Molecular Taxonomy and Evolution of Freshwater Crayfish of the Genus Cherax (Decapoda: Parastacidae) from northern Australia and New Guinea Rury Eprilurahman, B.Sc. A thesis submitted in fulfilment of the requirements for the degree of Master by Research Research Institute for the Environment and Livelihoods Charles Darwin University, Northern Territory, Australia Submitted August 2014 Accepted December 2014 ii Candidate Declaration I hereby declare that the work herein, now submitted as a thesis for the degree of Master by Research of the Charles Darwin University, is the result of my own investigations, and all references to ideas and work of other researchers have been specifically acknowledged. I hereby certify that the work embodied in this thesis has not already been accepted in substance for any degree, and is not being currently submitted in candidature for any other degree. Full Name : Rury Eprilurahman Signature Date : ......06/01/2015...... iii Abstract Taxonomic and evolutionary research of freshwater crayfish of the genus Cherax from tropical Australia and New Guinea (Papua-Indonesia and Papua New Guinea) is limited. The objective of this research is to place these Cherax species into a taxonomic and evolutionary framework following on from studies on Australian species using molecular genetic data with a special focus on species from Papua, Indonesia. This research presents an analysis of two data sets, one consisting of mt16S rRNA sequences from 134 samples and the second a combination of four genes (12S rRNA, 16S rRNA, COI, GAPDH) from a subset of these samples to test species boundaries and establish phylogenetic hypotheses for New Guinean and selected northern Australian species. These data supports the recognition of 25 Cherax species, including three recently described species and five new species discovered in this study from Papua, Indonesia. Two main clades were identified based on phylogenetic analyses. The New Guinean crayfish does not form a monophyletic group but share relationships with northern Australian species at different evolutionary depths, which is consistent with the geological history of the region. The diverse highland Cherax fauna of the Wissel Lakes form a well support monophyletic lineage but show minimal molecular divergence, which is at odds with their high morphological diversity. The results of this study fill a major gap in the understanding of the systematics and evolution history of the genus Cherax, one of the most diverse and widespread genera of freshwater crayfish. iv Contents Candidate Declaration ......................................................................................................... ii Abstract ................................................................................................................................ iii Contents ............................................................................................................................... iv List of Tables ....................................................................................................................... vi Table of Figures ................................................................................................................. vii Dedication ............................................................................................................................ ix Acknowledgements .............................................................................................................. x CHAPTER 1 ......................................................................................................................... 1 General Introduction ........................................................................................................... 1 1.1. Background ................................................................................................................ 1 1.2. Evolutionary History of Freshwater Crayfish ............................................................ 4 1.2.1. Family Parastacidae ............................................................................................ 5 1.2.2. Genus Cherax ..................................................................................................... 8 1.2.3. Northern Australian Cherax ............................................................................. 12 1.2.4. New Guinean Cherax ....................................................................................... 13 1.3. Molecular Systematics ............................................................................................. 18 1.3.1. DNA sequencing .............................................................................................. 19 1.3.2. Choices of molecular markers for phylogenetic inference ............................... 20 1.3.3. Phylogenetics.................................................................................................... 22 1.4. Geological History of Northern Australia and New Guinea Region........................ 25 1.4.1. Fluctuation of the sea levels and ancient river systems .................................... 26 1.4.2. Climate and geography of New Guinea and Australia ..................................... 28 1.4.3. Freshwater Habitats of New Guinea and northern Australia ........................... 33 1.4.3.1. Ayamaru and Aitinjo Lakes ...................................................................... 34 1.4.3.2. Wissel Lakes ............................................................................................. 36 1.4.3.3. Lake Habbema, Baliem and Ibele Rivers ................................................. 38 1.4.3.4. Fakfak and Bomberai Peninsula ............................................................... 40 1.4.3.5. Northern Australia .................................................................................... 41 1.5. Research problem statement and aims ..................................................................... 42 1.6. Thesis format ............................................................................................................ 43 CHAPTER 2 ......................................................................................................................................... 44 Evolution elevated: A phylogenetic analysis of New Guinean high land crayfish (Cherax: Decapoda: Parastacidae) reveals a decoupling of morphological and molecular evolution ............................................................................................................................ 44 2.1. Introduction ............................................................................................................. 44 2. 2. Materials and methods ............................................................................................. 48 2.2.1. Samples and sequences .................................................................................... 48 2.2.2. DNA extraction, PCR amplification, and sequencing ...................................... 50 2.2.3. Phylogenetic analyses....................................................................................... 51 2.2.4. Taxonomic analyses ......................................................................................... 52 2. 3. Results ..................................................................................................................... 61 v 2.3.1. Sequences ......................................................................................................... 61 2.3.2. Phylogenetic analysis ....................................................................................... 61 2.3.3. Molecular taxonomy......................................................................................... 66 2.3.4. Molecular clock analysis .................................................................................. 72 2. 4. Discussion ................................................................................................................ 75 2.4.1. Molecular taxonomy......................................................................................... 75 2.4.2. Molecular phylogeny ........................................................................................ 81 2.4.3. Zoogeography and divergence times ................................................................ 86 2. 5. Conclussion ............................................................................................................. 91 CHAPTER 3 ....................................................................................................................... 93 General Discussion ............................................................................................................. 93 3.1. Significance of research findings ............................................................................. 93 3.1.1. Cherax diversity – an overview ........................................................................ 98 3.2. Further study .......................................................................................................... 104 BIBLIOGRAPHY ............................................................................................................ 106 vi List of Tables Table 1.1. The recognised genera of the Parastacidae ...................................................... 6 Table 1.2. The classifications of the northern Australia species of Cherax essentially after Riek (1969), Short (1991; 1993), Short & Davie (1993), Austin (1996),