i EFFECTS OF LANDSCAPE ON GENETIC VARIATION OF STONE LAPPING MINNOW (Garra cambodgiensis (TIRANT, 1884)) POPULATIONS IN THE UPPER NAN RIVER CHAOWALEE JAISUK A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DOCTOR DEGREE OF SCIENCE IN AQUATIC SCIENCE FACULTY OF SCIENCE BURAPHA UNIVERSITY JANUARY 2018 COPYRIGHT OF BURAPHA UNIVERSITY ii iii ACKNOWLEDGEMENT First of all, I would like to express my deepest gratitude and sincere appreciation to my advisor, Dr.Wansuk Senanan, for her excellent advice, discussion, and stimulating suggestions throughout the research process and the dissertation writing. Without her patient instruction, insightful criticisms and expert guidance, the completion of this thesis would not have been possible. Secondly, I am grateful for the support from dissertation committee members, and colleagues from the Faculty of Sciences, Burapha University, the Faculty of Sciences and Agricultural Technology, Rajamangala University of Technology Lanna Nan Campus and the Nan Provincial Administrative Organization (PAO). The dissertation committee members, Dr.Wongpathom Kamolrat, Assoc. Prof. Dr.Vipoosit Manthachitra, Dr.Narinratana Kongjandtre and Dr.Salinee Khachonpisitsak provided valuable comments and suggestions to improve my dissertation research and writing. Dr.Prasarn Intacharoen from the Department of Aquatic Science and Mr.Suchart Chayhard from the Environmental science graduate program, Faculty of Sciences guided me through the use of GIS for my research. The Nan PAO provided the GIS map data. This research was funded by the 2017 graduate research program at National Research Council of Thailand, and the Department of Aquatic Science, Faculty of Science Burapha university. Laboratory support was provided by the Department of Aquatic Science, Faculty of Science, Burapha University and Biotechnology Center at Rajamangala University of Technology Lanna. I am very thankful for these opportunities. Finally, I greatly appreciate the loving support of my family and friends. Their support has sustained me through this intense intellectual journey. Chaowalee Jaisuk iv 54810252: MAJOR: AQUATIC SCIENCE; Ph.D. (AQUATIC SCIENCE) KEYWORDS: SPATIAL GENETIC VARIATION/ MICROSATELLITE VARIATION/ LANDSCAPE GENETICS/ PHYSICAL BARRIERS/ POPULATION GENETICS/ GARRA CAMBODGIENSIS/ UPPER NAN RIVER/ THAILAND/ TROPICAL STREAM FISH CHAOWALEE JAISUK: EFFECTS OF LANDSCAPE ON GENETIC VARIATION OF STONE LAPPING MINNOW (Garra cambodgiensis (TIRANT, 1884)) POPULATIONS IN THE UPPER NAN RIVER. ADVISORY COMMITTEE: WANSUK SENANAN, Ph.D. 125 P. 2018. Spatial genetic variation of stream-dwelling freshwater fish is typically affected by the historical and contemporary river landscape as well as life-history traits. Tropical river and stream landscapes have endured extended geological change and were less affected by the latest glaciation period. As a consequence, these systems tend to be extremely complex and may have shaped genetic diversity of fish populations in a unique manner. Such information on population structure for tropical aquatic systems, especially in freshwater ecosystem is lacking. These data are becoming important for designing appropriate management and conservation plans, as these aquatic systems are undergoing intense development and exploitation. Therefore, this dissertation research evaluated the effects of landscape features on population genetic diversity of Garra cambodgiensis, a tropical stream cyprinid, in the upper Nan River drainage basin, northern Thailand, using 5-11 hypervariable microsatellite loci. This research consisted of three elements focusing on two geographic scales, basin and sub-basin levels. First study described overall spatial genetic variation of populations from eight tributaries (six sub-basins) representing the entire upper Nan River drainage (Meed, Kon, Pua, Yao, Yang, Sa, Wa and Haeng rivers). Based on 11 microsatellite loci, I detected moderate genetic diversity within eight population samples (average number of alleles per locus across loci = 10.99±3.00; allelic richness = 10.12±2.44. Allelic richness within samples negatively correlated with stream order (P < 0.05). There was no evidence for recent bottleneck events in these populations. These populations in the upper Nan River drainage basin were genetically heterogeneous v (Global FST = 0.022, P < 0.01). The Bayesian clustering algorithms (TESS and STRUCTURE) suggested that four to five genetic clusters roughly coincide with sub-basins; (1) the headwater streams/ the main stem of the Nan River, (2) a middle tributary, (3) a southeastern tributary and (4) a southwestern tributary. I observed positive correlation between geographic distance and linearized FST (P < 0.05), and the genetic differentiation pattern can be moderately explained by the contemporary stream network (STREAMTREE analysis, R2 = 0.75). The MEMGENE analysis suggested genetic division between northern (genetic clusters 1 and 2) and southern (clusters 3 and 4) sub-basins. The second component examined the impacts of landscape features on genetic variation of G. cambodgiensis at a sub-basin level, the Nam Wa sub-basin. This sub-basin, with the Wa River being the major river, represents a complex landscape allowing for some in-depth evaluation. Samples came from five localities (SP, Pha, NW, HR and NS) along the Wa River, representing different land use types, elevations, stream orders and positions relative to a man-made dam, namely Nam Wa Dam. Based on 10 microsatellite loci, the genetic diversity of samples in the sub-basin level was lower than that observed in a basin level. The impacts of hierarchical structure of the stream on genetic variation was less noticeable. However, pairwise differences in elevation and pairwise geographic distance among sites were important explanatory variables contributing to the existing spatial genetic variation of G. cambodgiensis. The genetic impacts of a recently built large concrete dam, namely Nam Wa dam, on G. cambodgiensis populations in the Wa River was not apparent. However, genetic monitoring would be needed to assess long-term genetic impacts of this dam. The third component examined the effects of physical barriers on genetic variation of G. cambodgiensis from six locations above and below three physical barriers in the Wa River system, namely Sapun Waterfall (SPU, SPL, Pun Stream), Nakham Dam (NKU, NKL, Mang Stream) and Suwanua Dam (SWU, SWL, Wa River). Based on 5 microsatellite loci, sample located above the Sapun Waterfall (SPU) was most genetically distinct from other remaining samples, including SPL (FST = 0.097-0.307), and had the lowest genetic diversity. For the remaining samples, samples above and below the weirs were more genetically similar although the vi genetic distance values were significant for all sample pairs (FST = 0.051 for SWU-SWL; 0.024 for NKU-NKL). STRUCTURE analysis revealed unequal admixture from the NK samples in SWU and SWL, suggesting some restriction to movement downstream. The results suggested a large genetic impact of a large barrier (> 10 meter high) and more subtle genetic impacts of smaller concrete weirs (< 5 meter high). The barrier size should be an important consideration for the design for fish-friendly structures. In summary, a contemporary structure of a river network, pairwise difference in elevation and stream orders greatly shaped genetic population strucutre of G. cambodgiensis n the upper Nan River system. A high degree of genetic admixture in each location in the upper Nan River Basin highlighting the importance of natural flooding patterns and possible genetic impacts of supplementary stocking. At the sub-basin level in the Nam Wa sub-basin, isolated headwater populations may undergo recent bottlenecks. Any habitat change to disrupt the connectivity of the river should be avoided. Insights obtained from this research advance our knowledge of the interactions between the complexity of a tropical stream system and the ecology of stream-dwelling fish as well as provide guidance for current conservation and restoration efforts for this species. vii CONTENTS Page ABSTRACT ............................................................................................................. iv CONTENTS ............................................................................................................ vii LIST OF TABLES .................................................................................................. ix LIST OF FIGURES ................................................................................................ xii CHAPTER 1 INTRODUCTION ............................................................................................ 1 Statements and Significance of the Problems .......................................... 1 Objectives ................................................................................................ 4 Hypothesis................................................................................................ 4 Contribution to knowledge ...................................................................... 4 Scope of the study .................................................................................... 5 2 LITERATURE REVIEWS ............................................................................... 7 Genetics and Population .......................................................................... 7 The formation of population structure ..................................................... 9 Microsatellite