Michael T. Monaghan

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.

The Effect of Geographical Scale of Sampling on DNA Barcoding

Abstract 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 Barcodings 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.

DNA-based species delineation in tropical beetles using mitochondrial and nuclear markers

DNA barcoding has been successfully implemented in the identification of previously described species, and in the process has revealed several cryptic species. It has been noted that such methods could also greatly assist in the discovery and delineation of undescribed species in poorly studied groups, although to date the feasibility of such an approach has not been examined explicitly. Here, we investigate the possibility of using short mitochondrial and nuclear DNA sequences to delimit putative species in groups lacking an existing taxonomic framework. We focussed on poorly known tropical water beetles (Coleoptera: Dytiscidae, Hydrophilidae) from Madagascar and dung beetles (Scarabaeidae) in the genus Canthon from the Neotropics. Mitochondrial DNA sequence variation proved to be highly structured, with >95% of the observed variation existing between discrete sets of very closely related genotypes. Sequence variation in nuclear 28S rRNA among the same individuals was lower by at least an order of magnitude, but 16 different genotypes were found in water beetles and 12 genotypes in Canthon, differing from each other by a minimum of two base pairs. The distribution of these 28S rRNA genotypes in individuals exactly matched the distribution of mtDNA clusters, suggesting that mtDNA patterns were not misleading because of introgression. Moreover, in a few cases where sequence information was available in GenBank for morphologically defined species of Canthon, these matched some of the DNA-based clusters. These findings demonstrate that clusters of close relatives can be identified readily in the sequence variation obtained in field collected samples, and that these clusters are likely to correspond to either previously described or unknown species. The results suggest that DNA-assisted taxonomy will not require more than a short fragment of mtDNA to provide a largely accurate picture of species boundaries in these groups. Applied on a large scale, this DNA-based approach could greatly improve the rate of species discovery in the large assemblages of insects that remain undescribed.

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.

Population genetic structure of 3 alpine stream insects: influences of gene flow, demographics, and habitat fragmentation

Estimating scales of dispersal for benthic macroinvertebrates using neutral genetic markers requires consideration of genetic, demographic, and historical influences on population genetic structure. We used allozyme electrophoresis to investigate the population genetic structure of 3 species of alpine stream insects among major drainages of the Swiss Alps (Rhine, Inn, and Ticino rivers), among streams within each drainage, and within single streams. Within streams we examined reaches that were fragmented by lakes or resevoirs and unfragmented reaches. Rhithrogena loyolaea (Heptageniidae) exhibited little genetic differentiation (θ) within (θ = 0.01-0.03) and among (θ = 0.02-0.03) streams but significant differentiation among drainages (θ = 0.08), suggesting that dispersal occurs among stream fragments and among stream valleys. Allogamus auricollis (Limnephilidae) did not exhibit genetic differentiation at any scale, suggesting that dispersal occurs throughout the geographical range of the study. In contrast, Baetis alpinus (Baetidae) showed moderate to substantial differentiation both within (θ = 0.08-0.39) and among (θ = 0.06-0.09) streams. However, a distinct lack of genetic differentiation for B. alpinus among major drainages of the Alps (θ = 0.01) suggests that low θ values reflect historical rather than present-day levels of gene flow. We suggest that genetic population structure reflects a lack of equilibrium between gene flow and genetic drift, resulting from historical gene flow that continues to mask reduced dispersal and from recurring processes of recruitment that lead to random changes in genetic signatures. We conclude that demographic processes affect small-scale patterns and historical processes affect large-scale patterns. The simultaneous study of multiple spatial scales helps determine the relative importance of each. A synthesis of our results and data from published studies indicated that 4 consistent patterns of genetic differentiation emerged when multiple spatial scales were investigated. These patterns are indicative of taxon-specific dispersal ability within and among streams and whether taxa are in gene flow-genetic drift equilibrium.

Physical factors influencing fine organic particle transport and deposition in streams

The influence of physical factors on the transport and deposition of fine particulate organic matter (FPOM, 53–106 μm) in streams was investigated using 14C-labeled natural detritus. Field estimates of mean FPOM transport distance (SP) were calculated as the inverse of the longitudinal loss rate (kP) of particles. Deposition was determined by standardizing kP for depth and velocity and expressed as the mass transfer coefficient, vdep. SP varied by orders of magnitude (7–1000 m) within and among streams. As expected, vdep behaved more conservatively than SP but still varied by a factor of 18 (0.06–1.10 mm/s). Field-estimated deposition velocities were always less than the quiescent water fall velocity (vfall) but no consistent relationship existed between the 2 (r = 0.26, p = 0.53). Variability in SP was strongly associated with the cross-sectional area of the transient storage zone (AS; r = 0.93, p < 0.01) and the uptake length of water (SW; r = 0.84, p = 0.01). The transfer coefficient was highest in the 2 smallest streams (Q < 15 L/s) but was similar, and unrelated to stream size, among 6 experiments conducted in stream segments where Q exceeded 100 L/s. Variability in FPOM vdep was less related to physical characteristics than SP, although a significant, positive correlation was detected between vdep and AS/A. Evidence from this study suggests that the mechanisms assumed to govern particle transport in gravitational/hydrodynamic models may not be solely responsible for FPOM deposition in streams and that alternative processes, such as hyporheic filtration and biotic retention, may be important.

Trans-oceanic and endemic origins of the small minnow mayflies (Ephemeroptera, Baetidae) of Madagascar

We investigated the relative importance of dispersal and vicariance in forming the Madagascar insect fauna, sequencing approximately 2300 bp from three rRNA gene regions to investigate the phylogeny of Afrotropical small minnow mayflies (Ephemeroptera: Baetidae). Six lineages contained trans-oceanic sister taxa, and variation in genetic divergence between sister taxa revealed relationships that range from very recent dispersal to ancient vicariance. Dispersal was most recent and frequent in species that spend the larval stage in standing water, adding to evidence that these evolutionarily unstable habitats may select for ecological traits that increase dispersal in insects. Ancestral state likelihood analysis suggested at least one Afrotropical lineage had its origin in Madagascar, demonstrating that unidirectional dispersal from a continental source may be too simplistic. We conclude that the Malagasy mayfly fauna should be considered in a biogeographical context that extends beyond Madagascar itself, encompassing trans-oceanic dispersal within multiple lineages.

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.

Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater fishes

Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule‐coalescent (GMYC) model‐based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA‐barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endangered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large‐scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities.

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.