New mitogenomes of Runcinidae and Facelinidae: two understudied heterobranch families (Mollusca: Gastropoda).

Here, we present the mitochondrial sequences of two sea slugs (Heterobranchia): Runcina aurata and Facelina auriculata, the latter being the type species of the family. The mitochondrial genomes are 14,282 and 14,171bp in length, respectively, with a complete set of 13 PCGs, 2 rRNAs, and 22 tRNAs. None of the mitogenomes show gene reorganization, keeping the standard mitogenomic structure of Heterobranchia. Nucleotide composition differs significantly between them, with R. aurata showing the most AT-rich mitogenome (25.7% GC content) reported to date in Heterobranchia, and F. auriculata showing a rich GC content (35%) compared with other heterobranch mitochondrial genomes.

Novel molecular resources for single-specimen barcoding of enigmatic crustacean y-larvae.

Despite discovery more than 100years ago and documented global occurrence from shallow waters to the deep sea, the life cycle of the enigmatic crustacean y-larvae isincompletely understood and adult forms remain unknown. To date, only 2 of the 17 formally described species, all based on larval stages, have been investigated using an integrative taxonomic approach. This approach provided descriptions of the morphology of the naupliar and cyprid stages, and made use of exuvial voucher material and DNA barcodes. To improve our knowledge about the evolutionary history and ecological importance of y-larvae, we developed a novel protocol that maximises the amount of morpho-ecological and molecular data that can be harvested from single larval specimens. This includes single-specimen DNA barcoding and daily imaging of y-nauplii reared in culture dishes, mounting of the last naupliar exuviae on a slide as a reference voucher, live imaging of the y-cyprid instar that follows, and fixation, DNA extraction, amplification and sequencing of the y-cyprid specimen. Through development and testing of a suite of new primers for both nuclear and mitochondrial protein-coding and ribosomal genes, we showcase how new sequence data can be used to estimate the phylogeny of Facetotecta. We expect that our novel procedure will help to unravel the complex systematics of y-larvae and show how these fascinating larval forms have evolved. Moreover, we posit that our protocols should work on larval specimens from a diverse array of moulting marine invertebrate taxa.

Identification of Phyllosoma Larvae of the Lobsters Acantharctus and Biarctus (Crustacea: Scyllaridae) from the SW Indian Ocean.

The accurate assignment of cryptic larvae to species-level is a key aspect of marine ecological research and can be achieved through integrated molecular and morphological studies. A combination of two mitochondrial markers (COI and 16S) and a detailed morphological analysis was used to identify phyllosoma larvae of slipper lobster (Scyllaridae) species collected during a survey in the SW Indian Ocean. Two morphotypes were tentatively assigned to Acantharctus ornatus and Biarctus pumilus, both genera for which the larval morphology was unknown. Morphological revision of an adult specimen used to generate the putative A. ornatus sequences in GenBank revealed that it was misidentified and corresponds to B. dubius. The final phyllosoma stage of B. pumilus and subfinal and final stages of A. ornatus were described, clarifying prior misidentifications in the literature. Scyllarid biodiversity in the SW Indian Ocean is underestimated and sampling of deeper water layers is recommended to complete current knowledge of species and larval stages present in the region.

Integrative Taxonomy of Cyclocyprididae Kaufmann, 1900 (Ostracoda: Podocopa) with Description of a New Genus and Species.

The two widespread ostracod genera Cypria Zenker, 1854 and Physocypria Vávra, 1897 are traditionally distinguished based on the presence or absence of tubercles on the right valve margin. However, recent research based on soft body parts has uncovered new cryptic genera within Cypria and Physocypria. Following this line of research, a new Cyclocyprididae genus and species, Vizcainocypria viator gen. nov. sp. nov., is here described from individuals collected in rice fields and wetlands of the Iberian Peninsula. Vizcainocypria is compared with Cypria, Physocypria, Dentocypria Savatenalinton, 2017, Keysercypria Karanovic, 2011, Brasilocypria Almeida et al., 2023, and Claudecypria Almeida et al., 2023 based on morphological evidence. Besides the presence or absence of tubercles on the right valve, these genera can be distinguished according to their mandibular palp, second thoracopod, caudal ramus, and male hemipenis. Molecular analyses using mitochondrial (COX1), and nuclear (28S rDNA) genes provide further support for the differentiation of Cypria, Dentocypria, Physocypria and Vizcainocypria gen. nov. The present study highlights the importance of using an integrative taxonomy approach, combining shell and soft-body parts morphology and molecular data, to characterize the rich diversity of freshwater ostracods.

Single-specimen systematics resolves the phylogeny and diversity conundrum of enigmatic crustacean y-larvae.

Resolving the evolutionary history of organisms is a major goal in biology. Yet for some taxa the diversity, phylogeny, and even adult stages remain unknown. The enigmatic crustacean "y-larvae" (Facetotecta) are one particularly striking example. Here, we use extensive video-imaging and single-specimen molecular sequencing of >200 y-larval specimens to comprehensively explore for the first time their evolutionary history and diversity. This integrative approach revealed five major clades of Facetotecta, four of which encompass a considerable larval diversity. Whereas morphological analyses recognized 35 y-naupliar "morphospecies", molecular species-delimitation analyses suggested the existence of between 88 and 127 species. The phenotypic and genetic diversity between the morphospecies suggests that a more elaborate classification than the current one-genus approach is needed. Morphology and molecular data were highly congruent at shallower phylogenetic levels, but no morphological synapomorphies could be unambiguously identified for major clades, which mostly comprise both planktotrophic and lecithotrophic y-nauplii. We argue that lecithotrophy arose several times independently whereas planktotrophic y-nauplii, which are structurally more similar across clades, most likely display the ancestral feeding mode of Facetotecta. We document a remarkably complex and highly diverse phylogenetic backbone for a taxon of larval marine crustaceans, the full life cycle of which remains a mystery.

Mislabeling and nomenclatorial confusion of Typhlotanais sandersi Kudinova-Pasternak, 1985 (Crustacea: Tanaidacea) and establishment of a new genus.

Re-examination of historical collections allowed us to resolve the taxonomic status of Typhlotanais sandersi Kudinova-Pasternak, 1985, originally described based on a single specimen from Great-Meteor Seamount. The holotype of this species was considered lost and the species redescribed based on a second specimen from the type locality by Blazewicz-Paszkowycz (2007a), who placed Ty. sandersi on a newly established genus Typhlamia. Thorough morphological analysis of Typhlamia and Typhlotanais species and recently obtained genetic data of typhlotanaids from N Atlantic and NW Pacific waters allow us to conclude that the redescription of Ty. sandersi by Blazewicz-Paszkowycz (2007a) was based on a wrongly labelled specimen that, rather than a type of Ty. sandersi, represents in fact a new species of Typhlamia. The morphological comparison of the type species of Typhlotanais (Ty. aequiremis) with all 'long-bodied' typhlotanaid taxa with rounded pereonites margins (i.e., Typhlamia, Pulcherella, Torquella), and the use of genetic evidence, support the establishment of a new genus to accommodate: Ty. sandersi, Ty. angusticheles Kudinova-Pasternak, 1989, and a third species from N Atlantic waters, that is described here for the first time. Current knowledge on 'long-bodied' typhlotanaids with rounded pereonites is summarised and a taxonomical key for their identification provided.

Parasitoids (Hymenoptera) of Mealybug Pests (Hemiptera: Pseudococcidae) from Southern Brazil: Molecular and Morphological Characterization.

Parasitoids of three mealybug pests (Hemiptera: Pseudococcidae), Planococcus ficus (Signoret), Pseudococcus sociabilis Hambleton, and Pseudococcus viburni (Signoret) have been identified for the first time in Brazil. Mealybugs were collected in fruit-growing areas along southern Brazil during 2013-2016. An integrative approach, combining morphological and molecular methods, was used to identify the Brazilian parasitoids to the species level. Fifteen species were recorded, including 14 primary parasitoids belonging to Encyrtidae and Platygastridae and a single secondary parasitoid species belonging to Signiphoridae. The encyrtid parasitoids Acerophagus flavidulus (Brèthes), Anagyrus calyxtoi Noyes and Zaplatycerus sp., and the signiphorid secondary parasitoid Chartocerus axillaris De Santis are reported for the first time in Brazil.

A new species of Cypris (Crustacea: Ostracoda) from the Iberian Peninsula and the Balearic Islands, with comments on the first ostracod named using the Linnean system.

The genus Cypris, considered the oldest ostracod generic name erected using the Linnean system, comprises a reduced number of large-bodied species, mostly found in Africa and Asia. Only six of them are known to occur in Europe. Here we describe a new species, Cypris pretusi sp. nov., collected in small temporary streams and ponds along the Eastern Iberian Peninsula and Minorca (Balearic Islands). The new species is very close to the type species of the genus, Cypris pubera O.F. Müller, 1776, but differs from it in having a set of smaller subequal spines on the posterior edge of the valves, by the absence of conspicuous spines along the front edge, and by the beak-like frontal shape of its carapace in dorsal view, similar to Cypris decaryi Gauthier, 1933. Soft parts are very similar to the type species, but it differs in having shorter swimming setae on the second antennae. Molecular analyses of the COX1 region support its status as a species distinct from C. pubera and closer to Cypris bispinosa Lucas, 1849, also providing evidence for a separation of C. pubera s.l. in two clades, one of which is here considered to correspond to Cypris triaculeata Daday, 1892. We discuss the relationships of C. pretusi sp. nov. to other members of the genus and its possible origin from nearby biogeographic regions (probably Africa or Asia) and provide a key to species of Cypris found in Europe. We also discuss the relationship between Monoculus concha pedata (= M. conchaceus), the first ostracod named by Linnæus, and Cypris pubera, the type species of the genus, described by Müller in 1776 and considered by him the same species as the one first named by Linnæus.

Deep ocean seascape and Pseudotanaidae (Crustacea: Tanaidacea) diversity at the Clarion-Clipperton Fracture Zone.

Understanding the diversity and spatial distribution of benthic species is fundamental to properly assess the impact of deep sea mining. Tanaidacea provide an exceptional opportunity for assessing spatial patterns in the deep-sea, given their low mobility and limited dispersal potential. The diversity and distribution of pseudotanaid species is characterized here for the Clarion and Clipperton Fractures Zone (CCZ), which is the most extensive deposit field of metallic nodules. Samples were taken from the Belgian, German and French license areas, but also from the APEI 3 (Area of Particular Environmental Interest 3) of the Interoceanmetal consortium associates. The combination of morphological and genetic data uncovered one new pseudotanaid genus (Beksitanais n. gen.) and 14 new species of Pseudotanais (2 of them virtual taxa). Moreover, our results suggest that spatial structuring of pseudotanaid diversity is correlated with deep-sea features, particularly the presence of fractures and seamount chains crossing the CCZ. The presence of geographical barriers delimiting species distributions has important implications for the establishment of protected areas, and the APEI3 protected area contains only one third of the total pseudotanaid species in CCZ. The specimen collection studied here is extremely valuable and represents an important first step in characterizing the diversity and distribution of pseudotanaids within the Tropical Eastern Pacific.

Pancrustacean Evolution Illuminated by Taxon-Rich Genomic-Scale Data Sets with an Expanded Remipede Sampling.

The relationships of crustaceans and hexapods (Pancrustacea) have been much discussed and partially elucidated following the emergence of phylogenomic data sets. However, major uncertainties still remain regarding the position of iconic taxa such as Branchiopoda, Copepoda, Remipedia, and Cephalocarida, and the sister group relationship of hexapods. We assembled the most taxon-rich phylogenomic pancrustacean data set to date and analyzed it using a variety of methodological approaches. We prioritized low levels of missing data and found that some clades were consistently recovered independently of the analytical approach used. These include, for example, Oligostraca and Altocrustacea. Substantial support was also found for Allotriocarida, with Remipedia as the sister of Hexapoda (i.e., Labiocarida), and Branchiopoda as the sister of Labiocarida, a clade that we name Athalassocarida (="nonmarine shrimps"). Within Allotriocarida, Cephalocarida was found as the sister of Athalassocarida. Finally, moderate support was found for Hexanauplia (Copepoda as sister to Thecostraca) in alliance with Malacostraca. Mapping key crustacean tagmosis patterns and developmental characters across the revised phylogeny suggests that the ancestral pancrustacean was relatively short-bodied, with extreme body elongation and anamorphic development emerging later in pancrustacean evolution.

Integrative taxonomy methods reveal high mealybug (Hemiptera: Pseudococcidae) diversity in southern Brazilian fruit crops.

The Serra Gaúcha region is the most important temperate fruit-producing area in southern Brazil. Despite mealybugs (Hemiptera: Pseudococcidae) infesting several host plants in the region, there is a lack of information about the composition of species damaging different crops. A survey of mealybug species associated with commercial fruit crops (apple, persimmon, strawberry and grapes) was performed in Serra Gaúcha between 2013 and 2015, using both morphology and DNA analyses for species identification. The most abundant species were Pseudococcus viburni (Signoret), found on all four host plant species, and Dysmicoccus brevipes (Cockerell), infesting persimmon, vines and weeds. The highest diversity of mealybug species was found on persimmon trees, hosting 20 different taxa, of which Anisococcus granarae Pacheco da Silva & Kaydan, D. brevipes, Pseudococcus sociabilis Hambleton and Ps. viburni were the most abundant. A total of nine species were recorded in vineyards. Planococcus ficus (Signoret) and Pseudococcus longispinus (Targioni Tozzetti) were observed causing damage to grapes for the first time. A single species, Ps. viburni, was found associated with apples, while both Ps. viburni and Ferrisia meridionalis Williams were found on strawberry. Four of the mealybug species found represent new records for Brazil.

Tomato root microbiota and Phytophthora parasitica-associated disease.

BACKGROUND: Interactions between pathogenic oomycetes and microbiota residing on the surface of the host plant root are unknown, despite being critical to inoculum constitution. The nature of these interactions was explored for the polyphagous and telluric species Phytophthora parasitica. RESULTS: Composition of the rhizospheric microbiota of Solanum lycopersicum was characterized using deep re-sequencing of 16S rRNA gene to analyze tomato roots either free of or partly covered with P. parasitica biofilm. Colonization of the host root surface by the oomycete was associated with a shift in microbial community involving a Bacteroidetes/Proteobacteria transition and Flavobacteriaceae as the most abundant family. Identification of members of the P. parasitica-associated microbiota interfering with biology and oomycete infection was carried out by screening for bacteria able to (i) grow on a P. parasitica extract-based medium (ii), exhibit in vitro probiotic or antibiotic activity towards the oomycete (iii), have an impact on the oomycete infection cycle in a tripartite interaction S. lycopersicum-P. parasitica-bacteria. One Pseudomonas phylotype was found to exacerbate disease symptoms in tomato plants. The lack of significant gene expression response of P. parasitica effectors to Pseudomonas suggested that the increase in plant susceptibility was not associated with an increase in virulence. Our results reveal that Pseudomonas spp. establishes commensal interactions with the oomycete. Bacteria preferentially colonize the surface of the biofilm rather than the roots, so that they can infect plant cells without any apparent infection of P. parasitica. CONCLUSIONS: The presence of the pathogenic oomycete P. parasitica in the tomato rhizosphere leads to a shift in the rhizospheric microbiota composition. It contributes to the habitat extension of Pseudomonas species mediated through a physical association between the oomycete and the bacteria.

Temporal and spatial genetic differentiation in the crab Liocarcinus depurator across the Atlantic-Mediterranean transition.

Spatial genetic studies often require sampling broadly separated areas, difficult to access simultaneously. Although comparing localities surveyed at different time periods might result in spurious genetic differentiation, there is a general believe on the stability of genetic structure through time, particularly if sampled localities are isolated or very distant. By analysing spatial and temporal genetic differentiation of the portunid crab Liocarcinus depurator we assessed the contribution of historical and contemporary processes on population connectivity patterns across three main oceanographic discontinuities along the Atlantic-Mediterranean transition: Gibraltar Strait, Almeria-Oran Front and Ibiza Channel. A partial fragment of the cytochrome oxidase I gene was sequenced in 366 individuals collected from localities at both sides of each discontinuity during three time periods. Although localities showed genetic fluctuations through time, a significant gradient was detected along the coast for all sampling periods. Significant inter-annual differences identified within the Alicante area, north of the Almeria-Oran Front, were associated with shifts in the relative contribution of Atlantic and Mediterranean water masses. The persistence of a clinal pattern in the Atlantic-Mediterranean transition area together with local fluctuations suggests a complex balance of dispersal and selection.

DNA barcoding the phyllosoma of Scyllarides squammosus (H. Milne Edwards, 1837) (Decapoda: Achelata: Scyllaridae).

Scyllarides has the largest number of species with commercial importance within the Scyllaridae family. As for other achelate lobsters, however, little is known of the unique long-lived planktonic phyllosoma stages of any of these tropical and temperate species. Recently, a large and diverse collection of Scyllaridae phyllosoma, compiled from cruises along the Coral Sea and spanning several years, has been analysed. Molecular evidence from DNA-barcoding and phylogenetic analyses is provided here on the identity of S. squammosus phyllosoma larvae, including stages that were previously undescribed or poorly known. As a consequence, the growth and morphological changes that occur during the mid- to late-stages of S. squammosus larval development is now well-documented. Furthermore, an additional collection of S. squammosus larvae, described by Alain Michel and thought to be no longer extant, were discovered in the crustacean collection of the Muséum national d'Histoire naturelle, Paris. This new molecular and morphological information is complemented by a review of the literature. As a result, descriptions of key larval characters by a number of authors were evaluated and appear to suggest the existence of distinct groups of larvae within Scyllarides. From a combination of adult and larval morphology, and molecular data, the results presented here revealed inconsistencies with regard to the affinities of species assigned to Scyllarides. This new evidence will contribute to future studies addressing the phylogenetic relationships within the genus.

The evolution of a key character, or how to evolve a slipper lobster.

A new fossil lobster from the Cretaceous of Lebanon, Charbelicaris maronites gen. et sp. nov., is presented here, while the former species 'Cancrinos' libanensis is re-described as Paracancrinos libanensis comb. nov. P. libanensis is shown to be closer related to the contemporary slipper lobsters than to Cancrinos claviger (lithographic limestones, Jurassic, southern Germany). A finely-graded evolutionary scenario for the slipper-lobster morphotype is reconstructed based on these fossil species and extant forms. The evolutionary changes that gave rise to the current plate-like antennae of Scyllaridae, a key apomorphy of this group, are traced back through time. The antenna of what is considered the oldest slipper lobster became petaloid and consisted of about 20 fully articulated elements. For this group the name Scyllarida sensu lato tax. nov. is introduced. In a next evolutionary step, the proximal articles became conjoined and a lateral extension appeared on peduncle element 3. The entire distal petaloid region is conjoined already at the node of Verscyllarida tax. nov. In modern slipper lobsters, Neoscyllarida tax nov., the distal region is no longer petaloid in shape but asymmetrical. The study also emphasizes that exceptionally preserved fossils need to be documented with optimal documentation techniques to obtain all available information.

A salt bath will keep you going? Euryhalinity tests and genetic structure of caridean shrimps from Iberian rivers.

We assessed the role of euryhalinity and life-history traits on the population genetic structure of the four main caridean shrimp species from the Iberian Peninsula (Atyaephyra desmarestii, Dugastella valentina, Palaemon varians and Palaemon zariquieyi) able to complete their life cycle in freshwater/oligohaline habitats. Seawater exposure experiments indicated that A. desmarestii, D. valentina and P. zariquieyi are more sensitive to high salinity waters than P. varians and confirm the relationship between osmolality regulation and spatial distribution of species. The limited or no survival in seawater could explain the restricted distributions observed in D. valentina and P. zariquieyi, whereas the current A. desmarestii distribution could be due to either past river dynamics and/or human-mediated water transfers. Conversely, the high tolerance of P. varians to a large salinity range (euryhalinity) could explain its capacity to colonize geographically distant estuaries. In agreement with osmoregulation results, the phylogeography patterns of the cytochrome oxidase 1 (Cox 1) gene fragment revealed significant genetic differentiation among river systems whatever the species considered. Atyidae species presented higher nucleotide diversity levels than Palaemonidae species, while isolation-by-distance patterns were only found for the latter. Our results have important implications for the management and conservation of freshwater species, since the inter-catchment connectivity may affect the speciation processes.

Mealybug species from Chilean agricultural landscapes and main factors influencing the genetic structure of Pseudococcus viburni.

The present study aimed to characterize the distribution of mealybug species along Chilean agro-ecosystems and to determine the relative impact of host plant, management strategy, geography and micro-environment on shaping the distribution and genetic structure of the obscure mealybug Pseudococcus viburni. An extensive survey was completed using DNA barcoding methods to identify Chilean mealybugs to the species level. Moreover, a fine-scale study of Ps. viburni genetic diversity and population structure was carried out, genotyping 529 Ps. viburni individuals with 21 microsatellite markers. Samples from 16 localities were analyzed using Bayesian and spatially-explicit methods and the genetic dataset was confronted to host-plant, management and environmental data. Chilean crops were found to be infested by Ps. viburni, Pseudococcus meridionalis, Pseudococcus longispinus and Planococcus citri, with Ps. viburni and Ps. meridionalis showing contrasting distribution and host-plant preference patterns. Ps. viburni samples presented low genetic diversity levels but high genetic differentiation. While no significant genetic variance could be assigned to host-plant or management strategy, climate and geography were found to correlate significantly with genetic differentiation levels. The genetic characterization of Ps. viburni within Chile will contribute to future studies tracing back the origin and improving the management of this worldwide invader.

Geographical and Temporal Structures of Legionella pneumophila Sequence Types in Comunitat Valenciana (Spain), 1998 to 2013.

Legionella pneumophila is an accidental human pathogen associated with aerosol formation in water-related sources. High recombination rates make Legionella populations genetically diverse, and nearly 2,000 different sequence types (STs) have been described to date for this environmental pathogen. The spatial distribution of STs is extremely heterogeneous, with some variants being present worldwide and others being detected at only a local scale. Similarly, some STs have been associated with disease outbreaks, such as ST578 or ST23. Spain is among the European countries with the highest incidences of reported legionellosis cases, and specifically, Comunitat Valenciana (CV) is the second most affected area in the country. In this work, we aimed at studying the overall diversity of Legionella pneumophila populations found in the period from 1998 to 2013 in 79 localities encompassing 23 regions within CV. To do so, we performed sequence-based typing (SBT) on 1,088 L. pneumophila strains detected in the area from both environmental and clinical sources. A comparison with the genetic structuring detected in a global data set that included 20 European and 7 non-European countries was performed. Our results reveal a level of diversity in CV that can be considered representative of the diversity found in other countries worldwide.

Guiding Classical Biological Control of an Invasive Mealybug Using Integrative Taxonomy.

Delottococcus aberiae De Lotto (Hemiptera: Pseudococcidae) is a mealybug of Southern African origin that has recently been introduced into Eastern Spain. It causes severe distortions on young citrus fruits and represents a growing threat to Mediterranean citrus production. So far, biological control has proven unsatisfactory due to the absence of efficient natural enemies in Spain. Hence, the management of this pest currently relies only on chemical control. The introduction of natural enemies of D. aberiae from the native area of the pest represents a sustainable and economically viable alternative to reduce the risks linked to pesticide applications. Since biological control of mealybugs has been traditionally challenged by taxonomic misidentification, an intensive survey of Delottococcus spp. and their associated parasitoids in South Africa was required as a first step towards a classical biological control programme. Combining morphological and molecular characterization (integrative taxonomy) a total of nine mealybug species were identified in this study, including three species of Delottococcus. Different populations of D. aberiae were found on wild olive trees, in citrus orchards and on plants of Chrysanthemoides monilifera, showing intra-specific divergences according to their host plants. Interestingly, the invasive mealybug populations from Spanish orchards clustered together with the population on citrus from Limpopo Province (South Africa), sharing COI haplotypes. This result pointed to an optimum location to collect natural enemies against the invasive mealybug. A total of 14 parasitoid species were recovered from Delottococcus spp. and identified to genus and species level, by integrating morphological and molecular data. A parasitoid belonging to the genus Anagyrus, collected from D. aberiae in citrus orchards in Limpopo, is proposed here as a good biological control agent to be introduced into Spain.

Recombination in viruses: mechanisms, methods of study, and evolutionary consequences.

Recombination is a pervasive process generating diversity in most viruses. It joins variants that arise independently within the same molecule, creating new opportunities for viruses to overcome selective pressures and to adapt to new environments and hosts. Consequently, the analysis of viral recombination attracts the interest of clinicians, epidemiologists, molecular biologists and evolutionary biologists. In this review we present an overview of three major areas related to viral recombination: (i) the molecular mechanisms that underlie recombination in model viruses, including DNA-viruses (Herpesvirus) and RNA-viruses (Human Influenza Virus and Human Immunodeficiency Virus), (ii) the analytical procedures to detect recombination in viral sequences and to determine the recombination breakpoints, along with the conceptual and methodological tools currently used and a brief overview of the impact of new sequencing technologies on the detection of recombination, and (iii) the major areas in the evolutionary analysis of viral populations on which recombination has an impact. These include the evaluation of selective pressures acting on viral populations, the application of evolutionary reconstructions in the characterization of centralized genes for vaccine design, and the evaluation of linkage disequilibrium and population structure.