Odd-Paired is Involved in Morphological Divergence of Snail-Feeding Beetles.

Body shape and size diversity and their evolutionary rates correlate with species richness at the macroevolutionary scale. However, the molecular genetic mechanisms underlying the morphological diversification across related species are poorly understood. In beetles, which account for one-fourth of the known species, adaptation to different trophic niches through morphological diversification appears to have contributed to species radiation. Here, we explored the key genes for the morphological divergence of the slender to stout body shape related to divergent feeding methods on large to small snails within the genus Carabus. We show that the zinc-finger transcription factor encoded by odd-paired (opa) controls morphological variation in the snail-feeding ground beetle Carabus blaptoides. Specifically, opa was identified as the gene underlying the slender to stout morphological difference between subspecies through genetic mapping and functional analysis via gene knockdown. Further analyses revealed that changes in opa cis-regulatory sequences likely contributed to the differences in body shape and size between C. blaptoides subspecies. Among opa cis-regulatory sequences, single nucleotide polymorphisms on the transcription factor binding sites may be associated with the morphological differences between C. blaptoides subspecies. opa was highly conserved in a wide range of taxa, especially in beetles. Therefore, opa may play an important role in adaptive morphological divergence in beetles.

SPART: A versatile and standardized data exchange format for species partition information.

A wide range of data types can be used to delimit species and various computer-based tools dedicated to this task are now available. Although these formalized approaches have significantly contributed to increase the objectivity of species delimitation (SD) under different assumptions, they are not routinely used by alpha-taxonomists. One obvious shortcoming is the lack of interoperability among the various independently developed SD programs. Given the frequent incongruences between species partitions inferred by different SD approaches, researchers applying these methods often seek to compare these alternative species partitions to evaluate the robustness of the species boundaries. This procedure is excessively time consuming at present, and the lack of a standard format for species partitions is a major obstacle. Here, we propose a standardized format, SPART, to enable compatibility between different SD tools exporting or importing partitions. This format reports the partitions and describes, for each of them, the assignment of individuals to the "inferred species". The syntax also allows support values to be optionally reported, as well as original trees and the full command lines used in the respective SD analyses. Two variants of this format are proposed, overall using the same terminology but presenting the data either optimized for human readability (matricial SPART) or in a format in which each partition forms a separate block (SPART.XML). ABGD, DELINEATE, GMYC, PTP and TR2 have already been adapted to output SPART files and a new version of LIMES has been developed to import, export, merge and split them.

Role of Sex-Concordant Gene Expression in the Coevolution of Exaggerated Male and Female Genitalia in a Beetle Group.

Some sexual traits, including genitalia, have undergone coevolutionary diversification toward exaggerated states in both sexes among closely related species, but the underlying genetic mechanisms that allow correlated character evolution between the sexes are poorly understood. Here, we studied interspecific differences in gene expression timing profiles involved in the correlated evolution of corresponding male and female genital parts in three species of ground beetle in Carabus (Ohomopterus). The male and female genital parts maintain morphological matching, whereas large interspecific variation in genital part size has occurred in the genital coevolution between the sexes toward exaggeration. We analyzed differences in gene expression involved in the interspecific differences in genital morphology using whole transcriptome data from genital tissues during genital morphogenesis. We found that the gene expression variance attributed to sex was negligible for the majority of differentially expressed genes, thus exhibiting sex-concordant expression, although large variances were attributed to stage and species differences. For each sex, we obtained co-expression gene networks and hub genes from differentially expressed genes between species that might be involved in interspecific differences in genital morphology. These gene networks were common to both sexes, and both sex-discordant and sex-concordant gene expression were likely involved in species-specific genital morphology. In particular, the gene expression related to exaggerated genital size showed no significant intersexual differences, implying that the genital sizes in both sexes are controlled by the same gene network with sex-concordant expression patterns, thereby facilitating the coevolution of exaggerated genitalia between the sexes while maintaining intersexual matching.

Gene expression during genital morphogenesis in the ground beetle Carabus maiyasanus.

To investigate the developmental genetics of genital formation in the carabid beetle Carabus maiyasanus, we compared gene expression patterns among five stages using transcriptomic RNA sequencing data from abdominal segments and genitalia in the third (last) larval instar (including prepupa) and pupal stages. We identified 18 839 genes, of which 10 796 were differentially expressed among stages or between sexes. There were relatively few differentially expressed genes (DEGs) between the sexes (3%). The DEGs were clustered into six groups, mainly according to stage-specific expression patterns. Genes in clusters 1-3 showed high expression levels before pupation and low expression levels during the pupal period, whereas genes in clusters 4-6 showed high expression levels from the prepupal to the pupal stages. Genes related to the initial pupation process and differentiation of genital discs in Drosophila were involved in clusters 4 and 6 and showed low expression levels at early third instar and elevated expression levels from the early prepupal stage, suggesting that the pupation process and genital differentiation started in the prepupal stage. Clusters 4 and 5 included developmental genes related to organ size control, which may be important in the formation of internal genital structures during the pupal stage.

Genetic basis of species-specific genitalia reveals role in species diversification.

The diversity of genital morphology among closely related animals with internal fertilization is well known, but the genetic backgrounds are unclear. Here, we show that, in Carabus (Ohomopterus) beetles showing correlated evolution of male and female genital parts, only a few major quantitative trait loci (QTLs) determine differences in genital dimensions between sister species, and sequence divergence is pronounced in the genomic regions containing genital QTLs. The major QTLs for male and female genital dimensions reside in different locations within the same linkage group, implying that coevolution between the sexes is only loosely constrained and can respond to sexually antagonistic selection. The same genomic regions containing the major QTLs show elevated divergence between three pairs of parapatric species with marked differences in genital parts. Our study demonstrates that species diversification can follow coevolution of genitalia between the sexes, even without tight linkage of loci affecting male and female genital dimensions.

Triplicate parallel life cycle divergence despite gene flow in periodical cicadas.

Periodical cicadas comprise three species groups containing three pairs of 13- and 17-year life cycle species showing parallel divergence, along with a more anciently diverged 13-year species (Magicicda tredecim). The mechanism and genetic basis of this parallel divergence is unknown. Here we use orthologous transcriptome sequences to explore the demographic processes and genomic evolution associated with parallel life cycle divergence. The three 13- and 17-year species pairs have similar demographic histories, and the two life cycles diverged 200,000-100,000 years ago. Interestingly, these life cycle differences have been maintained despite substantial gene flow between 13- and 17-year species within species groups, which is possible during co-emergences. Sequence divergence between 13- and 17-year species in each species group (excluding M. tredecim) is minimal, and we find no shared divergent single-nucleotide polymorphisms (SNPs) or loci associated with all instances of life cycle divergence. The two life cycles may be controlled by highly limited genomic differences.

Genomic regions and genes related to inter-population differences in body size in the ground beetle Carabus japonicus.

Body size is a key trait in diversification among animal species, and revealing the gene regions responsible for body size diversification among populations or related species is important in evolutionary biology. We explored the genomic regions associated with body size differences in Carabus japonicus ground beetle populations by quantitative trait locus (QTL) mapping of F2 hybrids from differently sized parents from two populations using restriction site-associated DNA sequencing and de novo assembly of the beetle whole genome. The assembled genome had a total length of 191 Mb with a scaffold N50 of 0.73 Mb; 14,929 protein-coding genes were predicted. Three QTLs on different linkage groups had major effects on the overall size, which is composed chiefly of elytral length. In addition, we found QTLs on autosomal and X chromosomal linkage groups that affected head length and width, thoracic width, and elytral width. We determined the gene loci potentially related to control of body size in scaffolds of the genome sequence, which contained the QTL regions. The genetic basis of body size variation based on a small number of major loci would promote differentiation in body size in response to selection pressures related to variations in environmental conditions and inter-specific interactions.

Genomic divergence and lack of introgressive hybridization between two 13-year periodical cicadas support life cycle switching in the face of climate change.

Life history evolution spurred by post-Pleistocene climatic change is hypothesized to be responsible for the present diversity in periodical cicadas (Magicicada), but the mechanism of life cycle change has been controversial. To understand the divergence process of 13-year and 17-year cicada life cycles, we studied genetic relationships between two synchronously emerging, parapatric 13-year periodical cicada species in the Decim group, Magicicada tredecim and M. neotredecim. The latter was hypothesized to be of hybrid origin or to have switched from a 17-year cycle via developmental plasticity. Phylogenetic analysis using restriction-site-associated DNA sequences for all Decim species and broods revealed that the 13-year M. tredecim lineage is genomically distinct from 17-year Magicicada septendecim but that 13-year M. neotredecim is not. We detected no significant introgression between M. tredecim and M. neotredecim/M. septendecim thus refuting the hypothesis that M. neotredecim are products of hybridization between M. tredecim and M. septendecim. Further, we found that introgressive hybridization is very rare or absent in the contact zone between the two 13-year species evidenced by segregation patterns in single nucleotide polymorphisms, mitochondrial lineage identity and head width and abdominal sternite colour phenotypes. Our study demonstrates that the two 13-year Decim species are of independent origin and nearly completely reproductively isolated. Combining our data with increasing observations of occasional life cycle change in part of a cohort (e.g. 4-year acceleration of emergence in 17-year species), we suggest a pivotal role for developmental plasticity in Magicicada life cycle evolution.

A Rapid and Scalable Method for Multilocus Species Delimitation Using Bayesian Model Comparison and Rooted Triplets.

Multilocus sequence data provide far greater power to resolve species limits than the single locus data typically used for broad surveys of clades. However, current statistical methods based on a multispecies coalescent framework are computationally demanding, because of the number of possible delimitations that must be compared and time-consuming likelihood calculations. New methods are therefore needed to open up the power of multilocus approaches to larger systematic surveys. Here, we present a rapid and scalable method that introduces 2 new innovations. First, the method reduces the complexity of likelihood calculations by decomposing the tree into rooted triplets. The distribution of topologies for a triplet across multiple loci has a uniform trinomial distribution when the 3 individuals belong to the same species, but a skewed distribution if they belong to separate species with a form that is specified by the multispecies coalescent. A Bayesian model comparison framework was developed and the best delimitation found by comparing the product of posterior probabilities of all triplets. The second innovation is a new dynamic programming algorithm for finding the optimum delimitation from all those compatible with a guide tree by successively analyzing subtrees defined by each node. This algorithm removes the need for heuristic searches used by current methods, and guarantees that the best solution is found and potentially could be used in other systematic applications. We assessed the performance of the method with simulated, published, and newly generated data. Analyses of simulated data demonstrate that the combined method has favorable statistical properties and scalability with increasing sample sizes. Analyses of empirical data from both eukaryotes and prokaryotes demonstrate its potential for delimiting species in real cases.

Rarity and Incomplete Sampling in DNA-Based Species Delimitation.

DNA-based species delimitation may be compromised by limited sampling effort and species rarity, including "singleton" representatives of species, which hampers estimates of intra- versus interspecies evolutionary processes. In a case study of southern African chafers (beetles in the family Scarabaeidae), many species and subclades were poorly represented and 48.5% of species were singletons. Using cox1 sequences from >500 specimens and ∼100 species, the Generalized Mixed Yule Coalescent (GMYC) analysis as well as various other approaches for DNA-based species delimitation (Automatic Barcode Gap Discovery (ABGD), Poisson tree processes (PTP), Species Identifier, Statistical Parsimony), frequently produced poor results if analyzing a narrow target group only, but the performance improved when several subclades were combined. Hence, low sampling may be compensated for by "clade addition" of lineages outside of the focal group. Similar findings were obtained in reanalysis of published data sets of taxonomically poorly known species assemblages of insects from Madagascar. The low performance of undersampled trees is not due to high proportions of singletons per se, as shown in simulations (with 13%, 40% and 52% singletons). However, the GMYC method was highly sensitive to variable effective population size ([Formula: see text]), which was exacerbated by variable species abundances in the simulations. Hence, low sampling success and rarity of species affect the power of the GMYC method only if they reflect great differences in [Formula: see text] among species. Potential negative effects of skewed species abundances and prevalence of singletons are ultimately an issue about the variation in [Formula: see text] and the degree to which this is correlated with the census population size and sampling success. Clade addition beyond a limited study group can overcome poor sampling for the GMYC method in particular under variable [Formula: see text] This effect was less pronounced for methods of species delimitation not based on coalescent models.

Ecology has contrasting effects on genetic variation within species versus rates of molecular evolution across species in water beetles.

Comparative analysis is a potentially powerful approach to study the effects of ecological traits on genetic variation and rate of evolution across species. However, the lack of suitable datasets means that comparative studies of correlates of genetic traits across an entire clade have been rare. Here, we use a large DNA-barcode dataset (5062 sequences) of water beetles to test the effects of species ecology and geographical distribution on genetic variation within species and rates of molecular evolution across species. We investigated species traits predicted to influence their genetic characteristics, such as surrogate measures of species population size, latitudinal distribution and habitat types, taking phylogeny into account. Genetic variation of cytochrome oxidase I in water beetles was positively correlated with occupancy (numbers of sites of species presence) and negatively with latitude, whereas substitution rates across species depended mainly on habitat types, and running water specialists had the highest rate. These results are consistent with theoretical predictions from nearly-neutral theories of evolution, and suggest that the comparative analysis using large databases can give insights into correlates of genetic variation and molecular evolution.

Comparative transcriptomic analysis of two closely related ground beetle species with marked genital divergence using pyrosequencing.

Ground beetles of the subgenus Ohomopterus (genus Carabus) show marked divergence in species-specific male and female genital morphologies, which contributes to reproductive isolation among species. Characterizing the genetic basis of species-specific genital morphology is essential for understanding their diversification, but genomic information on Ohomopterus is not yet available. We analyzed mRNA extracted from abdominal sections of the last instar larvae and pupae of two sister species, Carabus (Ohomopterus) iwawakianus and C. (O.) uenoi, which show marked differences in genital morphology, to compare transcriptomic profiles using Roche 454 pyrosequencing. We obtained 1,608,572 high-quality reads and assembled them into 176,278 unique sequences, of which 66,049 sequences were combined into 12,662 clusters. Differential expression analyses for sexed pupae suggested that four and five clusters were differentially expressed between species for males and females, respectively. We also identified orthologous sequences of genes involved in genital development in Drosophila, which potentially affect genital development and species-specific genital morphology in Ohomopterus. This study provides the first large transcriptomic data set for a morphologically diversified beetle group, which can facilitate future studies on the genetic basis of species-specific genitalia.

Whole-community DNA barcoding reveals a spatio-temporal continuum of biodiversity at species and genetic levels.

A correlation of species and genetic diversity has been proposed but not uniformly supported. Large-scale DNA barcoding provides qualitatively novel data to test for correlations across hierarchical levels (genes, genealogies and species), and may help to unveil the underlying processes. Here we analyse sequence variation in communities of aquatic beetles across Europe (>5,000 individuals) to test for self-similarity of beta diversity patterns at multiple hierarchical levels. We show that community similarity at all levels decreases exponentially with geographic distance, and initial similarity is correlated with the lineage age, consistent with a molecular clock. Log-log correlations between lineage age, number of lineages, and range sizes, reveal a fractal geometry in time and space, indicating a spatio-temporal continuum of biodiversity across scales. Simulations show that these findings mirror dispersal-constrained models of haplotype distributions. These novel macroecological patterns may be explained by neutral evolutionary processes, acting continuously over time to produce multi-scale regularities of biodiversity.

Delimiting species using single-locus data and the Generalized Mixed Yule Coalescent approach: a revised method and evaluation on simulated data sets.

DNA barcoding-type studies assemble single-locus data from large samples of individuals and species, and have provided new kinds of data for evolutionary surveys of diversity. An important goal of many such studies is to delimit evolutionarily significant species units, especially in biodiversity surveys from environmental DNA samples. The Generalized Mixed Yule Coalescent (GMYC) method is a likelihood method for delimiting species by fitting within- and between-species branching models to reconstructed gene trees. Although the method has been widely used, it has not previously been described in detail or evaluated fully against simulations of alternative scenarios of true patterns of population variation and divergence between species. Here, we present important reformulations to the GMYC method as originally specified, and demonstrate its robustness to a range of departures from its simplifying assumptions. The main factor affecting the accuracy of delimitation is the mean population size of species relative to divergence times between them. Other departures from the model assumptions, such as varying population sizes among species, alternative scenarios for speciation and extinction, and population growth or subdivision within species, have relatively smaller effects. Our simulations demonstrate that support measures derived from the likelihood function provide a robust indication of when the model performs well and when it leads to inaccurate delimitations. Finally, the so-called single-threshold version of the method outperforms the multiple-threshold version of the method on simulated data: we argue that this might represent a fundamental limit due to the nature of evidence used to delimit species in this approach. Together with other studies comparing its performance relative to other methods, our findings support the robustness of GMYC as a tool for delimiting species when only single-locus information is available.

The effect of geographical scale of sampling on DNA barcoding.

Eight years after DNA barcoding was formally proposed on a large scale, CO1 sequences are rapidly accumulating from around the world. While studies to date have mostly targeted local or regional species assemblages, the recent launch of the global iBOL project (International Barcode of Life), highlights the need to understand the effects of geographical scale on Barcoding's goals. Sampling has been central in the debate on DNA Barcoding, but the effect of the geographical scale of sampling has not yet been thoroughly and explicitly tested with empirical data. Here, we present a CO1 data set of aquatic predaceous diving beetles of the tribe Agabini, sampled throughout Europe, and use it to investigate how the geographic scale of sampling affects 1) the estimated intraspecific variation of species, 2) the genetic distance to the most closely related heterospecific, 3) the ratio of intraspecific and interspecific variation, 4) the frequency of taxonomically recognized species found to be monophyletic, and 5) query identification performance based on 6 different species assignment methods. Intraspecific variation was significantly correlated with the geographical scale of sampling (R-square = 0.7), and more than half of the species with 10 or more sampled individuals (N = 29) showed higher intraspecific variation than 1% sequence divergence. In contrast, the distance to the closest heterospecific showed a significant decrease with increasing geographical scale of sampling. The average genetic distance dropped from > 7% for samples within 1 km, to < 3.5% for samples up to > 6000 km apart. Over a third of the species were not monophyletic, and the proportion increased through locally, nationally, regionally, and continentally restricted subsets of the data. The success of identifying queries decreased with increasing spatial scale of sampling; liberal methods declined from 100% to around 90%, whereas strict methods dropped to below 50% at continental scales. The proportion of query identifications considered uncertain (more than one species < 1% distance from query) escalated from zero at local, to 50% at continental scale. Finally, by resampling the most widely sampled species we show that even if samples are collected to maximize the geographical coverage, up to 70 individuals are required to sample 95% of intraspecific variation. The results show that the geographical scale of sampling has a critical impact on the global application of DNA barcoding. Scale-effects result from the relative importance of different processes determining the composition of regional species assemblages (dispersal and ecological assembly) and global clades (demography, speciation, and extinction). The incorporation of geographical information, where available, will be required to obtain identification rates at global scales equivalent to those in regional barcoding studies. Our result hence provides an impetus for both smarter barcoding tools and sprouting national barcoding initiatives-smaller geographical scales deliver higher accuracy.

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.

Inferring evolutionarily significant units of bacterial diversity from broad environmental surveys of single-locus data.

By far the greatest challenge for diversity studies is to characterize the diversity of prokaryotes, which probably encompasses billions of species, most of which are unculturable. Recent advances in theory and analysis have focused on multi-locus approaches and on combined analysis of molecular and ecological data. However, broad environmental surveys of bacterial diversity still rely on single-locus data, notably 16S ribosomal DNA, and little other detailed information. Evolutionary methods of delimiting species from single-locus data alone need to consider population genetic and macroevolutionary theories for the expected levels of interspecific and intraspecific variation. We discuss the use of a recent evolutionary method, based on the theory of coalescence within independently evolving populations, compared with a traditional approach that uses a fixed threshold divergence to delimit species.

Accelerated species inventory on Madagascar using coalescent-based models of species delineation.

High-throughput DNA sequencing has the potential to accelerate species discovery if it is able to recognize evolutionary entities from sequence data that are comparable to species. The general mixed Yule-coalescent (GMYC) model estimates the species boundary from DNA surveys by identifying independently evolving lineages as a transition from coalescent to speciation branching patterns on a phylogenetic tree. Applied here to 12 families from 4 orders of insects in Madagascar, we used the model to delineate 370 putative species from mitochondrial DNA sequence variation among 1614 individuals. These were compared with data from the nuclear genome and morphological identification and found to be highly congruent (98% and 94%). We developed a modified GMYC that allows for a variable transition from coalescent to speciation among lineages. This revised model increased the congruence with morphology (97%), suggesting that a variable threshold better reflects the clustering of sequence data into biological species. Local endemism was pronounced in all 5 insect groups. Most species (60-91%) and haplotypes (88-99%) were found at only 1 of the 5 study sites (40-1000 km apart). This pronounced endemism resulted in a 37% increase in species numbers using diagnostic nucleotides in a population aggregation analysis. Sample sizes between 7 and 10 individuals represented a threshold above which there was minimal increase in genetic diversity, broadly agreeing with coalescent theory and other empirical studies. Our results from > 1.4 Mb of empirical data suggest that the GMYC model captures species boundaries comparable to those from traditional methods without the need for prior hypotheses of population coherence. This provides a method of species discovery and biodiversity assessment using single-locus data from mixed or environmental samples while building a globally available taxonomic database for future identifications.

Speciation and DNA barcodes: testing the effects of dispersal on the formation of discrete sequence clusters.

Large-scale sequencing of short mtDNA fragments for biodiversity inventories ('DNA barcoding') indicates that sequence variation in animal mtDNA is highly structured and partitioned into discrete genetic clusters that correspond broadly to species-level entities. Here we explore how the migration rate, an important demographic parameter that is directly related to population isolation, might affect variation in the strength of mtDNA clustering among taxa. Patterns of mtDNA variation were investigated in two groups of beetles that both contain lineages occupying habitats predicted to select for different dispersal abilities: predacious diving beetles (Dytiscidae) in the genus Bidessus from lotic and lentic habitats across Europe and darkling beetles (Tenebrionidae) in the genus Eutagenia from sand and other soil types in the Aegean Islands. The degree of genetic clustering was determined using the recently developed 'mixed Yule coalescent' (MYC) model that detects the transition from between-species to within-population branching patterns. Lineages from presumed stable habitats, and therefore displaying lower dispersal ability and migration rates, showed greater levels of mtDNA clustering and geographical subdivision than their close relatives inhabiting ephemeral habitats. Simulations of expected patterns of mtDNA variation under island models showed that MYC clusters are only detected when the migration rates are much lower than the value of Nm=1 typically used to define the threshold for neutral genetic divergence. Therefore, discrete mtDNA clusters provide strong evidence for independently evolving populations or species, but their formation is suppressed even under very low levels of dispersal.