Haplowebs as a graphical tool for delimiting species: a revival of Doyle's "field for recombination" approach and its application to the coral genus Pocillopora in Clipperton.
ABSTRACT: BACKGROUND: Usual methods for inferring species boundaries from molecular sequence data rely either on gene trees or on population genetic analyses. Another way of delimiting species, based on a view of species as "fields for recombination" (FFRs) characterized by mutual allelic exclusivity, was suggested in 1995 by Doyle. Here we propose to use haplowebs (haplotype networks with additional connections between haplotypes found co-occurring in heterozygous individuals) to visualize and delineate single-locus FFRs (sl-FFRs). Furthermore, we introduce a method to quantify the reliability of putative species boundaries according to the number of independent markers that support them, and illustrate this approach with a case study of taxonomically difficult corals of the genus Pocillopora collected around Clipperton Island (far eastern Pacific). RESULTS: One haploweb built from intron sequences of the ATP synthase ? subunit gene revealed the presence of two sl-FFRs among our 74 coral samples, whereas a second one built from ITS sequences turned out to be composed of four sl-FFRs. As a third independent marker, we performed a combined analysis of two regions of the mitochondrial genome: since haplowebs are not suited to analyze non-recombining markers, individuals were sorted into four haplogroups according to their mitochondrial sequences. Among all possible bipartitions of our set of samples, thirteen were supported by at least one molecular dataset, none by two and only one by all three datasets: this congruent pattern obtained from independent nuclear and mitochondrial markers indicates that two species of Pocillopora are present in Clipperton. CONCLUSIONS: Our approach builds on Doyle's method and extends it by introducing an intuitive, user-friendly graphical representation and by proposing a conceptual framework to analyze and quantify the congruence between sl-FFRs obtained from several independent markers. Like delineation methods based on population-level statistical approaches, our method can distinguish closely-related species that have not yet reached reciprocal monophyly at most or all of their loci; like tree-based approaches, it can yield meaningful conclusions using a number of independent markers as low as three. Future efforts will aim to develop programs that speed up the construction of haplowebs from FASTA sequence alignments and help perform the congruence analysis outlined in this article.
Project description:Delimiting and describing species is fundamental to numerous biological disciplines such as evolution, macroecology, and conservation. Delimiting species as independent evolutionary lineages may and often does yield different outcomes depending on the species criteria applied, but methods should be chosen that minimize the inference of objectively erroneous species limits. Several protocols exploit single-gene or multi-gene coalescence statistics, assignment tests or other rationales related to nuclear DNA (nDNA) allele sharing to automatically delimit species. We apply seven different species delimitation protocols to a taxonomically confusing group of Malagasy lizards (Madascincus), and compare the resulting taxonomies with two newly developed metrics: the Taxonomic index of congruence C tax which quantifies the congruence between two taxonomies, and the Relative taxonomic resolving power index R tax which quantifies the potential of an approach to capture a high number of species boundaries. The protocols differed in the total number of species proposed, between 9 and 34, and were also highly incongruent in placing species boundaries. The Generalized Mixed Yule-Coalescent approach captured the highest number of potential species boundaries but many of these were clearly contradicted by extensive nDNA admixture between sympatric mitochondrial DNA (mtDNA) haplotype lineages. Delimiting species as phenotypically diagnosable mtDNA clades failed to detect two cryptic species that are unambiguous due to a lack of nDNA gene flow despite sympatry. We also consider the high number of species boundaries and their placement by multi-gene Bayesian species delimitation as poorly reliable whereas the Bayesian assignment test approach provided a species delimitation highly congruent with integrative taxonomic practice. The present study illustrates the trade-off in taxonomy between reliability (favored by conservative approaches) and resolving power (favored by inflationist approaches). Quantifying excessive splitting is more difficult than quantifying excessive lumping, suggesting a priority for conservative taxonomies in which errors are more liable to be detected and corrected by subsequent studies.
Project description:Scleractinia of the Maputaland reef complex (MRC) in South Africa exist at the margins of the Western Indian Ocean (WIO) coral distribution and are the only substantial hermatypic coral communities in South Africa. Pocillopora species occupy a conspicuous component of the MRC, and previous investigations identified three species of Pocillopora utilizing conventional taxonomy. Thus, our aims were four-fold: to elucidate Pocillopora species diversity using genetic techniques, primarily using species delimitation methods based on the ORF gene; to test for the presence of hybridisation within the Pocillopora community on the South-West margin of distribution in the Indian Ocean using two nuclear and two mitochondrial markers; to test the presence of cryptic species, using 13 microsatellite markers, finally, to elucidate the degree of genetic diversity within each Pocillopora species found and compare this to communities in lower latitudes. We illustrate taxonomic inconsistencies between these inventories and our phylogenetic data. The MRC harbours unique populations of Pocillopora, consisting of three species hypothetically co-occurring throughout the south WIO, namely: P. meandrina/P. eydouxi, commonly misidentified as P. verrucosa, P. verrucosa, sometimes correctly identified, but also commonly misidentified as P. damicornis sensu lato, and P. villosa, almost always misidentified as P. eydouxi. The hypothesis that hybrid swarms of Pocillopora occur in marginal environments such as the MRC was not supported, with low levels of introgressive hybridization reported instead. Analyses illustrate low genetic diversity at the species and population resolutions, suggesting a small founder population for each species. Nevertheless, these populations are demographically unique, exhibiting high levels of ITS2 haplotype endemism compared to higher latitude populations and the rest of the WIO. Pocillopora diversity on the MRC represents a unique assemblage and warrants further protection.
Project description:It can be challenging to identify scleractinian corals from the genus Pocillopora Lamarck 1816 in the field because of their large range of inter- and intra-specific morphological variation that co-occur with changes in the physical environment. This task is made more arduous in the context of a depth gradient, where light and water current could greatly affect the morphology of the corallum. Pocillopora verrucosa (Ellis & Solander 1786) in Taiwan was previously reported exclusively from shallow waters (<10 m in depth), but a recent observation of this species in the mesophotic zone (>40 m in depth) questions this bathymetric distribution. We used the mitochondrial open reading frame and the histone 3 molecular markers to investigate the vertical and horizontal spatial distribution of P. verrucosa around Ludao (Green Island), Taiwan. We genotyped 101 P. verrucosa-like colonies collected from four depth zones, ranging from 7 to 45 m, at three sites around the island. Of the 101 colonies sampled, 85 were genotyped as P. verrucosa, 15 as P. meandrina, and one specimen as an undescribed Pocillopora species. P. verrucosa was found at all depths, while P. meandrina and the undescribed Pocillopora specimen were limited to 15 m depth. P. verrucosa has a large bathymetric distribution around Ludao and could benefit from the refuge that the mesophotic zone offers. This study illustrates the difficulty of identifying Pocillopora corals in the field and emphasizes the relevance of molecular taxonomy as an important and complementary tool to traditional taxonomy for clarifying vertical and horizontal species distribution. Our results also illustrate the need in conservation biology to target species genetic diversity rather than just species diversity.
Project description:Species within the scleractinian genus Pocillopora Lamarck 1816 exhibit extreme phenotypic plasticity, making identification based on morphology difficult. However, the mitochondrial open reading frame (mtORF) marker provides a useful genetic tool for identification of most species in this genus, with a notable exception of P. eydouxi and P. meandrina. Based on recent genomic work, we present a quick and simple, gel-based restriction fragment length polymorphism (RFLP) method for the identification of all six Pocillopora species occurring in Hawai'i by amplifying either the mtORF region, a newly discovered histone region, or both, and then using the restriction enzymes targeting diagnostic sequences we unambiguously identify each species. Using this approach, we documented frequent misidentification of Pocillopora species based on colony morphology. We found that P. acuta colonies are frequently mistakenly identified as P. damicornis in K?ne'ohe Bay, O'ahu. We also found that P. meandrina likely has a northern range limit in the Northwest Hawaiian Islands, above which P. ligulata was regularly mistaken for P. meandrina.
Project description:Pocillopora damicornis (Linnaeus, 1758; Scleractinia, Pocilloporidae) has recently been found to comprise at least five distinct genetic lineages in Eastern Australia, some of which likely represent cryptic species. Due to similar and plastic gross morphology of these lineages, field identification is often difficult. Here we present a quick, cost effective genetic assay as well as three novel microsatellite markers that distinguish the two most common lineages found on the Great Barrier Reef. The assay is based on PCR amplification of two regions within the mitochondrial putative control region, which show consistent and easily identifiable fragment size differences for the two genetic lineages after Alu1 restriction enzyme digestion of the amplicons.
Project description:Processes of cnidarian evolution, including hybridization and phenotypic plasticity, have complicated the clear diagnosis of species boundaries within the phylum. Pocillopora acuta, a species of scleractinian coral that was recently split from the widespread Pocillopora damicornis species complex, occurs in at least two distinct morphs on the Great Barrier Reef. Contrasting morphology combined with evidence of differential bleaching thresholds among sympatrically distributed colonies suggest that the taxonomy of this recently described species is not fully resolved and may represent its own species complex. To examine the basis of sympatric differentiation between the two morphs, we combined analyses of micro- and macro-skeletal morphology with genome wide sequencing of the coral host, as well as ITS2 genotyping of the associated Symbiodinium communities. We found consistent differences between morphs on both the macro- and micro-skeletal scale. In addition, we identified 18 candidate functional genes that relate to skeletal formation and morphology that may explain how the two morphs regulate growth to achieve their distinct growth forms. With inconclusive results in endosymbiotic algal community diversity between the two morphs, we propose that colony morphology may be linked to bleaching susceptibility. We conclude that cryptic speciation may be in the early stages within the species P. acuta.
Project description:We aim to evaluate the genetic structure of an Atlantic Forest amphibian species, Scinax eurydice, testing the congruence among patterns identified and proposed by the literature for Pleistocene refugia, microrefugia, and geographic barriers to gene flow such as major rivers. Furthermore, we aim to evaluate predictions of such barriers and refugia on the genetic structure of the species, such as presence/absence of dispersal, timing since separation, and population expansions/contractions. We sequenced mitochondrial and nuclear genetic markers on 94 tissue samples from 41 localities. We inferred a gene tree and estimated genetic distances using mtDNA sequences. We then ran population clustering and assignment methods, AMOVA, and estimated migration rates among populations identified through mtDNA and nDNA analyses. We used a dated species tree, skyline plots, and summary statistics to evaluate concordance between population's distributions and geographic barriers and Pleistocene refugia. Scinax eurydice showed high mtDNA divergences and four clearly distinct mtDNA lineages. Species tree and population assignment tests supported the existence of two major clades corresponding to northeastern and southeastern Atlantic Forest in Brazil, each one composed of two other clades. Lineage splitting events occurred from late Pliocene to Pleistocene. We identified demographic expansions in two clades, and inexistent to low levels of migrations among different populations. Genetic patterns and demographic data support the existence of two northern Refuge and corroborate microrefugia south of the Doce/Jequitinhonha Rivers biogeographic divide. The results agree with a scenario of recent demographic expansion of lowland taxa. Scinax eurydice comprises a species complex, harboring undescribed taxa consistent with Pleistocene refugia. Two rivers lie at the boundaries among populations and endorse their role as secondary barriers to gene flow.
Project description:A previously reported mitochondrial DNA (mtDNA) phylogeny of Crematogaster (subgenus Decacrema) ants inhabiting Macaranga myrmecophytes indicated that the partners diversified synchronously and their specific association has been maintained for 20 million years. However, the mtDNA clades did not exactly match morphological species, probably owing to introgressive hybridization among younger species. In this study, we determined the congruence between nuclear simple sequence repeat (SSR, also called microsatellite) genotyping and mtDNA phylogeny to confirm the suitability of the mtDNA phylogeny for inferring the evolutionary history of Decacrema ants. Analyses of ant samples from Lambir Hills National park, northeastern Borneo, showed overall congruence between the SSR and mtDNA groupings, indicating that mtDNA markers are useful for delimiting species, at least at the local level. We also found overall high host-plant specificity of the SSR genotypes of Decacrema ants, consistent with the specificity based on the mtDNA phylogeny. Further, we detected cryptic genetic assemblages exhibiting high specificity toward particular plant species within a single mtDNA clade. This finding, which may be evidence for rapid ecological and genetic differentiation following a host shift, is a new insight into the previously suggested long-term codiversification of Decacrema ants and Macaranga plants.
Project description:Population genetics of the coral genus Pocillopora have been more intensively studied than those of any other reef-building taxon. However, recent investigations have revealed that the current morphological classification is inadequate to represent genetic lineages. In this study, we isolated and characterized novel microsatellite loci from morphological Pocillopora meandrina (Type 1) and Pocillopora acuta (Type 5). Furthermore, we characterized previously reported microsatellite loci. A total of 27 loci (13 novel loci) proved useful for population genetic analyses at two sites in the Ryukyu Archipelago, in the northwestern Pacific. Clonal diversity differed in each genetic lineage. Genetic structure suggested by microsatellites corresponded to clusters in a phylogenetic tree constructed from a mitochondrial open reading frame (mtORF). In addition, we found an unknown mitochondrial haplotype of this mtORF. These microsatellite loci will be useful for studies of connectivity and genetic diversity of Pocillopora populations, and will also support coral reef conservation.
Project description:Mitochondrial DNA (mtDNA) is widely used to clarify phylogenetic relationships among and within species, and to determine population structure. Due to the linked nature of mtDNA genes it is expected that different genes will show similar results. Phylogenetic incongruence using mtDNA genes may result from processes such as heteroplasmy, nuclear integration of mitochondrial genes, polymerase errors, contamination, and recombination. In this study we used sequences from two mitochondrial genes (cytochrome b and cytochrome oxidase subunit I) from the wild vectors of Chagas disease, Triatoma eratyrusiformis and Mepraia species to test for topological congruence. The results showed some cases of phylogenetic incongruence due to misplacement of four haplotypes of four individuals. We discuss the possible causes of such incongruence and suggest that the explanation is an intra-individual variation likely due to heteroplasmy. This phenomenon is an independent evidence of common ancestry between these taxa.