress towards the south, the diversity of the snail species in Faisalabad is highly reduced.

114 Table 4-21: Species Diversity Indices for Snails in Different Villages of Faisalabad. Villages N° H' N1 N2 E1 E5 t value p-value 204 R.B 15 2.22 9.24 7.40 0.82 0.78 3.68 0.00*** 07 J.B 12 2.08 7.96 6.38 0.84 0.77 3.37 0.00*** 119 J.B 11 2.05 7.76 6.66 0.85 0.84 3.88 0.00*** 214 G.B 12 2.13 8.40 6.79 0.86 0.78 3.72 0.00*** 121 J.B 8 1.87 6.47 5.66 0.90 0.85 4.07 0.00*** 123 J.B 8 1.55 4.72 4.11 0.75 0.84 2.71 0.02** 124 J.B 7 1.74 5.70 5.09 0.89 0.87 3.97 0.00*** 217 J.B 10 1.78 5.95 9.92 0.77 0.79 2.94 0.00** 202 R.B 15 2.36 10.63 8.56 0.87 0.78 4.25 0.00*** 208 R.B 13 2.14 8.48 6.78 0.83 0.77 3.57 0.00*** 103 J.B 13 2.29 9.91 8.29 0.89 0.82 4.47 0.00*** 214 R.B 10 1.91 6.77 5.50 0.83 0.78 3.27 0.01** 221 R.B 10 1.88 6.58 5.23 0.82 0.76 3.13 0.01** 219 R.B 12 1.83 6.26 4.63 0.74 0.69 2.62 0.01** 222 R.B 14 2.38 10.79 9.50 0.90 0.87 5.17 0.00*** 223 R.B 14 2.25 9.45 7.47 0.85 0.77 3.82 0.00*** 225 R.B 13 2.04 7.67 5.66 0.79 0.70 3.03 0.01** 235 R.B 10 1.98 7.27 5.84 0.86 0.77 3.52 0.00*** 295 R.B 7 1.61 5.03 4.27 0.83 0.81 3.05 0.01** 71 J.B 13 2.37 10.66 9.28 0.92 0.86 5.40 0.00*** 105G.B 13 2.15 8.59 7.07 0.84 0.80 3.76 0.00*** 65 G.B 14 2.34 10.43 8.88 0.89 0.84 4.69 0.00*** 56 G.B 11 1.63 5.12 2.98 0.68 0.48 1.93 0.04* 73 G.B 9 1.79 5.98 4.83 0.81 0.77 3.03 0.01**

115 Figure 4-23: Species Diversity Indices for Snails in Different Villages of Faisalabad.

Figure 4-24: Evenness for Snails in Different Villages of Faisalabad.

116 According to the Table 4.22 , all species was found in all villages linked with Rakh branch, Ghogera branch and Jinnah branch which are distributries of the irrigation system of province of Punjab. While the species diversity and the eveness was found maximum in R. B villages and minimum in J. B villages. However the diversity indices and eveness in all the villages is quite similar ranging from 2.27-2.46 and 0.80-0.87 respectively and the results are highly significant (Figure 4.25-4.26). The regression analysis of species diversity and evenness of snails in different irrigation canals in Faisalabad shows that there 97.03% relationship beween species diversity and species evenness in the irrigation canal linked to Rakh Branch, Ghogera Branch and Jhang Branch (Figure 4.27). The maximum number of snails was found in sugarcane, 6224, with the least number of snails in vegetables, 299. The number of snails in wheat, fodder and sugarcane ranges from 4347-6224. However the number of snails was 2795 in ditches (Table 4.23). The relative abundance of snails was maximum in sugarcane (32%) while least in the vegetables i.e., 2%. This may be due to the fact that sugarcane crop time is quite long when compared with the vegetables, due to which they have shorter rehabilitation time in vegetables as compared to the sugarcane. As in the summer the snails climb on the plant stalks and expose themselves to the stalks to escape the summer heat which increase the crop losses near harvest time (Desbiolles et al., 2003) and also reduce population density on the ground giving lesser stress to the individuals. A very short term disturbance effects are very long lasting for the snail survival as the land snails are generally poor dispersers (Baur and Baur, 1988), which may lead to local extinction for a long period of time (Hylander, 2004).The heterogeneity of the vegetation and a complex cover of top soil help co-existance of more species whereas there is a significant reduction of the gastropod abundance and diversity due to the homogenous grazingly (Labaune and Magnin 2002).

117 Table 4-22: Species Diversity Indices for Snails in Different Villages of Faisalabad. Months N° H' N1 N2 E5 t-value df p value Villages R.B 15 2.46 11.65 10.31 0.87 5.45 14 0.000*** Villages G.B 15 2.35 10.53 8.96 0.84 4.51 14 0.000*** Villages J.B 15 2.27 9.65 7.93 0.80 3.93 14 0.001***

Figure 4-25: Species Diversity Indices for Snails in Villages through Different Irrigation Canal of Faisalabad.

Figure 4-26: Evenness for Snails in Villages through Different Irrigation Canal of Faisalabad.

118 Figure 4-27: Regression Analysis of Species Diversity and Evenness of Snails in Different Irrigation Canals in Faisalabad

The percentage of snails in sugar cane is 32.27% followed by fodder and wheat i.e 29.16% and 22.53% respectively. In vegetables the snail percentage was least i.e. 1.55% (Table 4.24). The relative abundance of wheat and fodder is quite similar. The relative abundance of snails in ditches is nearly one half of sugarcane i.e. 14% (Figure 4.28). This is possibly because of the effect of different chemicals present in the irrigation water channels, turbidity, dissolved oxygen etc.

The maximum number of species was found in wheat crop with highest diversity index of 2.30 but having evenness with a value of 0.82. However the diversity index was least in the fodder with maximum eveness of 0.93 showing that it has least diversity of snail species which are evenly distributed. The diversity index of sugarcane and wheat ranges from 2.03-2.30 with eveness ranging from 0.80-0.82. The diversity index in fodder and vegetables ranges from 0.11- 0.17 while the diversity index of ditches is 0.55. The evenness of species in ditches, vegetales and fodder, range from 0.91-0.93 showing that despite lesser diversity the species are quite evenly distributed. The results are highly significant in all the crops while in ditches the results are non-significant (Table 4.25, Figure 4.29-4.30). There is a strong negative correlation between species diversity and evenness in the crops that clearly shows that when diversity is low the evenness was high and viceversa in the distribution of snails in the different crops. There is

119 95.3% relationship between species diversity and species evenness in different crops of Faisalabad (Figure 4.31).

120 Table 4-23: Distribution of Snails in Different Habitats of Agroecosystem. Sr.no Species/Habitats Wheat Sugarcane Fodder Vegetables Ditches Total 1 Ariophanta bistrialis ceylanica 555 1127 717 52 0 2451 2 Ariophanta bistrialis cyix 794 859 659 80 0 2392 Ariophanta bristrialis 3 taprobanensis 680 1105 727 46 0 2558 4 Ariophanta bristrialis 743 1583 898 52 0 3276 5 Ariophanta solata 236 439 510 0 0 1185 Ariophanta belangeri 6 bombayana 152 316 263 0 0 731 7 Oxychilus draparnaudi 244 70 219 37 0 570 8 Monacha catiana 169 74 377 12 0 632 9 Cernuella virgata 266 106 544 20 0 936 10 Pupoides abilabris 138 193 408 0 0 739 11 Physa fontinalis 0 0 0 0 955 955 12 Zooctecus insularis 221 340 303 0 0 864 13 Juvenile Zooctecus insularis 84 12 0 0 0 96 14 Cecilioides acicula 65 0 0 0 0 65 15 Oxyloma elegans 0 0 0 0 1840 1840 Total 4347 6224 5625 299 2795 19290

121 Figure 4-28: Relative Abundance of Snails in different Habitats of the Agroecosystem of Faisalabad.

122 Table 4-24: Pecent (%) Abundance and Distribution of Different Species in different Habitats of Faisalabad.

Species /Habitat Wheat Sugarcane Fodder Vegetables Ditches Total Ariophanta bistrialis ceylanica 12.8 18.1 12.7 17.4 0.0 12.7 Ariophanta bistrialis cyix 18.3 13.8 11.7 26.8 0.0 12.4 Ariophanta bristrialis taprobanensis 15.6 17.8 12.9 15.4 0.0 13.3 Ariophanta bristrialis 17.1 25.4 16.0 17.4 0.0 17.0 Ariophanta solata 5.4 7.1 9.1 0.0 0.0 6.1 Ariophanta belangeri bombayana 3.5 5.1 4.7 0.0 0.0 3.8 Oxychilus draparnaudi 5.6 1.1 3.9 12.4 0.0 3.0 Monacha catiana 3.9 1.2 6.7 4.0 0.0 3.3 Cernuella virgata 6.1 1.7 9.7 6.7 0.0 4.9 Pupoides albilabris 3.2 3.1 7.3 0.0 0.0 3.8 Physa fontinalis 0.0 0.0 0.0 0.0 34.2 5.0 Zooctecus insularis 5.1 5.5 5.4 0.0 0.0 4.5 Juvenile Zooctecus insularis 1.9 0.2 0.0 0.0 0.0 0.5 Cecilioides acicula 1.5 0.0 0.0 0.0 0.0 0.3 Oxyloma elegans 0.0 0.0 0.0 0.0 65.8 9.5 Total 22.5 32.3 29.2 1.6 14.5 100

Table 4-25: Species Diversity Indices for Snails in Different Crops of Faisalabad. Crops N° H' N1 N2 E5 t-value df p value Wheat 13 2.30 9.97 8.34 0.82 4.62 12 0.000*** Sugarcane 12 2.03 7.59 6.25 0.80 3.46 11 0.003*** Fodder 11 0.11 10.11 9.44 0.93 7.74 10 0.000*** Tomatoes and vegetables 7 0.17 6.16 5.73 0.92 5.00 6 0.001*** Ditches 2 0.55 1.90 1.82 0.91 3.16 1 0.098 NS

123 Figure 4-29: Species Diversity Indices for Snails in Different Crops of Faisalabad.

Figure 4-30: Evenness for Snails for Snails in Different Crops of Faisalabad.

124 Figure 4-31: Regression Analysis of Species Diversity and Evenness of Snails in different crops in Faisalabad

Species abundance distribution has been a point of interest for the scientists and researchers due to many reasons. This is because in this way the community can be undersood in a more better way rather than by just counting the species, as in this way heterogeneity and abundance can be incorporated which is the basis for the calculations (Magurran, 2004). Secondly in species abundance distribution, the rare-species tail can also provide information regarding the estimation of the numbers of species missed altogether and, therefore, of the true species richness. Chao (1984) estimated the number of singletons and doubletons in the sample with the help of a simple non parametric estimator. Various scientists found high percentages of singletons i.e. (11%, 12%, and 23%) (De Winter and Gittenberger, 1998; Fontaine et al., 2007b; Liew et al., 2010), leading to relatively high richness estimations. Thirdly, the species abundance distribution helps us to understand changes in species dominance induced due to the season (De Winter and Gittenberger, 1998) physical disturbance (Schilthuizen et al., 2005a) and to deduce the ecological processes developing the community structure.

The diversity indices are the highest for fauna in the sugarcane crop, followed by fodder, wheat and brassica, respectively. This may be due to the lesser use of chemicals i.e weedicides and insecticides in contrast to the other crops and the second probable reason is that sugarcane crop is available round the year in the field while in contrast to this the excessive use of

125 insecticides was recorded on other crops. Sugarcane was found to be the preferred crop by majority of faunal species followed by fodder, wheat and brassica (Inayat et al., 2010). Considering wheat a major crop and staple food in Pakistan, has less faunal diversity due to regular use of weedicides when compared with sugarcane crop, with greater diversity, has been confirmed by the studies of Cartea et al. (2009). The immediate environment and cropping patterns are very important in structuring the arthropod communities in crop varieties of the agroecosystem (Heong et al., 1991). There is maximum faunal similarity between wheat and brassica and in sugarcane and fodder. This is probably because both grew in the same season and are grown in the similar environmental conditions and result in similar faunal distribution while in ditches there were found snails belonging to a separate order, having preference for aquatic habitat (Inayat et al., 2010). The monthwise distribution of the Table 4.26, explains that there were maximum number of snails in the month of August, 3721, and the least number of snails were found in May, 1724. The number of snails found in the month of July, 3598, June, 3476, April ,3389, and March 3382 were nearly quite similar.

126 Table 4-26: Distribution of snails in different months. Sr.no Species/Habitats March April May June July August Total

1 Ariophanta bistrialis ceylanica 456 589 82 181 810 333 2451

2 Ariophanta bistrialis cyix 356 810 52 365 341 468 2392

3 Ariophanta bristrialis taprobanensis 535 614 43 516 478 372 2558

4 Ariophanta bristrialis 521 803 46 637 597 672 3276

5 Ariophanta solata 227 217 65 204 202 270 1185

6 Ariophanta belangeri bombayana 65 71 95 119 258 123 731

7 Oxychilus draparnaudi 77 47 159 127 127 33 570

8 Monacha catiana 152 41 183 145 62 49 632

9 Cernuella virgata 69 111 248 243 181 84 936

10 Pupoides albilabris 147 11 273 224 61 23 739

11 Physa fontinalis 214 55 145 41 219 281 955

12 Zooctecus insularis 84 12 138 225 160 245 864

13 Juvenile Zooctecus insularis 8 0 32 34 0 22 96

14 Cecilioides acicula 1 0 18 7 13 26 65

15 Oxyloma elegans 470 8 145 408 89 720 1840

Total 3382 3389 1724 3476 3598 3721 19290

127 The maximum relative abundance of snails was found in August and July,19% where the averge maximum temperature and average minimum temperaure was highest in August followed by July, however in May the relative bundance was just 9% where the averge maximum temperature and average minimum temperaure was lowest (Figure 4.23, Table 4.27).

The average maximum temperature was the least in May and the highest in the August. The humidity and rainfall were least in the month of August. Considering the average sunshine, there were least number of average sunshine hours in the month of August, 5 hours, however in May there were 8 hours of average sunshine (Table 4.28)

Figure 4-32: Relative Abundance of snails in different Months in Faisalabad.

128 Table 4-27: Pecent (%) Abundance and Distribution of Different Species in different Habitats of Faisalabad

Sr.no. Species/Habitats March April May June July August Total 1 Ariophanta bistrialis ceylanica 13.5 17.4 4.8 5.2 22.5 9.0 12.7 2 Ariophanta bistrialis cyix 10.5 23.9 3.0 10.5 9.5 12.6 12.4 3 Ariophanta bristrialis taprobanensis 15.8 18.1 2.5 14.8 13.3 10.0 13.3 4 Ariophanta bristrialis 15.4 23.7 2.7 18.3 16.6 18.1 17.0 5 Ariophanta solata 6.7 6.4 3.8 5.9 5.6 7.3 6.1 6 Ariophanta belangeri bombayana 1.9 2.1 5.5 3.4 7.2 3.3 3.8 7 Oxychilus draparnaudi 2.3 1.4 9.2 3.7 3.5 0.9 3.0 8 Monacha catiana 4.5 1.2 10.6 4.2 1.7 1.3 3.3 9 Cernuella virgata 2.0 3.3 14.4 7.0 5.0 2.3 4.9 10 Pupoides albilabris 4.4 0.3 15.8 6.4 1.7 0.6 3.8 11 Physa fontinalis 6.3 1.6 8.4 1.2 6.1 7.6 5.0 12 Zooctecus insularis 2.5 0.4 8.0 6.5 4.5 6.6 4.5 13 Juvenile Zooctecus insularis 0.2 0.0 1.9 1.0 0.0 0.6 0.5 14 Cecilioides acicula 0.0 0.0 1.0 0.2 0.4 0.7 0.3 15 Oxyloma elegans 13.9 0.2 8.4 11.7 2.5 19.4 9.5 Total 17.5 17.6 8.9 18.0 18.7 19.3 100

These results lead us to the conclusion that possibly there was a strong synergetic impact of these abiotic factors on the distribution of the snails as the juveniles member of most of the species are very sensitive to dessication (Asami, 1993) and a positive correlation exist between the moisture of the soil and gastropod distribution (Prior, 1985). The number and abundance depend on the presence of bryophyte layers, and is more intact in the wet areas as compared to the drier areas. This protects the litter layers underlying these bryophytes, on which the snails survive, and the moisture content of the soil. Generally, during sunny days there are dry and hot conditions, during sunny days which modify the characteristics of the top soil litter of the clearcuts (Chen et al., 1993) and as a result many snails are killed. The areas are generally cleared and prepared by sweeping up the well drained, steep lime stone slopes. This results in the vegetation cover loss, greater impact of sun radiations, loss of topsoil, resulting in desiccation, greater risk of fire, and in the reduction of the number of drought-intolerant snail species (Vermeulen and Whitten, 1999).

129 Table 4-28: Mean Envoirnmental Variables in Different Months. Envoirnmental variables March April May June July August Average Maximum Temperature (oC) 32 34 26 35 39 41 Average Minimum Temperature (oC) 22 25 19 26 27 28 Average Relative Humidity % 55 60 50 65 70 75 Average Rainfall (mm) 1 2 1 3 4 5 Average Sunshine (h) 8 9 8 9 6 5 Total Snails Specimens 3382 3389 1724 3476 3598 3721

The maximum species diversity index (2.49) was found in May, with maximum evenness in this month. It shows the diversity of species in this month which are evenly distributed. The least species diversity was found in April (1.90) and having the lowest value of eveness in April and July (0.79) (Table 4.29).

The species diversity in the month of March, May, June, July, and August ranges from 2.29 to 2.42. However, the evenness is 0.85 in March, which reduces in April after which a rise occurs in the month of May which gradually decrease to minimum in July as it is in April, with a rise in August (Figure 4.33-3.34). Regression analysis between species diversity and species evenness shows that there is 57.36% relationship between different months in agroecosystem of Faisalabad (Figure 4.35).

Table 4-29: Species Diversity Indices for Snails in Different Months in Faisalabad. Months N° H' N1 N2 E5 t-value df p value March 15 2.34 10.41 8.98 0.85 4.55 14 0.000*** April 13 1.90 6.67 5.49 0.79 2.96 12 0.01** May 15 2.49 12.08 10.53 0.86 5.69 14 0.000*** June 15 2.42 11.26 9.61 0.84 4.98 14 0.000*** July 14 2.30 10.01 8.09 0.79 4.21 13 0.001*** August 15 2.29 9.90 8.27 0.82 4.14 14 0.001*** N0 =Number of Snail Species H/ =Species Diversity E5= Evenness of species

130 Figure 4-33: Species Diversity Indices for Snails in Different Months in Faisalabad.

Figure 4-34: Evenness for Snails in Different Months in Faisalabad.

131 Figure 4-35: Regression Analysis of Species Diversity and Evenness of Snails in different months in Faisalabad.

132 Chokor and Oke (2009) reported that the snail diversity is decreasing in Nigeria due to deforestation as they found that the number of species, rather than species composition, was greatly reduced in the sunny places as there was a highly significant difference in species number between shady places and in the sun plot. Sunlight and moisture content of the soil renders major impact on the composition of terrestrial gastropod community in dry and sunny habitats. However, the other important possible driving factors are relative humidity and temperature but not sunlight (Suominen, 1999). Studies suggested fire, plant species, plant number, herb cover, slope aspect, and habitat humidity are the important ecological factors that predict the snail abundance at the sites and also be used as biological indicator of ecological changes (Anna, 2005). There are other ecological factors which are important to assess the diversity and distribution of the snail species such as bottom sediments, flow of water, diversity of macrophyte. The number of species depends on the number of bottom sediments, an increase in the flow velocity decreased the number of the species density and snail number while snail species richness is not effected by the vegetation density except Elodea canadensis and a few other species (Strzelec and Królczyk, 2004). The adverse climatic conditions do greatly effect pulmonate species except, Albinaria caerrulea, which survives in adverse conditions as they can prevent themselves from desiccation by showing a series of morphological and behavioural characteristics that can support their survival during adverse climatic conditions as there was no correlation between the climatic conditions and biochemical variables (Giokas, 2005).

More studies are required to evaluate the quality of environmental parameters of different villages and their impact on the snail diversity. The data of abiotic factors as independent variables and the number of snails as dependent variable were analyzed using linear regression, it was found that average minimum and average maximum temperature has a significant effect on the snail distribution while humidity, rainfall and sunshine has non significant effect of the snails distribution (Table 4.30, Figures 4.36, 4.37, 4.38, 4.39, 4.40).

133 Table 4-30: Regression Analysis of Envoirnmental Parameters and Numbers of Snails. (Y) (X) n r r2 slope Y-intercept t-value df P value No. of AverageMax. temp.(X) 6 0.877 0.77 122.35 -1005.95 3.66 4 0.01** snails AverageMin. temp.(X) 6 0.874 0.76 191.1 -1467.70 3.59 4 0.01** Average humidity(X) 6 0.772 0.6 61.26 -613.57 2.43 4 0.04NS Average Rainfall(X) 6 0.639 0.41 290.18 2441.20 1.66 4 0.09NS Average Sunshine(X) 6 -0.3 0.09 136.96 4242.22 -0.64 4 0.28NS

Figure 4-36: Effect of Maximum Temperature on the Number of Snails.

Figure 4-37: Effect of Minimum Temperature on the Number of Snails

134 Figure 4-38: Effect of Average Humidity on the Number of Snails.

Figure 4-39: Effect of Average Rainfall on the Number of Snails.

Figure 4-40: Effect of Average Sunshine on the Number of Snails.

135 4.4 SPECIES INTERACTION WITH HEAVY METALS AND OTHER SOIL FACTORS The maximum limit for Cadmium, according to National Environmental Quality Standards is 0.1 mg L-1. All the villages of Faisalabad under study have much less concentration of Cd than the allowed limit (Table 4.31, Figure 4.41).The maximum limit for Pb, according to NEQs is 0.5 mg L-1. All the villages of Faisalabad under study have much less concentration of Pb in soil than the allowed limit (Figure 4.42).The maximum limit for pH is 10 (GOP, 1997). The villages under study have very less pH, reaching maximum to 8.41 in 121 J.B. 222 R.B. with least value of 7.79 in 223 R.B. The pH of the soil in Faisalabad villages is from neutral to alkaline (Figure 4.43).The Electrical conductivity of the soil ranges from 0.75-5.22 dSm-1 with least EC in 202 R.B. and maximum in 73 G.B. (Figure 4.44).

136 Table 4-31: Comparison of Soil parameters with National Environmental Quality Standards (Pakistan). Soil Samples Villages EC Cd mg/L Pb mg/L pH dSm−1 204 R.B 0.0045 0.0400 8.1700 2.3200 07 J.B 0.0070 0.0430 7.9000 1.2100 119 J.B 0.0090 0.0460 8.1700 5.2200 214 G.B 0.0032 0.0510 8.2000 1.9200 121 J.B 0.0120 0.0340 8.4100 2.7100 123 J.B 0.0110 0.0370 7.9100 1.6100 124 J.B 0.0100 0.0480 8.1700 1.2300 217 J.B 0.0100 0.0370 8.1500 1.0800 202 R.B 0.0050 0.0450 8.0500 0.7500 208 R.B 0.0052 0.0420 8.0700 3.8500 103 J.B 0.0120 0.0330 8.1200 4.1000 214 R.B 0.0056 0.0320 8.3100 4.3200 221 R.B 0.0044 0.0430 8.1700 1.4700 219 R.B 0.0040 0.0410 8.2500 0.9300 222 R.B 0.0045 0.0330 8.4100 3.2100 223 R.B 0.0042 0.0300 7.7900 0.8200 225 R.B 0.0052 0.0440 8.2100 1.2000 235 R.B 0.0060 0.0350 7.9000 1.7600 295 R.B 0.0050 0.0420 8.1000 1.3300 71 J.B 0.0033 0.0430 7.9100 2.1000 105G.B 0.0031 0.0510 8.0000 3.1000 65 G.B 0.0051 0.0021 7.8000 2.2500 56 G.B 0.0045 0.0030 7.7100 5.1300 73 G.B 0.0032 0.0051 8.2000 3.9000 Pak. Environmental legislation 1997 0.1000 0.5000 10.000 3.9000

137 Figure 4-41: Comparison of Soil Cadmium concentration with National Environmental Quality Standards (Pakistan).

Figure 4-42: Comparison of Soil Lead Concentration with National Environmental Quality Standards (Pakistan).

Figure 4-43: Comparison of Soil pH with National Environmental Quality Standards (Pakistan).

138 Figure 4-44: Comparison of Soil Electrical Conductivity with National Environmental Quality Standards (Pakistan).

The number of snails is a dependent variable (Y) and the Cadmium concentration, Lead concentration, pH, Electrical conductivity of the soil is dependent variable.

The effect of temperature has a significant effect on the number of snails, while there is non significant effect of the Cadmium concentration, Lead concentration, pH and electrical conductivity of the soil on the number of snails (Table 4.32 Figure 4.45, 4.46, 4.47, 4.48).

Table 4-32: Regression Analysis of Soil Parameters and Number of snails. (Y) (X) N R r2 slope Y-intercept t-value Df P value No. of Cd. 24 -0.22 0.05 -20300.5 928.09 -1.06 22 0.15NS Snails Conc. Pb 24 -0.13 0.02 -2515.95 894.55 -0.63 22 0.27NS Conc. pH 24 0.085 0.01 -120.42 1777.51 -0.40 22 0.35NS EC 24 0.205 0.04 -39.29 897.82 -0.98 22 0.17NS

Figure 4-45: Effect of Average Cd concentration on the number of snails

139 Figure 4-46: Effect of Average lead concentration on the number of snails.

Figure 4-47: Effect of Average Electrical conductivity on the Number of Snails.

Figure 4-48: Effect of Average pH on the Number of Snails.

140 The fresh water snail abundance is greater in the wet season (July-September) than in dry season (December-February) experiencing less rainfall (Afshan et al., 2013). The community structure in molluscs is best expressed by soil moisture content (Machintosh 2002). Not only temperature and humidity, there are other physico-chemical factors which regulate distribution and abundance of the gastropods i.e pH, vegetation, pollution etc. (Ollerenshaw, 1958; Yilma, 1985). With the increase of the pollution if there is a decrease in the vegetation, the temperature of the envoirnment will increase facilitating the increase in the diversity of the thermophilic organisms. The heavy metal pollution reduces the microbial activity thereby allowing the accumulation of organic matter therefore increasing the species affinity to humidity (Grzés, 2009). According to Inayat et al. (2010) the diversity indices calculated in 2007-2008 were greater than that in 2008-2009 (226 species) and the reason for this is the rainfall, effecting temperature, residual effect of fertilizers, insecticides, weedicides and other environmental factors. Along with water, light is an additional physical factor that may affect macroinverteberate communities (Voshell, 2002). The rain fall patterns and fluctuation in the soil temperature may lead to the death of juvenile snails directly or due to indirect effect on the habitats. The change in the climatic conditions resulting in the decrease in rainfall and global warming has also been reported (Gerlach, 2007). The chemical factors such as dissolved oxygen, pH, hardness and nutrients are very important (Cushing and Allan, 2001) in determining the diversity and abundance of macroinverteberates. The snail densities are influenced by the factors like conductivity, dissolved oxygen, pH, rainfall and canopy cover Camara (2012).

Living communities show response to the entire range of biogeochemical factors in the envoirnment (Karr and Chu, 2000) as the seasonal variations are linked to the snail physiology and biochemical composition which is linked to the annual cycles of photoperiod, temperature, humidity, water availability (Cook, 2001; Storey,2002).

The presence of calcium is linked with the pH of the soil. The calcium reduces the acidity of the soil by increasing its pH, which increases the fertility of the soil http://EzineArticles.com/657433. The physico chemical factors like temperature, hardness, pH, altitude, size of water bodies, vegetation and pollution are among the significant aspects, which influence the distribution and abundance of mollusks (Dillon, 2000). The activity and distribution of land snails depends on several factors including precipitation, soil pH, soil Ca

141 content, canopy density, etc. The pH has a positive correlation with species richness and density (Burch, 1955; Hotopp, 2002; Aravind, 2005). Schiltuizen (2005a) reported that abundance of the snails is positively and strongly correlated with calcium carbonate and in turn with pH. Whereas pulmonate snails were significantly more abundant at disturbed localities than prosobranch snails, however, abundance for both groups was similar at undisturbed sites, along with this their abundance is usually strongly correlated with both, calcium carbonate concentration and pH.

Snails generally have high calcium requirement for their shells and reproduction (Graveland et al., 1994) and their abundance has a positive correlation with both pH and Calcium carbonate (CaCO3) (Waldén, 1995; Schilthuizen et al., 2003). However, in the regions where the soil is usually acidic, the snail richness is usually high but abundance of the snails is low.

The levels of the metals in the sampling sites were found to be higher than safe levels as mentioned by US EPA (2013) which must not exceed 0.005 ppm for Cd and for Pb the level ranges from 0-0.015 ppm. During this study the concentration for Cd ranges from 0.001-0.007 for Cd and for Pb the range was 0.0021 ppm to 0.051 ppm in the 24 villages sampled. Our study has revealed that there is no significant effect of the cadmium and lead concentration which is supported by the by Everard and Denny, (1984) but this increase in the concentration of heavy metals is in consequence of the population growth and technological advancement contaminationg the envoirnment and the situation becomes more adverse as they cannot be mineralized to their total innocuous forms (Bonaventura and Johnson, 1997). Due to this non- biodegradeability, these heavy metals tend to accumulate in the plants and animal tissues (Otitoloju et al., 2009). The macro inverteberates, that are extremely disturbed due to the changes in the soil chemistry, are strong bioindicators of the envoirnment (Paoletti 1999). These kinds of explorations of envoirnment use organisms like nematodes, earthworms, Collembola as well as molluscs (Otitoloju et al., 2009) and ants. However, the last two organisms are quite resistant to this type of contamination (Grzés, 2009) but still this type of contamination can lead to reduction in the diversity of species as changes in the compostion of the community eliminates the most sensitive species (Del Val et al., 1999 and Beyrem et al., 2007), while on the other hand the tolerance of the opportunistic species (Syrek et al., 2006) or invasive species (Piola and Johnston, 2008) is promoted. Not only these tolerant species are found in extremely contaminated sites, there is however a positive correlation between the abundance of some

142 groups of arthropods such as larvae of beetles of the subfamily Hoplinae and family Staphylinidae (Nahmani and Lavelle, 2002) and with an increase in the heavy metal pollution, there is an increase in the abundance of Protura, Diplura and Collembola (Migliorini et al. 2004). The same is in the case of the diversity of ant species which increases with the increase in heavy metal pollution (Grzés, 2009). The increase in the either abundance or richness and uniformity have not been commonly studied however, some studies reported these types of changes occur with the increase of pollution by heavy metals (Russell and Alberti, 1998, 2002). There is a record of physicochemical envoirment in the isotopic and elemental form in the shells and the tissues of the gastropods (Mills, et al., 2007). The snails are good biomonitors for heavy metals. There is a strong relation between sediments and soft tissues as significant correlations were found between sediments and the soft tissues for Cu (P<0.05) and Pb (P<0.01). The operculums and the shells have significant correlation, for Cd and Pb, with the sediments (Cheng, 2008). The pollution has an indirect effect on the diversity and abundance at the community level as well as at the individual level of the land snails by reducing the size of the shell belonging to the same species. Not only this these shell pose reproductive problems on the birds that consume these shell as a calcium supplement during their breeding season (Eeva et al., 2010) leading to biomagnification.

143 4.5 INTERACTION OF SNAIL SPECIES The analysis of variance of the species, month, and host crop is found highly significant. The association between the two variables of species and month is highly significant, the interaction between species with the host crop is found highly significant while the association between time and the host crop is also significant (Table 4.33).

Table 4-33: Analysis of Variance for Number of Specimen, using Adjusted SS for Test. Sources D.F Seq SS Adj SS Adj MS F value P- value Species 14 152149.4 152149.4 10867.8 32.71 0.000*** Months 5 7155.7 7155.7 1431.1 4.31 0.001*** Agroecosystem 3 55027.1 55027.1 18342.4 55.21 0.000*** Species *Months 70 73362.3 73362.3 1048 3.51 0.000*** Species *Agroecosystem 42 73580.6 73580.6 1751.9 5.27 0.000*** Months*Agroecosystem 15 9517.8 9517.8 634.5 1.91 0.019* Error 1290 428605.7 428605.7 332.3 Total 1439 799398.6

In the Table 4.34 the presence absence data for fifteen species during six months (March- August) has been used in the agroecosystem of Faisalabad. Using BASIC program SPASSOC.BAS, the variance ratio test, chi squares and association indices have been determined. An overall positive association is suggested (VR=1.796). However, W=10.7767 and falls within the range of 7.261-25 so we accept the null hypothesis of no association during different months.

However, there does seem to be some strong possibilities that true positive associations (1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, 4-5, 8-10) might exist (Table 4.34). There are negative associations between species 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-15, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12- 2-15, 3-7,3-8, 3-9,3-10,3-11,3-12, 3-15,4-7,4-8,4-9,4-10,4-11,4-12,4-15,5-7,5-8,5-9,5-10,5-11,5- 12,5-15, 6-8, 6-10, 7-14, 8-14, 9-11, 9-14, 9-15. This is clearly indicated in the Table 4.34 showing percentage of snails in different months of the study period that species 1, 2, 3,4,5 have clear cut high percentages as compared to species 7, 8, 9, 10, ,12, which may be due to the reason that they are competitive on the same feeding niches; however, species 11 and 15 are not much less in percentage due to which we can say that this negative relationship may be due to difference in the ecological niche as species 11 and 15 are only found in ditches.

144 Table 4-34: Interspecific association indices and test stastistics between fifteen snail species in different months. Multiple Species VR, Index of overall association= 1.796 W, Test statistic= 10.7767 Association Indices Species pair Association type Chi-Square Value Yate's Chi-Square Ochiai Dice Jaccard 1 2 Positive 6.000 0.960 1.000 1.000 1.000 1 3 Positive 6.000 0.960 1.000 1.000 1.000 1 4 Positive 6.000 0.960 1.000 1.000 1.000 1 5 Positive 6.000 0.960 1.000 1.000 1.000 1 6 Positive 1.200 0.000 0.775 0.750 0.600 1 7 Negative 1.200 0.000 0.516 0.500 0.333 1 8 Negative 1.200 0.000 0.516 0.500 0.333 1 9 Negative 0.600 0.150 0.671 0.667 0.500 1 10 Negative 1.200 0.000 0.516 0.500 0.333 1 11 Negative 0.600 0.150 0.671 0.667 0.500 1 12 Negative 0.600 0.150 0.671 0.667 0.500 1 13 Positive 0.240 0.960 0.447 0.333 0.200 1 14 Positive 0.240 0.960 0.447 0.333 0.200 1 15 Negative 0.600 0.150 0.671 0.667 0.500 2 3 Positive 6.000 0.960 1.000 1.000 1.000 2 4 Positive 6.000 0.960 1.000 1.000 1.000 2 5 Positive 6.000 0.960 1.000 1.000 1.000 2 6 Positive 1.200 0.000 0.775 0.750 0.600 2 7 Negative 1.200 0.000 0.516 0.500 0.333 2 8 Negative 1.200 0.000 0.516 0.500 0.333 2 9 Negative 0.600 0.150 0.671 0.667 0.500 2 10 Negative 1.200 0.000 0.516 0.500 0.333 2 11 Negative 0.600 0.150 0.671 0.667 0.500 2 12 Negative 0.600 0.150 0.671 0.667 0.500 2 13 Positive 0.240 0.960 0.447 0.333 0.200 2 14 Positive 0.240 0.960 0.447 0.333 0.200 2 15 Negative 0.600 0.150 0.671 0.667 0.500 3 4 Positive 6.000 0.960 1.000 1.000 1.000 3 5 Positive 6.000 0.960 1.000 1.000 1.000 3 6 Positive 1.200 0.000 0.775 0.750 0.600 3 7 Negative 1.200 0.000 0.516 0.500 0.333 3 8 Negative 1.200 0.000 0.516 0.500 0.333 3 9 Negative 0.600 0.150 0.671 0.667 0.500

145 3 10 Negative 1.200 0.000 0.516 0.500 0.333 3 11 Negative 0.600 0.150 0.671 0.667 0.500 3 12 Negative 0.600 0.150 0.671 0.667 0.500 3 13 Positive 0.240 0.960 0.447 0.333 0.200 3 14 Positive 0.240 0.960 0.447 0.333 0.200 3 15 Negative 0.600 0.150 0.671 0.667 0.500 4 5 Positive 6.000 0.960 1.000 1.000 1.000 4 6 Positive 1.200 0.000 0.775 0.750 0.600 4 7 Negative 1.200 0.000 0.516 0.500 0.333 4 8 Negative 1.200 0.000 0.516 0.500 0.333 4 9 Negative 0.600 0.150 0.671 0.667 0.500 4 10 Negative 1.200 0.000 0.516 0.500 0.333 4 11 Negative 0.600 0.150 0.671 0.667 0.500 4 12 Negative 0.600 0.150 0.671 0.667 0.500 4 13 Positive 0.240 0.960 0.447 0.333 0.200 4 14 Positive 0.240 0.96 0.447 0.333 0.200 4 15 Negative 0.600 0.150 0.671 0.667 0.500 5 6 Positive 1.200 0.000 0.775 0.750 0.600 5 7 Negative 1.200 0.000 0.516 0.500 0.333 5 8 Negative 1.200 0.000 0.516 0.500 0.333 5 9 Negative 0.600 0.150 0.671 0.667 0.500 5 10 Negative 1.200 0.000 0.516 0.500 0.333 5 11 Negative 0.600 0.150 0.671 0.667 0.500 5 12 Negative 0.600 0.150 0.671 0.667 0.500 5 13 Positive 0.240 0.960 0.447 0.333 0.200 5 14 Positive 0.240 0.960 0.447 0.333 0.200 5 15 Negative 0.600 0.150 0.671 0.667 0.500 6 7 Positive 0.667 0.000 0.667 0.667 0.500 6 8 Negative 0.667 0.000 0.333 0.333 0.200 6 9 Positive 0.000 0.750 0.577 0.571 0.400 6 10 Negative 0.667 0.000 0.333 0.333 0.200 6 11 Positive 0.000 0.750 0.577 0.571 0.400 6 12 Positive 3.000 0.750 0.866 0.857 0.750 6 13 Positive 1.200 0.000 0.577 0.500 0.333 6 14 Positive 1.200 0.000 0.577 0.500 0.333 6 15 Positive 0.000 0.750 0.577 0.571 0.400 7 8 Positive 0.667 0.000 0.667 0.667 0.500 7 9 Positive 3.000 0.750 0.866 0.857 0.750 7 10 Positive 0.667 0.000 0.667 0.667 0.500 7 11 Positive 0.000 0.750 0.577 0.571 0.400 7 12 Positive 3.000 0.750 0.866 0.857 0.750

146 7 13 Positive 1.200 0.000 0.577 0.500 0.333 7 14 Negative 1.200 0.000 0.000 0.000 0.000 7 15 Positive 0.000 0.750 0.571 0.571 0.400 8 9 Positive 0.000 0.750 0.577 0.571 0.400 8 10 Positive 6.000 2.667 1.000 1.000 1.000 8 11 Positive 0.000 0.750 0.577 0.571 0.400 8 12 Positive 0.000 0.750 0.577 0.571 0.400 8 13 Positive 1.200 0.000 0.577 0.500 0.333 8 14 Negative 1.200 0.000 0.000 0.000 0.000 8 15 Positive 3.000 0.750 0.866 0.857 0.750 9 10 Positive 0.000 0.750 0.577 0.571 0.400 9 11 Negative 1.500 0.094 0.500 0.500 0.333 9 12 Positive 0.375 0.094 0.750 0.750 0.600 9 13 Positive 0.600 0.150 0.500 0.400 0.250 9 14 Negative 2.400 0.150 0.000 0.000 0.000 9 15 Negative 1.500 0.094 0.500 0.500 0.333

147 The presence absence data for fifteen species in five habitats, in the agroecosystem of Faisalabad, has been used. Using BASIC program SPASSOC.BAS, the variance ratio test, chi squares and association indices have been determined. An overall positive association is suggested when V˃1 (VR=5.857143). However, W=29.28572 and doesnot fall within the range of 7.261-25 so we reject the null hypothesis of no association in different habitats.

However, there does seem to be some strong possibilities that true positive associations (1-2, 1-3, 1-4, 1-7,1-8,1-9, 2-3, 2-4, 2-7, 2-8, 2-9, 3-4, 3-7, 3-8, 3-9, 4-7, 4-8, 4-9, 5-6, 5-10, 5- 12, 6-10, 6-12, 7-8, 7-9, 8-9) might exist as shown in the table. On the other hand there are strong true negative association in (1-11, 1-15, 2-11, 2-15, 3-11, 3-15, 4-11,4-15, 7-11, 7-15, 8- 11, 8-15, 9-11, 9-15) (Table 4.35). Both of the species Physa fontinalis and Oxyloma elegans have been numbered 11 and 15 respectively. Both species are found in ditches or near water bodies while all other species are found in the agricultural crops due to which they have negative association as they cannot overlap the same habitat.

148 Table 4-35: Interspecific association indices and test stastistics between fifteen snail species in different habitats of agroecosystem. VR, Index of overall association=5.857143 W, Test statistic= 29.28572 Association Indices Species pair Association type Chi-Square Yate's Chi-Square Ochiai Dice Jaccard 1 2 Positive 5.000 0.703 1.000 1.000 1.000 1 3 Positive 5.000 0.073 1.000 1.000 1.000 1 4 Positive 5.000 0.703 1.000 1.000 1.000 1 5 Positive 1.875 0.052 0.866 0.857 0.750 1 6 Positive 1.875 0.052 0.866 0.857 0.750 1 7 Positive 5.000 0.703 1.000 1.000 1.000 1 8 Positive 5.000 0.703 1.000 1.000 1.000 1 9 Positive 5.000 0.703 1.000 1.000 1.000 1 10 Positive 1.875 0.052 0.866 0.857 0.750 1 11 Negative 5.000 0.703 0.000 0.000 0.000 1 12 Positive 1.875 0.052 0.866 0.857 0.750 1 13 Positive 0.833 0.052 0.707 0.667 0.500 1 14 Positive 0.313 0.703 0.500 0.400 0.250 1 15 Negative 5.000 0.703 0.000 0.000 0.000 2 3 Positive 5.000 0.703 1.000 1.000 1.000 2 4 Positive 5.000 0.703 1.000 1.000 1.000 2 5 Positive 1.875 0.052 0.866 0.857 0.750 2 6 Positive 1.875 0.052 0.866 0.857 0.750 2 7 Positive 5.000 0.703 1.000 1.000 1.000 2 8 Positive 5.000 0.703 1.000 1.000 1.000 2 9 Positive 5.000 0.703 1.000 1.000 1.000 2 10 Positive 1.875 0.052 0.866 0.857 0.750 2 11 Negative 5.000 0.073 0.000 0.000 0.000 2 12 Positive 1.875 0.052 0.866 0.857 0.750 2 13 Positive 0.833 0.052 0.707 0.667 0.500 2 14 Positive 0.313 0.703 0.500 0.400 0.250 2 15 Negative 5.000 0.703 0.000 0.000 0.000 3 4 Positive 5.000 0.703 1.000 1.000 1.000 3 5 Positive 1.875 0.052 0.866 0.857 0.750 3 6 Positive 1.875 0.052 0.866 0.857 0.750 3 7 Positive 5.000 0.703 1.000 1.000 1.000 3 8 Positive 5.000 0.703 1.000 1.000 1.000 3 9 Positive 5.000 0.703 1.000 1.000 1.000 3 10 Positive 1.875 0.502 0.866 0.857 0.750

149 3 11 Negative 5.000 0.703 0.000 0.000 0.000 3 12 Positive 1.875 0.052 0.866 0.857 0.750 3 13 Positive 0.833 0.052 0.707 0.667 0.5 3 14 Positive 0.313 0.703 0.500 0.400 0.250 3 15 Negative 5.000 0.703 0.000 0.000 0.000 4 5 Positive 1.875 0.052 0.866 0.857 0.750 4 6 Positive 1.875 0.052 0.866 0.857 0.750 4 7 Positive 5.000 0.703 1.000 1.000 1.000 4 8 Positive 5.000 0.703 1.000 1.000 1.000 4 9 Positive 5.000 0.703 1.000 1.000 1.000 4 10 Positive 1.875 0.052 0.866 0.857 0.750 4 11 Negative 5.000 0.703 0.000 0.000 0.000 4 12 Positive 1.875 0.052 0.866 0.857 0.750 4 13 Positive 0.833 0.052 0.707 0.667 0.500 4 14 Positive 0.313 0.703 0.500 0.400 0.250 4 15 Negative 5.000 0.703 0.000 0.000 0.000 5 6 Positive 5.000 1.701 1.000 1.000 1.000 5 7 Positive 1.875 0.052 0.866 0.857 0.750 5 8 Positive 1.875 0.052 0.866 0.857 0.750 5 9 Positive 1.875 0.052 0.866 0.857 0.750 5 10 Positive 5.000 1.701 1.000 1.000 1.000 5 11 Negative 1.875 0.052 0.000 0.000 0.000 5 12 Positive 5.000 1.701 1.000 1.000 1.000 5 13 Positive 2.222 0.313 0.816 0.800 0.667 5 14 Positive 0.833 0.052 0.577 0.500 0.333 5 15 Negative 1.875 0.052 0.000 0.000 0.000 6 7 Positive 1.875 0.052 0.000 0.000 0.000 6 8 Positive 1.875 0.052 0.866 0.857 0.750 6 9 Positive 1.875 0.052 0.866 0.857 0.750 6 10 Positive 5.000 1.701 1.000 1.000 1.000 6 11 Negative 1.875 0.052 0.000 0.000 0.000 6 12 Positive 5.000 1.701 1.000 1.000 1.000 6 13 Positive 2.222 0.313 0.816 0.800 0.667 6 14 Positive 0.833 0.052 0.577 0.500 0.333 6 15 Negative 1.875 0.052 0.000 0.000 0.000 7 8 Positive 5.000 0.703 1.000 1.000 1.000 7 9 Positive 5.000 0.703 1.000 1.000 1.000 7 10 Positive 1.875 0.052 0.866 0.857 0.750 7 11 Negative 5.000 0.703 0.000 0.000 0.000 7 12 Positive 1.875 0.052 0.866 0.857 0.750 7 13 Positive 0.833 0.052 0.707 0.667 0.500

150 7 14 Positive 0.313 0.703 0.500 0.400 0.250 7 15 Negative 5.000 0.703 0.000 0.000 0.000 8 9 Positive 5.000 0.703 1.000 1.000 1.000 8 10 Positive 1.875 0.052 0.866 0.857 0.750 8 11 Negative 5.000 0.703 0.000 0.000 0.000 8 12 Positive 1.875 0.052 0.866 0.857 0.750 8 13 Positive 0.833 0.052 0.707 0.667 0.500 8 14 Positive 0.313 0.703 0.500 0.400 0.250 8 15 Negative 5.000 0.703 0.000 0.000 0.000 9 10 Positive 1.875 0.052 0.866 0.857 0.750 9 11 Negative 5.000 0.703 0.000 0.000 0.000 9 12 Positive 1.875 0.052 0.866 0.857 0.750 9 13 Positive 0.833 0.052 0.707 0.667 0.500 9 14 Positive 0.313 0.703 0.500 0.400 0.250 9 15 Negative 5.000 0.703 0.000 0.000 0.000

151 The villages of 204 R.B, 219 R.B, 56 G.B, are approximately ninty percent similar and can be reffered as cluster 1, and the other group of villages of 07 J.B, 235 R.B, 119J.B, 123 J.B, 73G.B, 124J.B, 217 J.B, 221 R.B, 222R.B, 65 G.B 225 R.B, 295 R.B, 121 J.B, are approximately ninty percent similar and can be reffered as cluster 2. The third cluster of villages is 214 G.B, 103 J.B, 202 R.B, 223 R.B, 208 R.B is nearly 75 percent similar and can be reffered as cluster 3. The cluster 1 and 2 are nearly 80 percent similar and the village of 105 G.B is nearly 78 percent similar to the cluster 1 and 2. The village of 71 J.B is nearly 70% similar to the cluster 1, 2, and 3. The village of 214 R.B is least similar to the all clusters that is 64.72%. Still more investigations are required to evaluate the immediate envoirnmental conditions of the soil and water of these villages to understand reasons of similarity in the snail diversity (Figure 4.49).

There is greater than 90 percent similarity among the wheat, sugarcane, vegetables and fodder, while the similarity index of ditches is much less than the previously described crops i.e 35.58% (Table 4.36, Figure 4.50).

Dendrogram Single Linkage, Correlation Coefficient Distance

64.72

y 76.48 t i r a l i m i S 88.24

100.00 .B .B .B .B .B .B .B .B .B .B .B .B .B .B .B .B .B .B .B .B .B .B .B .B R R G J R J J G J J R R G R R J G G J R R R J R 4 9 6 7 5 9 3 3 4 7 1 2 5 5 5 1 5 4 3 2 3 8 1 4 0 1 5 0 3 1 2 7 2 1 2 2 6 2 9 2 0 1 0 0 2 0 7 1 2 2 2 1 1 1 2 2 2 2 2 1 1 2 1 2 2 2 2 Variables

Figure 4-49: Cluster analysis of Variables:Villages

152 Table 4-36: Cluster Analysis of the Variables: Wheat, Sugarcane, Fodder, Vegetables, Ditches. Correlation Coefficient Distance, Single Linkage Steps No. of Clusters Similarity Distance level Cluster joined New clusters No. of obs. in new cluster 1 4 96.0676 0.07865 1 4 1 2 2 3 95.5965 0.08807 1 2 1 3 3 2 94.4595 0.11081 1 3 1 4 4 1 35.5769 1.28846 1 5 1 5

Dendrogram Single Linkage, Correlation Coefficient Distance

35.58

y 57.05 t i r a l i m i S

78.53

100.00 Wheat Vegetables Sugarcane Fodder Ditches Variables

Figure 4-50: Cluster Analysis of the Variables: Wheat, Sugarcane, Fodder, Vegetables, Ditches.

153 There is more than 90% similarity in March and August in terms of species of snail, which is nearly 90% similar to the species composition in June and can be reffered as cluster 1 while the similarity between April and July is nearly 90% which can be reffered as cluster 2 while the species of snails in the month of May is highly different with least similarity i.e. 43.89% with the cluster 1 and 2. This may be due to the difference in the envoirnmental variables in May when compared with other months (Table 4.37, Figure 4.50).

Table 4-37: Cluster Analysis of the Variables: March, April, May, June, July, August.

Correlation Coefficient Distance, Single Linkage Steps No. of Similarity Distance Cluster joined New No. of obs. in Clusters level level clusters new cluster 1 5 93.0347 0.13931 1 6 1 2 2 4 91.2916 0.17417 2 5 2 2 3 3 90.5105 0.18979 1 4 1 3 4 2 88.0106 0.23979 1 2 1 5 5 1 43.8922 1.12216 1 3 1 6

Dendrogram Single Linkage, Correlation Coefficient Distance

43.89

y 62.59 t i r a l i m i S

81.30

100.00 March August June April July May Variables

Figure 4-51: Cluster Analysis of the Variables: March, April, May, June, July, August.

154 The explaination of increase in the species diversity due to heavy metal is still lacking , instead of this, scientists have pointed out the importance of the studies exploring the interactions between the fauna and the soil, i.e positive or negative association e.g predation or competition between edaphic organisms (Grzés, 2009). Due to this all macrofaunna community should be considered as indicators as they have wide ranges of adaptive mechanishms than a single group (Nahamani and Lavelle 2002, Koehler, 1996). The snails prefer certain habitats and migrate to these habitat, which may be in response to temperature (Bates et al. 2005), or to a chemical species (Le Bris et al., 2006), Alternatively, a gastropod may occupy a particular space because of its interaction with a potential predators or competitors (Micheli et al., 2002; Mullineaux et al., 2003). There are factors that may be physical, chemical or/and biological which likely affect gastropod species distributions to some extent. There are certain phylogenetic constraints on physiological adaptations that may restrict the habitats available to certain gastropod species (Mills et al., 2007) . For the characterization of the physicochemical environment, new techniques are being used to make feasible measurements on scale of the individual gastropods (Le Bris et al., 2006). There is a dire need of behavioral studies at multiple stages in species’ life histories, and under controlled conditions (Lee, 2004; Bates et al., 2005). However, in order to understand the role of gastropods in the ecosystem and to answer the questions regarding their assemblage in the discrete assemblages is associated with specific environments, and studies are required in the context of their interactions with other species in the community—prey, predators, competitors, and even potential facilitators (Mills et al., 2007). Due to the changing ecological communities i.e., the loss of native species and the translocation of invaders, there is a need of a broader understanding of community structure, which has both applied and theoretical importance (Keesing et al., 2010).

In India, there is little information on potential impact of climate change on land snails. Sen et al., (2012). There is a remarkable difference of the identified species in our study when comparing it with the studies already done by the Ali, (2005). There were only 3 species of the snails that had been identified, which may be due to the fact that he mainly focused on the diversity of snails in sugarcane fields. It is hoped that this work would be a step forward to investigate this highly ignored fauna to identify and document in Faisalabad, Pakistan.

155 Moreover, the rate of introduction of alien species such as snails and slugs appears to be on the increase in the region and was particularly noticed in Qatar (Al-Khayat, 2008) and the study will help to identify the alien species introduced and the effect they are causing on the local environment of Faisalabad.

156 CHAPTER 5

SUMMARY This study has been conducted at a micro spatial level in which an attempt has been made to identify the terrestrial snails in the agro-ecosystem of Faisalabad, not only on the basis of morphometry but also on the basis of molecular basis using RAPD markers. Along with this the diversity of the snails on the temporal and spatial scale has been studied. The information regarding the characterization of snails in agroecosystem of Pakistan is very meagre and fragmentary. This study has led us to detailed information of this little creature in 24 villages of Faisalabad with fifteen different species belonging to nine families.

The sampling was carried out for a period of six months from villages linked with Rakh branch, Jhang branch and Ghogera branch. During this period 19290 snails were randomly collected from four agroecosystems (sugarcane, wheat, fodder, vegetables fields) and ditches. The samples were studied under the microcope. The snails were isolated from the soil samples through sifting through screen of mesh size less than 1 mm. The identification of the specimens was made on the basis of number of whorls, coiling of the shell, umbilicus, shape, colour, shape of the aperture, presence or absence of operculum, height (mm), diameter (mm), and the diameter of the aperture (mm) using vernier caliper. The snail species were identified by using recent identification keys and diagrammatic description provided in them. Molecular characterization of snails has been done with the help of randomly amplified polymorphic DNA- polymerase chain reaction (RAPD-PCR) technique for understanding the biodiversity of snails in this region. Genetic characterization of 15 snail species was done by using 23 RAPD primers and out of which 15 RAPD primers produced polymorphic amplification. On the basis of Analysis of Molecular Variance there was found 11% variation among populations of the five habitats and 89% variation within populations in species population found in all the habitats. Genetic similarity among snail species was estimated by Nei’s genetic similarity indices showing a range of 0.5 to 0.74. Maximum genetic similarity was found between Ariophanta belangeri bombayana and Ariophanta bristrialis taprobanensis as well as Ariophanta belangeri bombayana and Ariophanta solata. Minimum genetic similarity based on Nei’s genetic indices was observed among Cernuella virgata and Ariophanta bistrialis cyix. Considering the

157 Zooctecus insularis and Juvenile Zooctecus insularis they are quite distant from each other in the cluster due to which it is expected that might belong to some new species, which need further investigations. In this study six species have been reported for the first time in Faisalabad.

The data of distribution and abundance was subject to different statistical tools i.e., shannon and wiener diversity index, index of overall association, two way analysis of variance, multiple regression, cluster analysis.

The maximum number of snails was found in sugarcane, 6224, with maximum relative abundance, 32%, and highest diversity index of 2.30. However, there were found least number of snails in vegetables, 299, with minimum relative abundance i.e. 2%. The diversity index was found least in fodder i.e. 0.11 followed by vegetables. The monthwise distribution of snails shows that there were maximum number of snails were found in the month of August, while in May there were least number of snails. The maximum relative abundance of snails was found in August and July, 19% in which the average maximum temperature and average minimum temperaure was highest in August followed by July, however in May the relative bundance was just 9% where the average maximum temperature and average minimum temperaure was lowest.

The number of snails found in the month of March, April, June, and July was quite similar. The maximum species diversity Index was found in May. However, least species diversity (1.90) was found in April. The evenness was also lowest in April and July. The species diversity in the month of March, May, June, July, and August are quite similar. There is more than 90% similarity in March, June and August in terms of species of snail and can be reffered as cluster 1 and the similarity between April and July is nearly 90% which can be reffered as cluster 2. The interaction among species was studied and an overall positive association was found among species.

The species diversity and the eveness was found maximum in R. B villages and minimum in J. B villages and the results are highly significant. The diversity indices showed that as we progress towards south, the diversity of the snail species in Faisalabad is highly reduced.

Different abiotic factors were also measured i.e. temperature, humidity, rainfall, sunshine, Cadmium concentration, Lead concentration, pH and electrical conductivity. It was found that

158 average minimum and average maximum temperature has a significant effect on the snail distribution while humidity, rainfall, sunshine, cadmium concentration, lead concentration, pH and electrical conductivity have no significant effect of the snails distribution.

The species interaction was studied and it was found that there is an overall positive association among species with no association in different months and a strong association in different habitats.

The association between the species and time is highly significant, the association between species with the host crop is found highly significant while the association between time and the host crop is also significant.

This study is of extreme importance to understand genetic diversity and conservation of snail species.

CONCLUSIONS AND RECOMMENDATIONS This is an effort to bring into notice the importance of this small creature which can be helpful in developing ecological models in future studies, as they play an important role as biodiversity indicators. There is a dire need of identification of the indicator species. The indicator species vary from place to place due to habitat sensitivity, geographical distribution and ease of identification. Fragmentry information may lead to wrong designation of indicator species. The fundamental information is the baseline data which may help to conduct the monitoring studies important for comparison. The recognition of the indicator species helps in studying the wild malacofaunna being affected by agricultural practices. The decline in the lower fauna is a warning to wider communities. In this way there can be the development of conservation guidelines by the decision makers and legislators.

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