Article

Microsatellite development and detection of admixture among three sympatric Haploblepharus species (Carcharhiniformes: Scyliorhinidae)

Authors:
  • CPAWS-BC (Canadian Parks and Wilderness Society - British Columbia)
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Abstract

• Haploblepharus is an understudied genus comprising four recognized scyliorhinid species that are endemic to southern Africa. Species identification within this group has historically been problematic due to a high degree of morphological conservatism among congeners, further complicated by the possibility of interspecific hybridization. • This study describes the development of two microsatellite panels comprising 10 polymorphic markers for the puffadder shyshark, Haploblepharus edwardsii . The markers were characterized in 35 H. edwardsii specimens and tested for cross‐species utility in Haploblepharus fuscus , Haploblepharus pictus , and the more distantly related scyliorhinid Halaelurus natalensis . • Genetic diversity statistics were estimated for each species, and the presence of population differentiation was tested for in H. edwardsii and H. pictus . Furthermore, interspecific genetic differentiation was examined to infer the potential use of these markers for species identification as well as for detecting signatures of admixture among Haploblepharus species. • All microsatellite markers were polymorphic in each species, with polymorphism information contents ranging from 0.43 to 0.62. Population differentiation was only evident for H. pictus , where genetic discontinuity was detected among geographically distant sampling sites. • Statistically significant differentiation (fixation index between populations F ST = 0.091 to 0.382) was found between all species; however, the level of differentiation between H. fuscus and H. pictus was low in comparison and seemingly at a population level rather than at a species level. Species assignment using Bayesian clustering analysis resulted in approximately 57% of 88 genotyped specimens being unambiguously assigned to a distinct genetic cluster that confirmed accurate taxonomic assignment. • Overall, the low levels of differentiation together with the presence of distinct and admixed genetic clusters suggests a recent divergence and possible contemporary hybridization within the genus Haploblepharus. As such, conservation strategies should be focused on the generic level until such a time that Haploblepharus species can be readily identified.

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... However, as heteroplasmy has previously been shown to resolve to homoplasmy within a few generations [91,92], the shared characteristic of ambiguous sites among Haploblepharus species could be explained by the occurrence of contemporary hybridisation and paternal leakage (i.e., the bi-parental inheritance of mitochondrial DNA) [77,93]. Although the inability to delineate species was not the intended outcome of this study, the previously reported overlap of morphological features [16,18] and admixed species clusters [19], in conjunction with the presence of nucleotide ambiguities, highlights the ongoing evolutionary mechanisms at play within this morphologically conserved genus. It is important to note that the presence of heteroplasmy is mentioned herein with caution. ...
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This paper examines two assumptions that have formed the basis for much of the past and present work on hybrid zones. These assumptions derive from the observation that crosses between genetically divergent individuals (e.g., from different subspecies, species, etc.) often give rise to genotypes that are less fertile or less viable than those produced from crosses between genetically similar individuals. The first assumption is that natural hybridization will not affect the evolutionary history of the hybridizing forms because there is a low probability of producing novel genotypes with higher relative fitness. The second viewpoint is more extreme in that it assumes that all hybrid genotypes will be less fit. Even if rare gene flow does occur it will thus not contribute to patterns of diversification or adaptation because the hybrids will always be selected against. Examples from both plant and animal hybridization are discussed that are not consistent with these assumptions. Numerous instances of natural hybridization are used to demonstrate that extremely low fertility or viability of early-generation hybrids (e.g., F 1, F 2, B 1) does not necessarily prevent extensive gene flow and the establishment of new evolutionary lineages. In addition, it is demonstrated that various hybrid genotypes have equivalent or higher fitness than their parents in certain habitats.
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Interspecies hybridisation in nature is a well-studied phenomenon, but it has not been analysed using genetic markers in the class Chondrichthyes (sharks, rays and chimeras). Two black-tip whaler shark species (Australian, Carcharhinus tilstoni; Common, C. limbatus) have overlapping distributions in Australia, distinct mitochondrial DNA sequence (ND4, COI, control region) and distinct morphological features such as length at sexual maturity, length at birth and number of vertebrae. A mismatch was observed between species identification using mtDNA sequence and species identification using morphological characters. To test whether hybridisation between the two species was responsible, a nuclear gene with species-specific mutations was sequenced. Extensive interspecies hybridisation was found to be occurring. Hybrids were found from five locations on the eastern Australian coastline, spanning 2,000 km. If hybrid fitness is low and hybrids are common, then fisheries recruitment may be overestimated and the productivity of the black-tip shark fishery may be well below that required to support commercial exploitation. To guard against identification errors, the likelihood of hybridisation and subsequent introgression should be assessed prior to using mtDNA (e.g. barcoding) to identify shark species. The C. limbatus-C. tilstoni species complex provides a unique opportunity to investigate the ability of sharks to adapt to environmental change, in particular, the impact of hybridization on species distributions which favour C. tilstoni along the north and C. limbatus along the south eastern Australian coastline.
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This paper examines the alpha level taxonomy of the genus Haploblepharus Garman, 1913 (Chondrichthyes; Scyliorhinidae). Three species are endemic to South Africa, with one species occurring in Namibia and South Africa. Haploblepharus pictus exhibits considerable colour variation which has led to some confusion between that species and H. edwardsii, resulting in a significant range extension for H. pictus. A neotype is designated for H. edwardsii, and problematic Haploblepharus juveniles are tentatively assigned to H. kistnasamyi. A novel species identification key is presented for Haploblepharus, and a comprehensive review of the taxonomy (including type material and synonyms) and distribution of all taxa is presented. The genus Haploblepharus contains four contemporary taxa: H. edwardsii (Schinz, 1822), H. pictus (Müller & Henle, 1838), H. fuscus Smith, 1950, and H. kistnasamyi Human & Compagno, 2006.
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The common approach to the multiplicity problem calls for controlling the familywise error rate (FWER). This approach, though, has faults, and we point out a few. A different approach to problems of multiple significance testing is presented. It calls for controlling the expected proportion of falsely rejected hypotheses — the false discovery rate. This error rate is equivalent to the FWER when all hypotheses are true but is smaller otherwise. Therefore, in problems where the control of the false discovery rate rather than that of the FWER is desired, there is potential for a gain in power. A simple sequential Bonferronitype procedure is proved to control the false discovery rate for independent test statistics, and a simulation study shows that the gain in power is substantial. The use of the new procedure and the appropriateness of the criterion are illustrated with examples.
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We describe a model-based clustering method for using multilocus genotype data to infer population structure and assign individuals to populations. We assume a model in which there are K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned (probabilistically) to populations, or jointly to two or more populations if their genotypes indicate that they are admixed. Our model does not assume a particular mutation process, and it can be applied to most of the commonly used genetic markers, provided that they are not closely linked. Applications of our method include demonstrating the presence of population structure, assigning individuals to populations, studying hybrid zones, and identifying migrants and admixed individuals. We show that the method can produce highly accurate assignments using modest numbers of loci—e.g., seven microsatellite loci in an example using genotype data from an endangered bird species. The software used for this article is available from http://www.stats.ox.ac.uk/~pritch/home.html.
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Species diversity is declining more rapidly in freshwater ecosystems than in any other, but the consequences for genetic diversity, and hence evolutionary potential, are poorly understood. In part this reflects limited use and development of modern molecular tools and genetic approaches to address conservation questions in rivers, lakes and wetlands. As widespread, diverse and functionally important organisms, freshwater macroinvertebrates are ideal candidates for genetic approaches to reveal, for example, the conservation consequences of demographic histories and past disturbances. However, the availability of microsatellite markers for this group is very limited. Using next generation sequencing, microsatellite markers were developed for Isoperla grammatica (Poda, 1761), Amphinemura sulcicollis (Stephens, 1836) and Baetis rhodani (Pictet, 1843) to enable conservation genetic investigations of these widespread invertebrate species. Fifty‐two robust microsatellite loci were developed (18, 21 and 13 per species), all with high levels of allelic diversity (7–27, 3–16, 5–13 alleles per loci, respectively). These tools will allow assessment of genetic structure, dispersal and demographic resilience in these model species as a function of environmental change and variation, thereby aiding freshwater monitoring and conservation. The authors urge further capacity building to support genetic applications to the conservation biology of other aquatic organisms. Copyright © 2016 John Wiley & Sons, Ltd.
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Closely related species can provide valuable insights into evolutionary processes through comparison of their ecology, geographic distribution and the history recorded in their genomes. In the Indo-Pacific, many reef fishes are divided into sister species that come into secondary contact at biogeographic borders, most prominently where Indian Ocean and Pacific Ocean faunas meet. It is unclear whether hybridization in this contact zone represents incomplete speciation, secondary contact, an evolutionary dead-end (for hybrids) or some combination of the above. To address these issues, we conducted comprehensive surveys of two widely-distributed surgeonfish species, Acanthurus leucosternon (N = 141) and A. nigricans (N = 412), with mtDNA cytochrome b sequences and ten microsatellite loci. These surgeonfishes are found primarily in the Indian and Pacific Oceans, respectively, but overlap at the Christmas and Cocos-Keeling Islands hybrid zone in the eastern Indian Ocean. We also sampled the two other Pacific members of this species complex, A. achilles (N = 54) and A. japonicus (N = 49), which are known to hybridize with A. nigricans where their ranges overlap. Our results indicate separation between the four species that range from the recent Pleistocene to late Pliocene (235,000 to 2.25 million years ago). The Pacific A. achilles is the most divergent (and possibly ancestral) species with mtDNA dcorr ≈ 0.04, whereas the other two Pacific species (A. japonicus and A. nigricans) are distinguishable only at a population or subspecies level (ΦST = 0.6533, P < 0.001). Little population structure was observed within species, with evidence of recent population expansion across all four geographic ranges. We detected sharing of mtDNA haplotypes between species and extensive hybridization based on microsatellites, consistent with later generation hybrids but also the effects of allele homoplasy. Despite extensive introgression, 98% of specimens had concordance between mtDNA lineage and species identification based on external morphology, indicating that species integrity may not be eroding. The A. nigricans complex demonstrates a range of outcomes from incomplete speciation to secondary contact to decreasing hybridization with increasing evolutionary depth.
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Inferences of population structure and more precisely the identification of genetically homogeneous groups of individuals are essential to the fields of ecology, evolutionary biology, and conservation biology. Such population structure inferences are routinely investigated via the program STRUCTURE implementing a Bayesian algorithm to identify groups of individuals at Hardy-Weinberg and linkage equilibrium. While the method is performing relatively well under various population models with even sampling between subpopulations, the robustness of the method to uneven sample size between subpopulations and/or hierarchical levels of population structure has not yet been tested despite being commonly encountered in empirical datasets. In this study, I used simulated and empirical microsatellite datasets to investigate the impact of uneven sample size between subpopulations and/or hierarchical levels of population structure on the detected population structure. The results demonstrated that uneven sampling often leads to wrong inferences on hierarchical structure and downward biased estimates of the true number of subpopulations. Distinct subpopulations with reduced sampling tended to be merged together, whilst at the same time, individuals from extensively sampled subpopulations were generally split, despite belonging to the same panmictic population. Four new supervised methods to detect the number of clusters were developed and tested as part of this study and were found to outperform the existing methods using both evenly and unevenly sampled datasets. Additionally, a sub-sampling strategy aiming to reduce sampling unevenness between subpopulations is presented and tested. These results altogether demonstrate that when sampling evenness is accounted for, the detection of the correct population structure is greatly improved. This article is protected by copyright. All rights reserved.
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A new species of catshark, Haploblepharus kistnasamyi sp. nov. (Class Chondrichthyes, Order Carcharhiniformes, Family Scyliorhinidae) is described from kwaZulu-Natal, South Africa. The type series includes the holotype, RUSI 39835, and two paratypes, RUSI 6075 and RUSI 6077. This species was previously recognised as a variant of H. edwardsii (Schinz 1822), which occurs along the southern coast of South Africa. The colour pattern of H. kistnasamyi is superficially similar to H. edwardsii although distinct; however H. kistnasamyi is distinguished from all other Haploblepharus in having a stockier build compared to its congeners, a less depressed head and trunk, and a compressed caudal peduncle.
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Hybridization is an important evolutionary mechanism in plants and has been increasingly documented in animals. Difficulty in reconstruction of reticulate evolution, however, has been a longstanding problem in phylogenetics. Consequently, hybrid speciation may play a major role in causing topological incongruence between gene trees. The incongruence, in turn, offers an opportunity to detect hybrid speciation. Here we characterized certain distinctions between hybridization and other biological processes, including lineage sorting, paralogy,and lateral gene transfer,that are responsible for topological incongruence between gene trees. Consider two incongruent gene trees with three taxa, A, B, and C, where B is a sister group of A on gene tree 1 but a sister group of C on gene tree 2. With a theoretical model based on the molecular clock, we demonstrate that time of divergence of each gene between taxa A and C is nearly equal in the case of hybridization (B is a hybrid) or lateral gene transfer, but differs significantly in the case of lineage sorting or paralogy. After developing a bootstrap test to test these alternative hypotheses, we extended the model and test to account for incongruent gene trees with numerous taxa. Computer simulation studies supported the validity of the theoretical model and bootstrap test when each gene evolved at a constant rate. The computer simulation also suggested that the model remained valid as long as the rate heterogeneity was occurring proportionally in the same taxa for both genes. Although the model could not test hypotheses of hybridization versus lateral gene transfer as the cause of incongruence, these two processes may be distinguished by comparing phylogenies of multiple unlinked genes.
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Christmas Island is located at the overlap of the Indian and Pacific Ocean marine provinces and is a hot spot for marine hybridization. Here, we evaluate the ecological framework and genetic consequences of hybridization between butterflyfishes Chaeto-don guttatissimus and Chaetodon punctatofasciatus. Further, we compare our current findings to those from a previous study of hybridization between Chaetodon trifascia-tus and Chaetodon lunulatus. For both species groups, habitat and dietary overlap between parental species facilitate frequent heterospecific encounters. Low abundance of potential mates promotes heterospecific pair formation and the breakdown of assor-tative mating. Despite similarities in ecological frameworks, the population genetic signatures of hybridization differ between the species groups. Mitochondrial and nuclear data from C. guttatissimus 3 C. punctatofasciatus (1% divergence at cyt b) show bidi-rectional maternal contributions and relatively high levels of introgression, both inside and outside the Christmas Island hybrid zone. In contrast, C. trifasciatus 3 C. lunula-tus (5% cyt b divergence) exhibit unidirectional mitochondrial inheritance and almost no introgression. Back-crossing of hybrid C. guttatissimus 3 C. punctatofasciatus and parental genotypes may eventually confound species-specific signals within the hybrid zone. In contrast, hybrids of C. trifasciatus and C. lunulatus may coexist with and remain genetically distinct from the parents. Our results, and comparisons with hybrid-ization studies in other reef fish families, indicate that genetic distance between hybridizing species may be a factor influencing outcomes of hybridization in reef fish, which is consistent with predictions from terrestrially derived hybridization theory.
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The identification of the genetic structure of populations from multilocus genotype data has become a central component of modern population-genetic data analysis. Application of model-based clustering programs often entails a number of steps, in which the user considers different modeling assumptions, compares results across different pre-determined values of the number of assumed clusters (a parameter typically denoted K), examines multiple independent runs for each fixed value of K, and distinguishes among runs belonging to substantially distinct clustering solutions. Here, we present Clumpak (Cluster Markov Packager Across K), a method that automates the post-processing of results of model-based population structure analyses. For analyzing multiple independent runs at a single K value, Clumpak identifies sets of highly similar runs, separating distinct groups of runs that represent distinct modes in the space of possible solutions. This procedure, which generates a consensus solution for each distinct mode, is performed by the use of a Markov clustering algorithm that relies on a similarity matrix between replicate runs, as computed by the software Clumpp. Next, Clumpak identifies an optimal alignment of inferred clusters across different values of K, extending a similar approach implemented for a fixed K in Clumpp, and simplifying the comparison of clustering results across different K values. Clumpak incorporates additional features, such as implementations of methods for choosing K and comparing solutions obtained by different programs, models, or data subsets. Clumpak, available at http://clumpak.tau.ac.il, simplifies the use of model-based analyses of population structure in population genetics and molecular ecology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Article
Coastal and demersal chondrichthyans, such as the small-spotted catshark, are expected to exhibit genetic differentiation in areas of complex geomorphology like the Mediterranean Basin because of their limited dispersal ability. To test this hypothesis, we used a fragment of the mitochondrial cytochrome c oxidase subunit I gene and 12 nuclear microsatellite loci in order to investigate the genetic structure and historical demography of this species, and to identify potential barriers to gene flow. Samples were collected from the Balearic Islands, the Algerian Basin, the Ionian Sea, the Corinthian Gulf and various locations across the Aegean Sea. Additional sequences from the Atlantic and the Levantine Basin retrieved from GenBank were included in the mitochondrial DNA analysis. Both mitochondrial and nuclear microsatellite DNA data revealed a strong genetic subdivision, mainly between the western and eastern Mediterranean, whereas the Levantine Basin shared haplotypes with both areas. The geographic isolation of the Mediterranean basins seems to enforce the population genetic differentiation of the species, with the deep sea acting as a strong barrier to its dispersal. Contrasting historical demographic patterns were also observed in different parts of the species' distribution, most notably a population growth trend in the western Mediterranean/Atlantic area and a slight decreasing one in the Aegean Sea. The different effects of the Pleistocene glacial periods on the habitat availability may explain the contrasting demographic patterns observed. The current findings suggest that the small-spotted catshark exhibits several genetic stocks in the Mediterranean, although further study is needed.Heredity advance online publication, 3 December 2014; doi:10.1038/hdy.2014.107.
Article
The unpredictable and low cross-amplification success of microsatellite loci tested for congeneric amphibian species has mainly been explained by the size and complexity of amphibian genomes, but also by taxonomy that is inconsistent with phylogenetic relationships among taxa. Here, we tested whether the cross-amplification success of nine new and 11 published microsatellite loci cloned for an amphibian source species, the fire salamander (Salamandra salamandra), correlated with the genetic distance across all members of True Salamanders (genera Chioglossa, Lyciasalamandra, Mertensiella and Salamandra that form a monophyletic clade within the family of Salamandridae) serving as target species. Cross-amplification success varied strongly among the species and showed a highly significant negative relationship with genetic distance and amplification success. Even though lineages of S. salamandra and Lyciasalamndra have separated more than 30 Ma, a within genus amplification success rate of 65% was achieved for species of Lyciasalamandra thus demonstrating that an efficient cross-species amplification of microsatellite loci in amphibians is feasible even across large evolutionary distances. A decrease in genome size, on the other hand, paralleled also a decrease in amplified loci and therefore contradicted previous results and expectations that amplification success should increase with a decrease in genome size. However, in line with other studies, our comprehensive dataset clearly shows that cross-amplification success of microsatellite loci is well explained by phylogenetic divergence between species. As taxonomic classifications on the species and genus level do not necessarily mirror phylogenetic divergence between species, the pure belonging of species to the same taxonomic units (i.e. species or genus) might be less useful to predict cross-amplification success of microsatellite loci between such species.
Article
Compilation and analysis of information from the literature regarding cross-species microsatellite amplification and polymorphism success, and relating this to source-target species genetic distance as estimated by pairwise cytochrome b (cytb) divergence, enabled an in-depth investigation of factors affecting avian cross-species microsatellite amplification. Source-target species cytb distances provided accurate estimates of cross-species microsatellite amplification/polymorphism success rates not only in birds, but also in taxa where microsatellites cross-amplify across contrasting levels of taxonomic classification (frogs and cetaceans). As cytb is one of the most commonly sequenced DNA regions, pairwise cytb genetic distances should therefore be useful for predicting cross-species microsatellite success across a range of taxonomic groups. While the most important factor affecting cross-species microsatellite amplification/polymorphism success was a negative association with source-target species genetic distance, associations with additional features affecting cross-species amplification/polymorphism success included: decreasing PCR annealing temperature significantly increasing the chance of successful cross-species amplification, and a significant positive association between source species polymorphism and the proportion of target species in which a locus revealed polymorphism. No association between cross-species amplification and repeat motif (di-, tri-, or tetranucelotide) or repeat structure (perfect, imperfect, or compound) was observed. A set of nine loci which cross-amplified across an unusually broad range of passerine bird species were also identified, and could serve as a good starting point for cross-species amplification testing in passerine species for which insufficient loci are available.
Article
In many studies involving microsatellites cross-species amplification, primers designed for one (source) species are used to amplify homologous loci in related (target) species. However, it is not clear how closely related the species must be to attain significant success. Genetic divergence is a clear and easy way to assess similarity between species and provides an accurate measure of their evolutionary distance. Eight Mediterranean target species of the family Serranidae were analysed using twelve primers developed for Serranus cabrilla. Additionally, two mitochondrial genes (12S rRNA and 16S rRNA) were chosen on the basis of their extensive use in phylogenetic and evolutionary analyses to compute genetic divergence between the species. Significant negative correlations were found between genetic divergence and both cross-species amplification and maintained polymorphism of microsatellite markers, which could be generalized by gathering information from different fish studies. The success of obtaining amplifiable and polymorphic microsatellite loci can be a priori approximated knowing the mtDNA genetic divergence between a given source and target species using our inferred regression equations.