Joan Pons

Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics

Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags (‘barcodes’). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three ‘bait’ sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species ‘barcodes’ that currently use the cox1 gene only.

Beyond barcodes: complex DNA taxonomy of a South Pacific Island radiation

DNA barcodes can provide rapid species identification and aid species inventories in taxonomically unstudied groups. However, the approach may fail in recently diverged groups with complex gene histories, such as those typically found on oceanic islands. We produced a DNA-based inventory of taxonomically little known diving beetles (genus Copelatus) in the Fiji archipelago, where they are a dominant component of the aquatic invertebrate fauna. Sampling from 25 localities on five islands and analysis of sequences from one nuclear (328 bp histone 3) and three mitochondrial (492 bp rrnL, 786 bp cox1, 333 bp cob) gene regions revealed high haplotype diversity, mainly originated since the Pleistocene, and subdivided into three major phylogenetic lineages and 22 statistical parsimony networks. A traditional taxonomic study delineated 25 morphologically defined species that were largely incongruent with the DNA-based groups. Haplotype diversity and their spatial arrangement demonstrated a continuum of relatedness in Fijian Copelatus, with evidence for introgression at various hierarchical levels. The study illustrates the difficulties for formal classification in evolutionarily complex lineages, and the potentially misleading conclusions obtained from either DNA barcodes or morphological traits alone. However, the sequence profile of Fijian Copelatus provides an evolutionary framework for the group and a DNA-based reference system for the integration of ecological and other biodiversity data, independent of the Linnaean naming system.

The towering orogeny of New Guinea as a trigger for arthropod megadiversity

Early studies on Melanesian mountain systems provided insights for fundamental evolutionary and ecological concepts. These island-like systems are thought to provide opportunities in the form of newly formed, competition-free niches. Here we show that a hyperdiverse radiation of freshwater arthropods originated in the emerging central New Guinea orogen, out of Australia, about 10 million years ago. Further diversification was mainly allopatric, with repeated more recent colonization of lowlands as they emerged in the form of colliding oceanic island arcs, continental fragments and the Papuan Peninsula, as well as recolonization of the central orogen. We unveil a constant and ongoing process of lineage accumulation while the carrying capacity of the island is about to be reached, suggesting that lineage diversification speed now exceeds that of landmass/new ecological opportunity formation. Therefore, the central orogeny of New Guinea acts as a motor of diversification for the entire region.

Mitochondrial Cox1 Sequence Data Reliably Uncover Patterns of Insect Diversity But Suffer from High Lineage-Idiosyncratic Error Rates

Background The demand for scientific biodiversity data is increasing, but taxonomic expertise is often limited or not available. DNA sequencing is a potential remedy to overcome this taxonomic impediment. Mitochondrial DNA is most commonly used, e.g., for species identification (“DNA barcoding”). Here, we present the first study in arthropods based on a near-complete species sampling of a family-level taxon from the entire Australian region. We aimed to assess how reliably mtDNA data can capture species diversity when many sister species pairs are included. Then, we contrasted phylogenetic subsampling with the hitherto more commonly applied geographical subsampling, where sister species are not necessarily captured. Methodology/Principal Findings We sequenced 800 bp cox1 for 1,439 individuals including 260 Australian species (78% species coverage). We used clustering with thresholds of 1 to 10% and general mixed Yule Coalescent (GMYC) analysis for the estimation of species richness. The performance metrics used were taxonomic accuracy and agreement between the morphological and molecular species richness estimation. Clustering (at the 3% level) and GMYC reliably estimated species diversity for single or multiple geographic regions, with an error for larger clades of lower than 10%, thus outperforming parataxonomy. However, the rates of error were higher for some individual genera, with values of up to 45% when very recent species formed nonmonophyletic clusters. Taxonomic accuracy was always lower, with error rates above 20% and a larger variation at the genus level (0 to 70%). Sørensen similarity indices calculated for morphospecies, 3% clusters and GMYC entities for different pairs of localities was consistent among methods and showed expected decrease over distance. Conclusion/Significance Cox1 sequence data are a powerful tool for large-scale species richness estimation, with a great potential for use in ecology and β-diversity studies and for setting conservation priorities. However, error rates can be high in individual lineages.

Localization of tandemly repeated DNA sequences in beetle chromosomes by fluorescentin situ hybridization

In situ hybridization to chromosomes and nuclei ofTenebrio molitor shows the massive presence of a species-specific satellite DNA in all chromosomes and six sites of rDNA in mitotic chromosomes. These sites are located in two autosomal pairs and in the X and Y chromosomes. In a related species,Misolampus goudoti, in which two different families of highly repetitive DNA have been previously characterized, one family is located in centromeric regions of all chromosomes with the exception of chromosome Y, while the other repeated DNA family is present both in centromeric and distal regions of all chromosomes. rRNA genes in this species are present in a medium-sized autosomal pair only. These results show that molecular cytogenetics can be applied to coleopteran chromosomes and open the way for a physical mapping of DNA sequences in these organisms. The results also provide insights into the type of meiotic association of the X and Y chromosomes in Coleoptera and the distribution of repeated DNAs within the genome of these insects.

Using Exon and Intron Sequences of the Gene Mp20 to Resolve Basal Relationships in Cicindela (Coleoptera:Cicindelidae)

The genus Cicindela (Coleoptera: Cicindelidae) is a species-rich cosmopolitan group of tiger beetles useful for comparing clade diversification worldwide. Knowledge about relationships of major groups is important for this analysis but basal nodes in Cicindela have been difficult to resolve with standard mtDNA markers. Here we developed the Mp20 gene, a single-copy nuclear marker coding for a muscle-associated protein in insects, for phylogenetic analysis of basal groups of Cicindela. Nearly full-length sequences were obtained for 51 cicindelids, including major taxonomic groups from all continents. Sequences of Mp20 were between 1.2 and 1.7 kb and spanning three introns. Phylogenetic signal of exon and intron sequences was compared with that from four gene regions of mtDNA (COI, COIII, Cytb, 16S rRNA; 2.4 kb total). Because introns differed in length, sequence alignment was conducted using various procedures of phenetic and parsimony-based character coding of indels to assess their phylogenetic information content, but major nodes were recovered consistently. Mp20 sequences contributed two thirds of the total support of the combined analysis, with most signal from the introns. We found major clades of Cicindela to be geographically largely coincident with continental regions, confined to Australasia, the Holarctic, the Indian subcontinent, Africa, and South and Central America. Clock estimates using various maximum-likelihood (ML) branch length calculations resulted in roughly similar divergence times whether Mp20 exon, introns, or mtDNA were used, and they were not greatly affected by different procedures for coding and optimizing indel characters. Based on existing clock calibrations in Cicindela, basal splits of continental lineages occurred in the mid-Miocene, placing the radiation of basal groups of Cicindela to a period when their open-vegetation habitats expanded globally.

Size, frequency, and phylogenetic signal of multiple-residue indels in sequence alignment of introns

Indels in DNA sequences frequently affect more than a single nucleotide, creating problems for alignment, character coding and phylogenetic analysis. However, the size and frequency of multiple‐residue indels is not usually tested, and with popular alignment packages their reconstruction is indirectly acheived by reducing the affine (gap extension) cost. We explored the length distribution of indels in intron sequences of the gene Mp20 by modifying the gap opening and gap extension costs. Given a “known” tree for the study group, global homology levels were greatest under low gap cost, with gap extension costs of roughly 0.4‐fold the opening cost. Different approaches to gap coding and weighting suggested that taxonomic congruence was correlated with high frequencies of multiple‐position indels, with a maximum indel length of 2–5 bp and few indels above 15 bp, but also including a proportion of indels > 100 bp. Only a small minority of indels could be reconstructed as single‐position indels. Consequently, tree topologies improved when homologous multinucleotide indels were recoded as binary characters which are otherwise highly homoplastic and weighted characters in single‐position coding. In tree‐generating alignment procedures as implemented in POY, where gap penalty determines the character weight during tree search, the problem of assigning inappropriately high weight to multiple‐residue indels could partly be overcome by setting the extension costs to about 0.4‐fold lower than gap opening costs. We conclude that multiple consecutive gap positions are not independent characters and hence methods for parsimony reconstruction of long indels are required. Finally, we also observed a general lack of correlation between taxonomic and character congruence, demonstrating the difficulties of applying congruence criteria to decide among competing alignments. This highlights the value of recent model‐based alignment procedures which can implement the statistical distributions of indel size classes, and do not rely on potentially circular strategies for optimizing overall congruence.

Deep mtDNA subdivision within Linnean species in an endemic radiation of tiger beetles from New Zealand (genus Neocicindela)

The invertebrate fauna of New Zealand is of great interest as a geologically tractable model for the study of species diversification, but direct comparisons with closely related lineages elsewhere are lacking. Integrating population-level analyses with studies of taxonomy and clade diversification, we performed mtDNA analysis on Neocicindela (Cicindelidae, tiger beetles) for a broad sample of populations from 11 of 12 known species and 161 specimens (three loci, 1883 nucleotides), revealing 123 distinct haplotypes. Phylogenetic reconstruction recovered two main lineages, each composed of 5-6 Linnean species whose origin was dated to 6.66 and 7.26 Mya, while the Neocicindela stem group was placed at 10.82 ± 0.48 Mya. Species delimitation implementing a character-based (diagnostic) species concept recognized 19 species-level groups that were in general agreement with Linnean species but split some of these into mostly allopatric subgroups. Tree-based methods of species delimitation using a mixed Yule-coalescence model were inconclusive, and recognized 32-51 entities (including singletons), splitting existing species into up to 8 partially sympatric groups. These findings were different from patterns in the Australian sister genus Rivacindela, where character-based and tree-based methods were previously shown to produce highly congruent groupings. In Neocicindela, the pattern of mtDNA variation was characterized by high intra-population and intra-species haplotype divergence, the coexistence of divergent haplotypes in sympatry, and a poor correlation of genetic and geographic distance. These observations combined suggest a scenario of phylogeographic divergence and secondary contact driven by orogenetic and climatic changes of the Pleistocene/Pliocene. The complex evolutionary history of most species of Neocicindela due to the relative instability of the New Zealand biota resulted in populations of mixed ancestry but not in a general loss of genetic variation.

Islands beneath islands: phylogeography of a groundwater amphipod crustacean in the Balearic archipelago

BackgroundMetacrangonyctidae (Amphipoda, Crustacea) is an enigmatic continental subterranean water family of marine origin (thalassoid). One of the species in the genus, Metacrangonyx longipes, is endemic to the Balearic islands of Mallorca and Menorca (W Mediterranean). It has been suggested that the origin and distribution of thalassoid crustaceans could be explained by one of two alternative hypotheses: (1) active colonization of inland freshwater aquifers by a marine ancestor, followed by an adaptative shift; or (2) passive colonization by stranding of ancestral marine populations in coastal aquifers during marine regressions. A comparison of phylogenies, phylogeographic patterns and age estimations of clades should discriminate in favour of one of these two proposals.ResultsPhylogenetic relationships within M. longipes based on three mitochondrial DNA (mtDNA) and one nuclear marker revealed five genetically divergent and geographically structured clades. Analyses of cytochrome oxidase subunit 1 (cox1) mtDNA data showed the occurrence of a high geographic population subdivision in both islands, with current gene flow occurring exclusively between sites located in close proximity. Molecular-clock estimations dated the origin of M. longipes previous to about 6 Ma, whereas major cladogenetic events within the species took place between 4.2 and 2.0 Ma.ConclusionsM. longipes displayed a surprisingly old and highly fragmented population structure, with major episodes of cladogenesis within the species roughly correlating with some of the major marine transgression-regression episodes that affected the region during the last 6 Ma. Eustatic changes (vicariant events) -not active range expansion of marine littoral ancestors colonizing desalinated habitats-explain the phylogeographic pattern observed in M. longipes.

The complete mitochondrial genome of the subterranean crustacean Metacrangonyx longipes (Amphipoda): A unique gene order and extremely short control region

Metazoan mitochondrial genomes usually consist of the same gene set, but some taxonomic groups show a considerable variety in gene order and nucleotide composition. The mitochondrial genomes of 37 crustaceans are currently known. Within the malacostracan superorder Peracarida, only three partial mitogenome sequences and the complete sequence of Ligia oceanica (Isopoda) are available. Frequent translocation events have changed the mitochondrial gene order in crustaceans, providing an opportunity to study the patterns and mechanisms of mitogenome rearrangement and to determine their impact on phylogenetic reconstructions. Here we report the first complete nucleotide sequence of an amphipod species, Metacrangonyx longipes, belonging to a phylogenetically enigmatic family occurring in continental subterranean waters. The genome has 14,113 bp and contains the usual 13 protein coding genes and two rRNA subunits, but only 21 out of the typical 22 tRNA genes of Metazoa. This is the shortest mitogenome described thus far for a crustacean and also one of the richest in AT (76.03%). The genome compactness results from a very small control region of 76 bp, the occurrence of frequent gene overlap, and the absence of large non-coding fragments. Six of the protein-coding genes have unusual start codons. Comparison of individual protein coding genes with the sequences known for other crustaceans suggests that nad2, nad6, nad4L and atp8 show the highest divergence rates. M. longipes shows a unique crustacean mitogenome gene order, differing even from the condition found in Parhyale hawaiiensis (Amphipoda), whose coding sequence has also been completed in the present study.