Haplotype diversity and phylogenetic relationships among the Iberian barbels (Barbus,Cyprinidae) reveal two evolutionary lineages

The phylogenetic relationships and haplotype diversity of all Iberian barbels were examined by analyzing the complete mitochondrial cytochrome b gene sequence (1141 bp) of 72 specimens from 59 Iberian localities. Phylogenetic findings demonstrated a clear distinction between two mitochondrial lineages and confirmed the existence of two previously considered subgenera: Barbus and Luciobarbus: The first subgenus, Barbus, is represented on the Iberian Peninsula by Barbus haasi and Barbus meridionalis. The second subgenus, Luciobarbus, includes the remaining endemic Iberian species: Barbus comizo, Barbus bocagei, Barbus microcephalus, Barbus sclateri, Barbus guiraonis, and Barbus graellsii. Mean haplotype divergence between these subgenera was 10.40%, providing evidence of a clear subdivision within the Iberian barbels. Our results conflict with those reported in a recent study, based on 307 cytochrome b base pairs, that failed to identify any division within the genus Barbus in the Iberian Peninsula. The inclusion of nine further species belonging to this genus (used as outgroups) allowed us to establish a closer relationship of the Iberian species of the subgenus Barbus with other European taxa than with the Iberian Luciobarbus, which was found to cluster with North African, Caucasian, and Greek species. At the population level, no biogeographic structure was shown by specimens of each species (only 5.98% of the variation was attributable to differences among populations of each species). Given the discrete amount of divergence found among the Luciobarbus species, the formation of current hydrographic basins during the Plio-Pleistocene seems to have played a major role in their isolation and evolution.     

Morphological and biometric revision of the cleithra, opercular and pharyngeal bones of Iberian teleosts belonging to the genus Barbus (Pisces, Cyprinidae)

Species of the genus Barbus present in the Iberian Peninsula are an interesting study group, due to its diversity and complex taxonomic position. In this work, 299 specimens of eight species were studied. All of them are endemic to the Iberian Peninsula, except for Barbus meridionalis. Cleithra, opercular and pharyngeal bones were extracted from each specimen. These bones were morphologically compared and measured, obtaining biometric indices. From these indices, the biometric differences of each bone were analysed with a principal component analysis. Later, a principal component analysis and a discriminant analysis were performed considering the three bones together. The morphological differences and similarities are congruous with the biometric results. In addition, this osteological comparison partially agrees with the present taxonomic position of these species, being a contribution to the systematics and phylogeny of genus.     

Molecular identification (RAPD) of the eight species of the genus Barbus (Cyprinidae) in the Iberian Peninsula

Random amplified polymorphic DNA (RAPD) analysis has been used to identify the eight Barbus species of the Iberian Peninsula, whose morphological differentiation is difficult. Ten random primers were employed to generate RAPD markers. Different RAPD profiles were observed for the different species. Four species-specific markers were found in B. bocagei , seven in B. comizo , five in B. graellsii , three in B. guiraonis , eight in B. haasi , 13 in B. meridionalis , four in B. microcephalus and four in B. sclateri. Evidence is presented that RAPD markers constitute useful tools for accurate taxonomic identification of Spanish barbels which is one of the first prerequisites in effective conservation programmes.     

Recent radiation of Iberian barbel fish (Teleostei, Cyprinidae) inferred from cytochrome b genes

Nucleotide sequence fragments of the mitochondrial DNA gene encoding cytochrome b were examined in 26 individuals belonging to the seven species of Barbus endemic to the Iberian Peninsula: Barbus haasi, B. bocagei, B. graellsii, B. sclateri, B. comiza, B. guiraonis, and B. microcephalus. Six of the seven currently recognized species can be distinguished on the basis of their cytochrome b nucleotide sequences. Sequence divergence estimates for Spanish species of Barbus (0-6.5%) are, in general, low in comparison to those reported for other fish species, and hybrid individuals were found. All of these observations suggest a recent radiation. The inferred phylogenetic tree has two main clades, one including B. graellsii, B. guiraonis, and B. microcephalus, and the other the remaining species groups.     

Rapid radiation of the Mediterranean Luciobarbus species (Cyprinidae) after the Messinian salinity crisis of the Mediterranean Sea, inferred from mitochondrial phylogenetic analysis

Phylogenetic relationships of 64 freshwater Barbus s.s. species distributed in basins around the Mediterranean Sea were assessed using cytochrome b sequences. Our results are in concordance with previous morphological and genetic studies, which proposed that these species belong to two major lineages (or subgenera): Barbus and Luciobarbus . We were particularly interested in phylogenetic relationships among species of the Luciobarbus lineage that are primarily found in the southern Mediterranean region from the Iberian Peninsula to the Middle East. In the Luciobarbus lineage, species that were previously attributed to the Capoeta genus were clustered. In this study, we observed short internodes between monophyletic groups having a geographical agreement around the Mediterranean. However, groups from the opposite sides of the Mediterranean Sea (Iberian Peninsula– Capoeta , north-western Africa–Middle East) seem to be phylogenetically close. We therefore infer that rapid radiation of Luciobarbus species in the Late Miocene better fits our data rather than gradual founder events in the southern Mediterranean. We propose that the biogeographical event along an east–west route, responsible for the present distribution of Luciobarbus species, was the 'Lago Mare' phase of the Mediterranean Sea that provided a rapid dispersal route over extensive distances. This provides new insights into the speciation pattern of this group, and may be of general use in the study of freshwater species in these regions.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 207–222.     

MtDNA markers reveal the existence of allopatric evolutionary lineages in the threatened lampreys <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> (L.) and <Emphasis Type="Italic">Lampetra planeri</Emphasis> (Bloch) in the Iberian glacial refugium

The Iberian Peninsula has been identified as an important glacial refugium during the Pliocene and Pleistocene epochs for the genus Lampetra, providing intermittent refuge and postglacial opportunities for colonization and expansion. We used mitochondrial DNA markers to investigate the processes that have shaped present-day genetic constitution of the genus Lampetra within the Iberian Peninsula. We surveyed 1,173 bp of the cytochrome b gene and 829 bp of the genes ATPase subunits 6 and 8 in 233 individuals of Lampetra fluviatilis (L.) and Lampetra planeri (Bloch) from 21 localities along their distribution range in the Iberian Peninsula. We identified four highly divergent allopatric evolutionary lineages that evolved by fragmentation during the Pliocene and Pleistocene likely driven by environmental factors, namely regional geomorphic events. The high level of genetic divergence between the four lineages suggests that sufficient time has apparently passed by to originate a complex of incipient or cryptic resident species and allows the definition of four evolutionary significant units (ESUs) for L. planeri and one ESU for L. fluviatilis. These findings have important consequences for the interpretation of refugia biological diversity and have major implications on the conservation of these threatened species.     

Phylogenetic relationships of Iberian cyprinids: systematic and biogeographical implications.

The phylogenetic relationships among all Iberian endemic cyprinids were inferred using the complete nucleotide sequence of the cytochrome b gene. The inferred molecular phylogeny included representatives from Central European, Asian and North African species, and is highly congruent with previous phylogenies based on osteological characters. Iberian cyprinids were grouped into only five, very speciose lineages (with the exception of the monotypic Anaecypris): Barbus, Luciobarbus, Chondrostoma, Leuciscus and Anaecypris. The existence of such a relatively small number of Iberian cyprinid lineages can be explained by the historical isolation of the Iberian Peninsula. North African and Asian barbels are the sister group of Iberian Luciobarbus, supporting a south-eastern route of colonization of the Iberian Peninsula for this subgenus. With leuciscins, Anaecypris hispanica was considered a relict species as it could not be related to any other Iberian cyprinid. The phylogenetic relationships among the main lineages of Iberian cyprinids based on cytochrome b sequence data supported the traditional division of the Cyprinidae into two subfamilies: Cyprininae and Leuciscinae.     

Evolutionary and biogeographical patterns within Iberian populations of the genus Squalius inferred from molecular data

Origins and biogeography of the chub, genus Squalius (formerly within the genus Leuciscus), in the Iberian Peninsula were inferred from comparison between patterns of geographic distribution and phylogenetic relationships among populations belonging to 14 European Squalius species. The phylogeny recovered was based on the complete sequence of the mitochondrial cytochrome b gene. Squalius species were grouped into three major clades. The basal clade included species distributed across the ancient Paratethys Sea. The second clade included species from Central and East Europe and the northern areas of the Mediterranean basin towards Minor Asia. The third clade included species from the Mediterranean Peninsulas (Iberian, Italy, and Balkans). The Iberian Squalius species do not constitutes a monophyletic group. Our data indicate that the Iberian Peninsula was colonized at least twice by two different monophyletic lineages, a meridional group and a Central Europe group. The amount of species diversity found in the Iberian Peninsula and the phylogenetic relationships among these species, together with their geographic distribution, suggest that the Central Europe lineage colonized the Iberian Peninsula at a latter time. Our data indicate that the northeastern Iberian lineage is phylogenetically close to Greek populations of Squalius cephalus, while the second lineage formed a monophyletic group including Squalius pyrenaicus, Squalius carolitertii, Squalius aradensis, and Squalius torgalensis. The speciation process that generated these species and the geographic structure of their populations, principally in S. pyrenaicus, can be attributed to paleogeographical events like the ancient endorrheism and the development of hydrographic basins.     

Phylogeny and evolution of the genus Barbus in the Iberian Peninsula as revealed by allozyme electrophoresis

Variation of 14 enzyme systems encoded by 31 presumptive loci in different barbel species was studied using starch gel electrophoresis. Eighteen samples representing 11 Barbus tetraploid taxa were taken, including 10 populations from the Iberian Peninsula, six from other parts of western and southern Europe, one from northern Africa and one diploid species as outgroup from Asia Minor. The genetic analysis reassessed of the taxonomic status of the Iberian barbels into two distinct species groups. The first group included B. bocagei, B. comiza, B. graellsii, B. gulraonis, B. microcephalus , and B. sclateri , that aligned with B. callensis from northern Africa and with B. apoensis from Asia Minor; the other group included B. haasi and B. merldlonahs that was related to the European species, B. barbus, B. plebejus and B. peloponnesius . These groups are probably not monophyletic. It is suggested that the isolation of the Iberian Peninsula from Europe since the Oligocene-Miocene may explain the genetic affinities of the Iberian barbels with those of North African rather than with the European group.     

The Iberian Peninsula: ancient history of a hot spot of mite harvestmen (Arachnida: Opiliones: Cyphophthalmi: Sironidae) diversity

The Iberian Peninsula represents a hot spot of cyphophthalmid (mite harvestman) disparity, with four of the eight genera currently recognized in the family Sironidae represented in the region – a generic diversity and morphological disparity not found in any other region of the World so far. From these, two genera (Iberosiro and Odontosiro) are monotypic, and are restricted to the western side of the peninsula. Parasiro is restricted to the north‐east region, from the Catalonian Coastal Ranges and both sides of the Eastern Pyrenees, in areas where the annual rainfall surpasses 1000 mm, and mostly restricted to areas with Paleozoic and Variscan rocks, with other species of the genus extending to Corsica, Sardinia, and the Italian Peninsula. A second species of the genus Paramiopsalis, Paramiopsalis eduardoi sp. nov. from Fragas do Eume, is described here along with a re‐diagnosis of the genus. Paramiopsalis species, together with Odontosiro, inhabit the north‐west corner of the Iberian Peninsula, an area with some of the highest recorded annual rainfall, and with Paleozoic rocks from the Iberian Massif or Variscan granitoid rocks. A phylogenetic analysis of the members of the family Sironidae using four molecular markers, despite not including all of the Iberian genera, clearly shows the non‐monophyly of the Iberian Cyphophthalmi, indicating that the Iberian Peninsula is home to multiple ancient lineages of mite harvestmen. The two Paramiopsalis species form a sister clade to the Balkan genus Cyphophthalmus, whereas Parasiro constitutes the first lineage of the sironids represented.     

Comparative phylogeography of endemic cyprinids in the south-west Iberian Peninsula: evidence for a new ichthyogeographic area

The comparative phylogeography and evolutionary history of three native cyprinid genera, Squalius (subfamily Leuciscinae), Chondrostoma (subfamily Leuciscinae) and Barbus (subgenus Luciobarbus ; subfamily Cyprininae), were examined focusing mainly in the South-Western region of the Iberian Peninsula, where recently described endemic species are present with considerably restricted distribution areas. In order to accomplish that the variation at the complete cytochrome b gene (1140 bp) was analysed for specimens from the South-Western region, and also for representatives of the three genera from all over the Iberian Peninsula. Data indicate different evolutionary histories, with distinct time periods of colonization between the two cyprinid subfamilies in the Iberian Peninsula. Four new Iberian ichthyogeographic areas are accordingly proposed based on congruent phylogeographic and geological evidences: the South-Western, the South-Eastern, the Atlantic and the Mediterranean. Evidence was provided for the older isolation of the South-Western area in the Miocene during the Endorheic Drainages phase, designating a clearly defined and distinct ichthyogeographic area. A new molecular clock calibration is proposed for the subgenus Luciobarbus .     

Understanding the biogeography of a group of earthworms in the Mediterranean basin--the phylogenetic puzzle of Hormogastridae (Clitellata: Oligochaeta)

Traditional earthworm taxonomy is hindered due to their anatomical simplicity and the plasticity of the characteristics often used for diagnosing species. Making phylogenetic inferences based on these characters is more than difficult. In this study we use molecular tools to unravel the phylogeny of the clitellate family Hormogastridae. The family includes species of large to mid-sized earthworms distributed almost exclusively in the western Mediterranean region where they play an important ecological role. We analyzed individuals from 46 locations spanning the Iberian Peninsula to Corsica and Sardinia, representing the four described genera in the family and 20 species. Molecular markers include mitochondrial regions of the cytochrome c oxidase subunit I gene (COI), 16S rRNA and tRNAs for Leu, Ala, and Ser, two nuclear ribosomal genes (nearly complete 18S rRNA and a fragment of 28S rRNA) and two nuclear protein-encoding genes (histones H3 and H4). Analyses of the data using different approaches corroborates monophyly of Hormogastridae, but the genus Hormogaster is paraphyletic and Hormogaster pretiosa appears polyphyletic, stressing the need for taxonomic revisionary work in the family. The genus Vignysa could represent an early offshoot in the family, although the relationships with other genera are uncertain. The genus Hemigastrodrilus is related to the Hormogaster elisae complex and both are found in the Atlantic drainage of the Iberian Peninsula and France. From a biogeographic perspective Corsica and Sardinia include members of two separate hormogastrid lineages. The species located in Corsica and Northern Sardinia are related to Vignysa, whereas Hormogaster pretiosa pretiosa, from Southern Sardinia, is closely related to the Hormogaster species from the NE Iberian Peninsula. A molecular dating of the tree using the separation of the Sardinian microplate as a calibration point (at 33 MY) and assuming a model of vicariance indicates that the diversification of Hormogastridae may be ancient, ranging from 97 to 67 Ma.     

Diversification within glacial refugia: tempo and mode of evolution of the polytypic fish Barbus sclateri

A diversity of evolutionary processes can be responsible for generating and maintaining biodiversity. Molecular markers were used to investigate the influence of Plio-Pleistocene climatic oscillations on the evolutionary history of taxa restricted to the freshwaters of a classical glacial refugium. Population genetic, phylogenetic and phylogeographical methods allowed the inference of temporal dynamics of cladogenesis and processes shaping present-day genetic constitution of Barbus sclateri , a polytypic taxon found in several independent river drainages in southern Iberian Peninsula. Results from different analyses consistently indicate several range expansions, high levels of allopatric fragmentation, and admixture following secondary contacts throughout its evolutionary history. Using a Bayesian demographical coalescent model on mitochondrial DNA sequences calibrated with fossil evidence, all cladogenetic events within B. sclateri are inferred to have occurred during the Pleistocene and were probably driven by environmental factors. Our results suggest that glaciation cycles did not inhibit cladogenesis and probably interacted with regional geomorphology to promote diversification. We conclude that this polytypic taxon is a species complex that recently diversified in allopatry, and that Pleistocene glaciation–deglaciation cycles probably contributed to the generation of biological diversity in a classical glacial refugium with high endemicity.     

Phylogeography and species delineation of the genus Phoxinus Rafinesque, 1820 (Actinopterygii: Leuciscidae) in the Iberian Peninsula

The genus Phoxinus is comprised of at least 15 currently recognized species inhabiting Eurasia. Morphological traits have been traditionally used to delineate species in Phoxinus; however, the high level of phenotypic plasticity observed in the genus has confounded this process. Molecular genetic analyses have revealed a higher than expected genetic structure within Phoxinus. Here, we analyzed both nuclear and mitochondrial molecular genetic markers to infer the phylogeography and divergence times of Phoxinus in the Iberian Peninsula. Our results show that the Iberian lineages of Phoxinus were polyphyletic. They also support the co‐existence of three species in the Iberian Peninsula, two corresponding to two previously recognized species (Phoxinus bigerri and Phoxinus septimaniae) and a third undescribed species (Phoxinus sp.). Phoxinus bigerri is structured into western Cantabrian, eastern Cantabrian, and Artibai basins. We hypothesize that this structure is a consequence of glaciation–deglaciation cycles during the Pleistocene. While the presence of P. septimaniae in the Iberian Peninsula is possibly the result of human translocation, that of Phoxinus sp. in lower Ebro rivers may be attributed to past fluvial captures. Our study represents the first report to show a relationship among Phoxinus populations from central Pyrenean rivers of Spain and France. Furthermore, we found genetic hybridization between Phoxinus sp. and P. septimaniae in the shared localities, a likely consequence of anthropogenic activities. Overall, our findings provide insight into the genetic structure of Iberian Phoxinus populations, including the presence of an undescribed species and the putative introduction of some species that may have implications for conservation.     

Biogeographic hypothesis explaining the diversity of the genus Amphimallon Berthold, 1827, in the Mediterranean Basin (Coleoptera,Scarabaeidae, Melolonthinae,Rhizotrogini)

In this paper, the analysis of the current distribution of species, or species-groups, of the Western–Palaearctic genus Amphimallon Berthold, 1827, based on a phylogenetic hypothesis recently provided and relevant paleogeographical events that occurred in the Mediterranean Basin from Late Paleogene (35 Myr BP) to the present, supports a biogeographical scenario to explain species diversity within this genus. The center of origin of this genus is hypothesized to be in the Iberian Peninsula. Microplate fragments drifting from the Iberian Peninsula, successive landmass suture events between the Eastern and Western Mediterranean Basins, separation of Tethys and Paratethys Oceans, re-establishment of marine connections, uplift of the Alps, and drying out of the Mediterranean are hypothesized as the principal events allowing dispersal of populations throughout Mediterranean Basin and biogeographical vicariances within the genus Amphimallon.     

A new species of Haplophyllum A. Juss. (Rutaceae) from the Iberian Peninsula: evidence from morphological, karyological and molecular analyses

BACKGROUND AND AIMS: The discovery of a new species, Haplophyllum bastetanum F.B. Navarro, V.N. Suárez-Santiago & Blanca sp. nov., in the south-east of Spain has prompted the comparative study of species of the Iberian Peninsula, and others related, through morphological, cytogenetic, molecular, distributional and ecological characterization. METHODS: The morphological study involved a quantitative analysis of the species present in the Iberian Peninsula and a comparative analysis of the morphological characteristics between H. bastetanum and other related species. Mitotic analyses were made with root meristems taken from germinating seeds. Phylogenetic analyses of the internal transcribed spacer sequences of nuclear ribosomal DNA were performed using neighbour-joining (NJ) and maximum-parsimony methods. KEY RESULTS: Haplophyllum bastetanum is a diploid species (2n = 18) distinguished primarily for its non-trifoliate glabrous leaves, lanceolate sepals, dark-green petals with a dorsal band of hairs, and a highly hairy ovary with round-apex locules. The other two Iberian species (H. linifolium and H. rosmarinifolium) are tetraploid (2n = 36) and have yellow petals. Both phylogenetic methods generated a well-supported clade grouping H. linifolium with H. rosmarinifolium. In the NJ tree, the H. linifolium-H. rosmarinifolium clade is a sister group to H. bastetanum, while in the parsimony analysis this occurred only when the gaps were coded as a fifth base and the characters were reweighted according to the rescaled consistency index. This latter group is supported by the sequence divergence among taxa. CONCLUSIONS: The phylogenies established from DNA sequences together with morphological and cytogenetic analyses support the separation of H. bastetanum as a new species. The results suggest that the change in the number of chromosomes may be the key mechanism of speciation of the genus Haplophyllum in the Iberian Peninsula. An evolutionary scheme for them is propounded.     

Genetic analysis based on plastidial and ribosomal sequences of the endemic bi-edaphic taxon Jurinea pinnata (Lag.) DC. (Compositae) in the Guadix-Baza Basin

Jurinea pinnata (Lag.) DC. is one of the three species of the genus that grows in the Iberian Peninsula. This species grows on gypsum and dolomite, substrates rich in endemic taxa. Actually, the genus Jurinea Cass. is particularly rich in endemic taxa associated with special substrates. The island-like distribution of J. pinnata makes it an interesting case for the study of genetic differentiation processes. The geographical proximity of J. pinnata and J. humilis makes this evolutionary puzzle even more difficult to solve. These facts could affect the genetic attributes of the species as far as diversity and differentiation are concerned. Our field research involved the sampling of individuals from eight populations of J. pinnata. Of them, six were located in the Guadix-Baza Basin. We used sequences of ribosomal and plastid DNA to perform the genetic analyses. Results revealed no differences between individuals occurring on the two kinds of substrates and evidences of hybridization between J. pinnata and J. humilis. Furthermore, the phylogenetic analysis revealed a possible polyphyletic origin for the adaptation to special substrates within the Jurinea genus, while monophyly was observed in species from the Iberian Peninsula. This could indicate a more general adaptation to arid or saline environments.     

Genetic Identification of Western Mediterranean Sturgeons and its Implication for Conservation

To date, the only native sturgeon species in Western Europe was believed to be Acipenser sturio. However, this species is currently restricted to the Gironde River (Southern France), and it poses serious difficulties for rearing in captivity and for using in recovery programme. Furthermore, it has been questioned whether A. sturio is in fact the only species within the rivers of Western Europe, as A. naccarii, a species previously considered endemic of the Adriatic region, has been reported from the Iberian Peninsula in recent years. Here, we have used forensic techniques to obtain DNA from several museum specimens of sturgeons caught in the Spanish Guadalquivir River and in other European rivers. We analysed DNA sequences from these specimens for five genetic markers (three nuclear and two mitochondrial regions), which were subsequently compared with sequences obtained from A. sturioand A. naccarii. Our study demonstrates that A. naccarii coexists withA. sturio, from the Adriatic Sea to the Iberian Peninsula, a finding that could be taken into account in future sturgeon recovery programmes in Western Europe.     

Phylogenetic relationships among the Ibero-African cobitids (Cobitis, cobitidae) based on cytochrome b sequence data

We estimated the phylogenetic relationships of all Ibero-African spined loaches of the genus Cobitis using the complete mitochondrial cytochrome b gene (1140bp). We analysed 93 individuals of seven cobitid species found in all the principal drainages of the Iberian Peninsula and North Africa. A molecular phylogeny was used to revise current systematics of the Ibero-African Cobitis species and to infer a biogeographical model for Cobitis in the Western Mediterranean area during the Cenozoic period. Phylogenetic analysis provided support for the monophyly of two mtDNA clades: Clade A or Italian Clade with the Italian species (C. bilineata, C. zanandreai), and Clade B or the Ibero-African Clade. The Ibero-African Clade included all species endemic for the Iberian Peninsula (C. vettonica, C. calderoni, and C. paludica) and North Africa (C. maroccana). The species C. paludica does not constitute a natural group, and could be divided into at least four monophyletic mtDNA lineages with moderate to high bootstrap values and posterior probability support. Phylogenetic relationships of the Ibero-African species were not resolved satisfactorily, but in all analyses C. calderoni from Northern Iberian Peninsula was basal. We have re-calibrated a molecular clock for the genus Cobitis (0.68% per million year by pairwise) using populations inhabiting both sides of the Gibraltar Strait. Application of this Cobitis mtDNA clock provides evidence that the Messinian salinity crisis played a primary role in the diversification of some Ibero-African cobitid species. The basal polytomies of the Ibero-African Clade might suggest that all mtDNA lineages diversified rapidly.     

Diptera of forensic importance in the Iberian Peninsula: larval identification key

A revision of the species and families of sarcosaprophagous flies (Diptera: Calliphoridae, Sarcophagidae, Muscidae, Fanniidae, Drosophilidae, Phoridae, Piophilidae and Stratiomyidae) suitable for forensic purposes in the Iberian Peninsula is presented. Morphological characteristics that allow the accurate identification of third instars of the species present in the Iberian Peninsula are described and presented in the form of a diagnostic key. For larval Calliphoridae, characteristics such as the spines of the body segments were useful for the genus Calliphora whereas features of the anal segment and the cephalopharyngeal skeleton were useful for larvae of Lucilia. Identification of three Chrysominae species present in the Iberian Peninsula is included. For larval Sarcophagidae, characters such as the arrangement and shape of spiracular openings, structures of the anal segment and the cephalopharyngeal skeleton were used for the first time. A new record of Sarcophaga cultellata Pandellé, from a human corpse, is also included as well as recent incursions into the European cadaveric entomofauna such as Synthesiomyia nudiseta (van der Wulp) and Hermetia illucens (Linnaeus). This work provides useful new information that could be applied to forensic investigations in the Iberian Peninsula and in southern Europe.