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Molecular Analysis of Necrophagous Diptera of the Iberian Peninsula

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Molecular Analysis of Necrophagous Diptera of the Iberian Peninsula UNIVERSIDAD DE MURCIA ESCUELA INTERNACIONAL DE DOCTORADO Molecular Analysis of Necrophagous Diptera of the Iberian Peninsula Análisis Molecular de Dípteros Necrófagos de la Península Ibérica D. Alberto Fuentes López 2018 Molecular analysis of necrophagous Diptera of the Iberian Peninsula Análisis molecular de dípteros necrófagos de la península Ibérica Tesis Doctoral Alberto Fuentes López 2018 Escuela Internacional de Doctorado Universidad de Murcia Directores: Dr. José Galián Albaladejo Dr. Elena Romera Lozano Support projects El trabajo presentado en esta tesis se ha realizado en el Área de Biología Animal del Departamento de Zoología y Antropología Física de la Universidad de Murcia bajo la dirección de los Doctores José Galián Albaladejo y Elena Romera Lozano, como parte de los proyectos “Análisis molecular de dípteros necrófagos de la península Ibérica” (CGL2011-25298) financiado la Secretaría de Estado de Investigación, Desarrollo e Innovación y “Animal Phylogeny and Evolution” (19908-GERM-15) financiado por la Fundación Séneca en la convocatoria de Grupos de Excelencia Regional. El autor ha disfrutado de un contrato asociado a un proyecto CDTI “Selección y Evaluación de Semioquímicos para la Atracción de Dípteros” (IDI-20160234). Durante el desarrollo del mismo ha realizado una estancia de tres meses en el Departamento de Biología Animal de la Facultad de Ciencias de la Universidad de Lisboa, (Portugal), bajo la supervisión de la Doctora María Teresa Rebelo, y con el apoyo económico del programa Erasmus + Prácticas de la Universidad de Murcia. Contents Resumen .................................................................................................................................... 13 Introduction ............................................................................................................................... 31 Objectives .................................................................................................................................. 43 Chapter I. Molecular identification of forensically important fly species in Spain using COI barcodes ..................................................................................................................................... 49 Chapter II. DNA-based and Taxonomic identification of forensically important Sarcophagidae (Diptera) in Southeastern Spain. ..................................................................... 95 Chapter III. Diversity of the forensically important fly species Calliphora vicina, (Diptera: Calliphoridae) in the Iberian Peninsula using COI, 16S and ITS2 sequences ......................... 115 Chapter IV. Species of the genus Lucilia (Diptera: Calliphoridae) with forensic interest in northern Spain: a molecular and morphogeometrical approach .......................................... 133 Conclusions .............................................................................................................................. 173 Bibliography ............................................................................................................................. 177 Tables Index Table 1.1: Correspondence between preliminary identification (morphology or BLAST) with BOLD Identification of forensically important fly species. Number of specimens for each species and Barcode Index Number (BINs) assigned by BOLD are given. Samples that have finally change the first identification are shaded ...................................................................... 59 Supplementary Table 1.1: Locality, GenBank reference, BIN assigned by BOLD and final identification of all new sequences of the present study. ................................................... 70 Supplementary Table 1.2: Code assigned by BOLD, location of the samples and recognized species used in phylogeographic analysis of L. caesar and L. illustris. ............................... 82 Supplementary Table 1.3: Code assigned by BOLD, location of the samples and recognized species used in phylogeographic analysis of S. lehmanni and S. variegata. .................... 89 Table 2.1: Species collected, number of forensic case, location name, place of the corpse/trap, number of specimens and date of collection. ...................................................... 99 Table 3.1: Sequences used to build the matrix of the three genes, total of sequences for each matrix and localities that where the sequences come from. ................................................. 121 Table 4.1: Description of the landmarks used on the wings. .................................................. 139 Table 4.2: Description of the landmarks used on the heads. ................................................. 140 Table 4.3: Geographical location and sex of the specimens. Analyses in which each one is included is indicated by ●. ....................................................................................................... 141 Figures Index Figure 1: Chapter 5 of Sun Tźu's Chinese book on Forensic Medicine deals with a case stabbing solved by use of insects. From Benecke (2001) .................................................................................................... 31 Figure 2: The typical blow fly life cycle. Times given in the chart are for 21 degrees (by The Cleveland Natural History Museum, 2011 ................................................................................................................. 33 Figure 3: Pig carcass in the different stages of decomposition. From left to right: Fresh, Bloat, Active Decomposition, Advanced Decomposition and Remains. From www.academia.dk ............................. 34 Figure 4: Isomegalen diagram of Aldrichina graham larvae from hatching to peak feeding state. Time is plotted against temperature where each line represents developmental larval length in mm (3-19). From Wang et al. (2018) ............................................................................................................................ 35 Figure 5: Schematic use of the DNA barcoding technique. From www.biome-id.com .......................... 37 Figure 6: Structure of the mitochondrial DNA molecule. From Zhao et al, (2013) ................................. 38 Figure 7: Example of landmarks position. From Cheverud (2017) ........................................................... 40 Figure 1.1: Chosen sampling localities in Spain. ....................................................................................... 53 Figure 1.2: NJ tree of forensically important fly species. Bars show different ID and species delimitation methods. Discrepancies among them are showed shaded. Identification changes are shown in right margin. ............................................................................................................................... 63 Figure 1.3: Haplotype network of L. caesar (red) and L. illustris (green). Asterisk indicates the haplotypes recovered in our sampling. ..................................................................................................... 64 Figure 1.4: Haplotype network of S. variegata (greenish colours) and S. lehmanni (reddish colours). Asterisk indicates the haplotypes recovered in our sampling, Sequences located in an unexpected place according to its identification in database are indicated by hashtag. The haplotype colour indicates country of origin of the sequence ........................................................... 68 Figure 2.1: Location of sampling points. ................................................................................................. 100 Figure 2.2: NJ tree of forensically important Sarcophagidae species. Bars show different ID and species delimitation methods. Discrepancies among them are showed shaded. .............................................. 103 Figure 2.3: Species distribution in the studied area .............................................................................. 106 Figure 2.4: Haplotype network of S. tibialis ............................................................................................ 107 Figure 2.5: Haplotype network of S. argyrostoma. ................................................................................ 108 Figure 3.1: Sampling localities from Spain and Portugal. The localities are: (1) Barañain, (2), Lubiano, (3) San Sebastián, (4) Arrigorriaga, (5) Santander, (6) Oviedo, (7) Navia, (8) Lugo, (9) Betanzos, (10) Pontevedra, (11) Ourense, (12) Fontoura, (13) Vila Real, (14) Bragança, (15) Porto, (16) Montemor-o- Novo, (17) Mérida, (18) São Luis, (19) Moura, (20) Mértola, (21) Sagres, (22) Faro, (23) Punta Umbría, (24) Jabugo and (25) Dos Hermanas. ...................................................................................................... 118 Figure 3.2: Geographical areas chosen for the RASP analyses. Names and localities of each areas are: “A” Basque Country area (Barañain, Lubiano, San Sebastián and Arrigorriaga), “B” Asturias-Cantabria area (Santander, Oviedo and Navia), “C” Galicia area (Lugo, Betanzos, Pontevedra and Ourense), “D” North Portugal area (Fontoura, Bragança, Vila Real and Porto), “E” Extremadura area (Mérida, Montemor-o-Novo and Moura), “F” South Portugal area (São Luis, Mértola, Sagres and Faro) and “G” Andalucía area (Jabugo, Punta Umbría and Dos Hermanas).................................................................. 120 Figure 3.3: Haplotype network build with PopArt for the ITS2 gen. ..................................................... 122 Figure 3.4: Haplotype
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