Molecular approaches to the assessment of biodiversity in limnic gastropods (Cerithioidea, Thiaridae) with perspectives on a Gondwanian origin DISSERTATION zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Lebenswissenschaftlichen Fakult¨at der Humboldt-Universit¨at zu Berlin von Dipl. Biol. France Gimnich Pr¨asident der Humboldt-Universit¨at zu Berlin: Prof. Dr. Jan-Hendrik Olbertz Dekan der Lebenswissenschaftlichen Fakult¨at: Prof. Dr. Richard Lucius Gutachter/innen: Prof. Dr. Hannelore Hoch Prof. Dr. Matthias Glaubrecht Prof. Dr. Stefan Richter Tag der m¨undlichen Pr¨ufung: 13.07.2015 Meinen Eltern Selbst¨andigkeitserkl¨arung Hiermit versichere ich an Eides statt, dass die vorgelegte Arbeit, abgesehen von den ausdr¨ucklich bezeichneten Hilfsmitteln, pers¨onlich, selbstst¨andig und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt wurde. Daten und Konzepte, die aus anderen Quellen direkt oder indirektubernommen ¨ wurden, sind unter Angaben von Quellen kenntlich gemacht. Diese Arbeit hat in dieser oder ¨ahnlichen Form keiner anderen Pr¨ufungsbeh¨orde vorgelegen und ich habe keine fr¨uheren Promotionsversuche unternom- men. F¨ur die Erstellung der vorliegenden Arbeit wurde keine fremde Hilfe, insbesondere keine entgeltliche Hilfe von Vermittlungs- bzw. Beratungsdiensten in Anspruch genom- men. Contents I Contents SUMMARY 1 ZUSAMMENFASSUNG 3 1 General Introduction 5 1.1Biogeography.................................. 5 1.2Studyspecies.................................. 6 1.3Speciesbiogeography.............................. 7 1.4Previouswork.................................. 9 1.5 Goals and structure of the present thesis ................... 10 2 General material and methods 12 2.1 Sampling ..................................... 12 2.1.1 Sampling sites .............................. 12 2.1.2 Sampling methods ........................... 13 2.2Geneticmethods................................ 14 2.2.1 DNAisolation,PCRandsequencing................. 14 2.3Analysesofmoleculardata........................... 15 2.3.1 Sequence assembly and alignment ................... 15 2.3.2 Phylogeneticanalyses.......................... 15 3 Australian thiarids - ancient relicts or recent invaders? 17 3.1Specificintroduction.............................. 17 3.2Specificmaterialandmethods......................... 19 3.2.1 Phylogeneticanalyses.......................... 19 3.2.2 Divergencetimeestimates....................... 21 3.3Results...................................... 23 3.3.1 Phylogeneticanalyses.......................... 23 3.3.2 MolecularDating............................ 27 3.4Discussion.................................... 30 4 A biogeographical revision of Australian thiarids 36 4.1Specificintroduction.............................. 36 4.2Specificmaterialandmethods......................... 37 4.2.1 Distribution maps ............................ 37 4.2.2 Photosofshells............................. 39 4.2.3 Geneticdata.............................. 39 4.3Results...................................... 40 4.3.1 Systematicrevision........................... 43 4.4Discussion.................................... 81 5 Historical DNA - Museum specimens as a source of genetic data 85 5.1Specificintroduction.............................. 85 5.2Specificmaterialandmethods......................... 87 5.2.1 Historical DNA extractions ...................... 87 5.2.2 Primerdesign&PCR.......................... 87 5.2.3 Phylogeneticanalysis.......................... 88 5.3Resultsanddiscussion............................. 89 5.3.1 Extraction ................................ 89 5.3.2 Primerdesignandphylogeneticanalysis............... 89 5.3.3 Systematicrelevanceoftheresults.................. 92 5.3.4 Summary and conclusion ........................ 94 6 Contrasting patterns revealed by AFLPs 95 6.1Specificintroduction.............................. 95 6.2Specificmaterialandmethods......................... 96 6.2.1 Sample choice .............................. 96 6.2.2 Amplified Fragment Length Polymorphism (AFLP) ......... 96 6.2.3 Automaticmasking-markerselection................101 6.2.4 Analyses.................................104 6.3Results......................................104 6.3.1 AMAREoutput.............................104 6.3.2 Neighbour-joining tree .........................105 6.4Discussion....................................107 7 General discussion 109 REFERENCES 117 APPENDIX 135 A List of sampling localities sorted by sampling year 135 B Mastermix and PCR profiles 147 C Additional analyses of phylogenetic trees 149 D AFLP Analyses 163 E Historical DNA sample list 171 F Abbreviations 173 Contents III List of Figures 1 Climate map of Australia ............................ 8 2 Disjunct distribution of “Thiara” australis and Plotiopsis balonnensis ... 9 3 Sampling sites in Australia 2011 ........................ 12 4 Chart of sampling treatment .......................... 13 5 RAxML topology based on combined COI and 16S sequences ........ 24 6 RAxML topology based on combined 28S and H3 sequences ........ 25 7 BEASTchronogrambasedon28Sdata.................... 28 8 BEAST chronogram based on combined COI and 16S sequences ...... 29 9 StagesofGondwanaBreakup......................... 31 10 Species-Areacladogram............................ 33 11 Freshwater zoogeographical regions ...................... 37 12 DrainagesystemsinAustralia......................... 38 13 RAxMLtopologybasedonCOIsequences.................. 40 14 Chartsshowingtheextentofspeciesmis-assignments............ 41 15 Distribution map of Thiara amarula ..................... 44 16 Distribution map of “Thiara” australis .................... 45 17 Shells of “Thiara” australis .......................... 46 18 Shells of Thiara rudis .............................. 50 19 Distribution map of Thiara rudis ....................... 50 20 M. tuberculata and “S.” denisoniensis from locality where they co-occur . 52 21 Distribution map of Melanoides tuberculata .................. 52 22 Shell of Melanoides tuberculata from the East coast of Australia . 53 23 Distribution map of Melasma onca ...................... 55 24 Shells of Melasma onca ............................. 55 25 Distribution map of Plotiopsis balonnensis .................. 57 26 Shells of Plotiopsis balonnensis ........................ 59 27 P. balonnensis and “Thiara” australis from locality where they co-occur. 59 28 Distribution map of Pseudoplotia scabra ................... 66 29 Shells of Pseudoplotia scabra .......................... 66 30 Distribution map of Ripalania queenslandica ................. 68 31 Distribution map of Sermyla venustula .................... 70 32 Shells of Sermyla venustula .......................... 72 33 Distribution map of Stenomelania cf. aspirans ................ 74 34 Distribution map of “Stenomelania” denisoniensis .............. 76 35 Shells of “Stenomelania” denisoniensis .................... 76 36 Geographical range overlap of “Thiara” australis and P. balonnensis .... 83 37 PrimerpositionsinhistoricalDNAapproach................. 88 38 Shellsbeforeandaftertheextractionprocedure............... 89 39 Bayesian consensus phylogram based on 16S sequences including hDNA . 91 40 Occurrences and shells of R. queenslandica in and outside of Australia . 92 41 AFLPrestrictionandligation......................... 97 42 AFLPadaptors................................. 98 43 AFLP selective amplification .......................... 99 44 FlowchartofAMAREprogram........................103 45 Neighbour-joining tree of AFLP data .....................106 46 Summary of geographic ranges .........................114 47 BayesianinferencephylogrambasedonconcatenatednDNA........149 48 MaximumParsimonytopologybasedonconcatenatedmtDNA.......150 49 Majorityruleconsensustreebasedonconcatenatednucleardata......151 50 Bayesian inference based on combined mtDNA dataset ...........152 51 Bayesian inference phylogram based on the concatenated data (combined mitochondrialandnuclearDNA).......................153 52 BEAST chronogram based on 28S sequences using one calibration point . 154 53 BEAST chronogram based on combined mtDNA sequences using one cali- brationpoint..................................155 54 RAxMLtreebasedon16SsequencesofthehDNAdataset.........156 56 Maximum parsimony tree based on 16S sequences of the hDNA dataset . 157 55 Maximum likelihood tree based on 16S sequences of the hDNA dataset . 158 57 RAxMLtreebasedonthelargedatasetof16Ssequences..........162 58 PCR profile for preselective amplification in AFLP analysis .........163 59 PCR profile for selective amplification in AFLP analysis ..........164 Contents V List of Tables 1 Biogeography of Cerithioidean families .................... 18 2 Taxa included in the phylogenetic analyses .................. 20 3 Fossil record outline of Potamididae and Hemisinidae ............ 22 4 Additional outgroups for BEAST analyses .................. 23 5 ResultsofaBayesionestimationofdivergencetimes............. 27 6 Listofcorrectandwrongpre-IDspertaxa.................. 42 7 Material examined of Thiara amarula ..................... 44 8 Material examined of “Thiara” australis ................... 47 9 Material examined of Thiara rudis ...................... 51 10 Material examined of Melanoides tuberculata ................. 53 11 Material examined of Melasma onca ..................... 56 12 Material examined of Plotiopsis balonnensis ................. 60 13 Material examined of Pseudoplotia scabra ................... 67 14