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Cryptic species, cryptic endosymbionts, and geographical variation in chemical defences in the bryozoan Bugula neritina.

Molecular markers often offer the only means to discriminate between species and to elucidate the specificity of many community interactions, both of which are key to the understanding of ecological patterns. Western Atlantic populations of the bryozoan Bugula neritina vary in the palatability of their larvae to predators: individuals south of Cape Hatteras produce chemical deterrents to fish predators that are absent in more northern individuals. We use mitochondrial cytochrome oxidase c subunit I (COI) sequences to show that the differences in palatability between populations correlate with the geographical distributions of two cryptic species within B. neritina. Furthermore, these cryptic species differ in their associations with bacteria that may confer chemical resistance to predation. Small subunit rRNA primers specific to a subset of gamma-proteobacteria amplified only the bacterium Endobugula sertula from the southern cryptic species. Endobugula sertula produces a family of chemical compounds (bryostatins) that may deter predators of its animal host. In contrast, the same primers amplified an array of gamma-proteobacteria from the unprotected northern cryptic bryozoan species, but never E. sertula. In combination, these findings suggest that the geographical variation in palatability observed in the larvae of B. neritina is not the result of local adaptation of a single species to regions of differing predation pressure, but rather results from the comparison of cryptic species that differ in the presence or absence of a bacterium that may provide protection against predators. The ability to identify the cryptic Bugula species and their differing relationships with bacterial associates provides an example of the important role molecular techniques may play in addressing ecological questions.

Animals↗

Can skull morphology be used to predict ecological relationships between bat species? A test using two cryptic species of pipistrelle.

Can ecological relationships between bat species be predicted largely on the basis of morphology? This question was addressed by investigating skull morphology of two cryptic species of the pipistrelle bat. Since 45 Pipistrellus pipistrellus apparently eats larger prey than 55 P. pipistrellus, we predicted that it would have a larger overall skull size, a larger dentary apparatus, and a larger gape. To test these predictions, variables were measured from skulls of the two cryptic species, and comparisons made between them. In accordance with our predictions, overall skull size was larger in 45 P. pipistrellus than in 55 P. pipistrellus, and 45 P. pipistrellus had a longer lower jaw and the distance between the jaws at maximum gape was larger. In addition, 45 P. pipistrellus had longer upper canines, which may allow it to pierce harder prey items than 55 P. pipistrellus. Only some aspects of dietary differences between the two cryptic species could be explained by differences in skull morphology, and we suggest that empirical data, at least on diet and habitat use, are also required to explain mechanisms of resource partitioning among species in bat communities.

Analysis of Variance↗

Evidence for subdivision of the root-endophyte Phialocephala fortinii into cryptic species and recombination within species.

The genetic structure of the root-endophyte Phialocephala fortinii was analyzed in three study sites using 11 single-copy RFLP probes. A total of 541 strains isolated from surface-sterilized, fine roots (diameter 0.5-3 mm) of Norway spruce (Picea abies) were examined. The average gene diversity (H) was high in all three study sites. Cluster analysis showed that up to four well-separated clusters of multi-locus haplotypes were present within the sites. Significant population subdivision was detected among these clusters, suggesting that groups of multi-locus haplotypes were reproductively isolated and that P. fortinii is a species complex composed of several cryptic species. This hypothesis was supported by ISSR-PCR which showed clusters consistent with those of the multi-locus haplotypes identified by RFLP analysis. In contrast, ITS sequence analysis did not allow to separate the species as clearly. The index of association (IA) did not deviate significantly from zero within any cryptic species, suggesting that recombination occurs within these species. Cryptic species occurred sympatrically. Thalli of two cryptic species were detected in the same 5-mm-long root segment in one instance. No significant differentiation was observed among populations of the same cryptic species in forest stands located approximately 5 km from each other. This finding is consistent with significant gene flow over this spatial scale. In addition, several isolates with both identical multi-locus haplotype and identical ISSR fingerprint were found at each study site indicating genotype flow or a recent common history between study sites.

Ascomycota↗

Independent origins of tetraploid cryptic species in the fern Ceratopteris thalictroides.

Ceratopteris thalictroides (L.) Brongn is a tetraploid fern species that contains at least three cryptic species, the south, the north and the third type. In this study we combined data from both chloroplast DNA (cpDNA) and nuclear DNA sequences of three diploid species and three cryptic species of C. thalictroides to unravel the origin of the cryptic species, particularly of the reticulate relationships among the diploid and tetraploid taxa in the genus Ceratopteris. Of the three diploid species examined, C. cornuta had cpDNA identical to that of the tetraploid third type plants, and this diploid species is a possible maternal ancestor of the tetraploid third type. Analysis of the homologue of the Arabidopsis thaliana LEAFY gene (CLFY1) identified ten alleles in the genus Ceratopteris, with six alleles found in C. thalictroides. The unrooted tree of the CLFY1 gene revealed four clusters. Each cryptic species showed fixed heterozygosity at the CLFY1 locus and had two alleles from different clusters of the CLFY1 tree. Consideration of the cpDNA sequences, CLFY1 genotypes of the cryptic species and CLFY1 gene tree in concert suggested that the cryptic species of C. thalictroides had originated through independent allopolyploidization events involving C. cornuta and two unknown hypothetical diploid species.

Base Sequence↗

An analysis of species boundaries and biogeographic patterns in a cryptic species complex: the rotifer--Brachionus plicatilis.

Since the advent of molecular phylogenetics, there is increasing evidence that many small aquatic and marine invertebrates--once believed to be single, cosmopolitan species--are in fact cryptic species complexes. Although the application of the biological species concept is central to the identification of species boundaries in these cryptic complexes, tests of reproductive isolation do not frequently accompany phylogenetic studies. Because different species concepts generally identify different boundaries in cryptic complexes, studies that apply multiple species concepts are needed to gain a more detailed understanding of patterns of diversification in these taxa. Here we explore different methods of empirically delimiting species boundaries in the salt water rotifer Brachionus plicatilis by comparing reproductive data (i.e., the traditional biological species concept) to phylogenetic data (the genealogical species concept). Based on a high degree of molecular sequence divergence and largely concordant genetic patterns in COI and ITS1, the genealogical species hypothesis indicates the existence of at least 14 species--the highest estimate for the group thus far. A test of the genealogical species concept with biological crosses shows a fairly high level of concordance, depending on the degree of reproductive success used to draw boundaries. The convergence of species concepts in this group suggests that many of the species within the group may be old. Although the diversity of the group is higher than previously understood, geographic distributions remain broad. Efficient passive dispersal has resulted in global distributions for many species with some evidence of isolation by distance over large geographic scales. These patterns concur with expectations that micro-meiofauna (0.1-1mm) have biogeographies intermediate to microbial organisms and large vertebrates. Sympatry of genetically distant strains is common.

Animals↗

Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator.

Astraptes fulgerator, first described in 1775, is a common and widely distributed neotropical skipper butterfly (Lepidoptera: Hesperiidae). We combine 25 years of natural history observations in northwestern Costa Rica with morphological study and DNA barcoding of museum specimens to show that A. fulgerator is a complex of at least 10 species in this region. Largely sympatric, these taxa have mostly different caterpillar food plants, mostly distinctive caterpillars, and somewhat different ecosystem preferences but only subtly differing adults with no genitalic divergence. Our results add to the evidence that cryptic species are prevalent in tropical regions, a critical issue in efforts to document global species richness. They also illustrate the value of DNA barcoding, especially when coupled with traditional taxonomic tools, in disclosing hidden diversity.

Animal Feed↗

A review of the use of ribosomal DNA (rDNA) to differentiate among cryptic Anopheles species.

Cryptic species complexes are groups of closely related species that are difficult or impossible to distinguish by morphological traits. These complexes are known from a wide variety of arthropods and are common among the well-studied, medically-important insects. For example, many of the anopheline vectors of malaria parasites are members of cryptic species complexes. Complexes typically include both vector and non-vector species, and two or more member species are often found sympatrically. Until the late 1950, only two such Anopheles complexes were known, the A. gambiae complex from Africa and the A. maculipennis complex from Europe. Today, dozens of Anopheles cryptic species complexes are recognized, and accumulating evidence suggests that most important malaria vectors are likely to be members of such complexes. A variety of methods have been developed for identifying the species of individual specimens from these complexes, although until recently only those based on species-specific allozymes and polytene chromosome inversions were widely used. The limitations inherent in these methods have been circumvented with DNA-based procedures, which are especially useful because both sexes and all developmental stages can be identified, and DNA can be recovered from samples stored by a wide variety of simple methods. Several DNA-based identification techniques have been developed, including hybridization assays based on species-specific repeat sequences, and diagnostic PCR fragments produced either by the use of random PCR primers or by amplifying DNA with primers based on known species-specific sequences. In this review we discuss the relative marks of different methods of cryptic species identification, with emphasis on the use of ribosomal DNA as a target for species-diagnostic PCR assays.

Animals↗

Allozyme analysis of cryptic species in the Asplenium niduscomplex from West Java, Indonesia.

In various fern species, a large amount of rbcL sequence variation has been reported, and it is possible that these species contain several reproductively isolated cryptic species. In our previous study on Asplenium nidus L., it was suggested that the plants growing in Mt. Halimun National Park, West Java, Indonesia, consist of several cryptic species based on the results of crossing experiments among rbcL sequence types. In this study, we examined allozyme polymorphisms of five rbcL sequence types found in West Java in order to test the hypothesis that the assemblages of A. nidus delimited based on the rbcL sequences are separate Mendelian populations and gene flow is disrupted by reproductive isolation from one another. The calculated fixation indices suggested that the individuals in each rbcL type are randomly crossing at least in the investigated localities. Nevertheless, these rbcL-based assemblages were genetically differentiated in allozymes that are encoded in their nuclear genomes, and it is also suggested that gene flow is disrupted even between sympatrically distributed pairs of rbcL sequence types. Therefore, our findings support the view that the five rbcL sequence types in West Java are potential cryptic species.

Journal Article↗

Species concepts and malaria parasites: detecting a cryptic species of Plasmodium.

Species of malaria parasite (phylum Apicomplexa: genus Plasmodium) have traditionally been described using the similarity species concept (based primarily on differences in morphological or life-history characteristics). The biological species concept (reproductive isolation) and phylogenetic species concept (based on monophyly) have not been used before in defining species of Plasmodium. Plasmodium azurophilum, described from Anolis lizards in the eastern Caribbean, is actually a two-species cryptic complex. The parasites were studied from eight islands, from Puerto Rico in the north to Grenada in the south. Morphology of the two species is very similar (differences are indistinguishable to the eye), but one infects only erythrocytes and the other only white blood cells. Molecular data for the cytochrome b gene reveal that the two forms are reproductively isolated; distinct haplotypes are present on each island and are never shared between the erythrocyte-infecting and leucocyte-infecting species. Each forms a monophyletic lineage indicating that they diverged before becoming established in the anoles of the eastern Caribbean. This comparison of the similarity, biological and phylogenetic species concepts for malaria parasites reveals the limited value of using only similarity measures in defining protozoan species.

Animals↗

Differential tolerance among cryptic species: a potential cause of pollutant-related reductions in genetic diversity.

Differential mortality of cryptic species (i.e., morphologically similar but genetically distinct sibling species) may contribute to observed reductions in genetic diversity at contaminated sites if the members of a complex of cryptic species exhibit differential responses to the contaminants that are present. We conducted toxicity bioassays with both polynuclear aromatic hydrocarbon and metal contamination on Cletocamptus fourchensis and C. stimpsoni from two intensively sampled locations. Previous molecular and detailed morphological analyses segregated these as cryptic species from the cosmopolitan C. deitersi. We found that these species occur together at two field sites and that they exhibit unique toxic responses to heavy metals, suggesting differential tolerances at contaminated sites. These findings suggest that reported losses of genetic diversity at contaminated sites may represent a reduction in species diversity rather than a loss of the presumed less-tolerant genotypes within a species. They also suggest that members of a cryptic species complex should not be used in laboratory toxicity tests unless populations are genetically characterized. Future studies using genetic diversity as a marker of contaminant effects should consider the possibility of undetected cryptic species.

Animals↗

A molecular phylogeny of nuclear and mitochondrial sequences in Hymenolepis nana (Cestoda) supports the existence of a cryptic species.

Since isolates of Hymenolepis nana infecting humans and rodents are morphologically indistinguishable, the only way they can be reliably identified is by comparing the parasite in each host using molecular tools. In the current study, isolates of H. nana from rodent and human hosts from a broad geographical range were sequenced at the ribosomal first internal transcribed spacer (ITS1), the mitochondrial cytochrome c oxidase subunit 1 (C01) gene and the nuclear paramyosin gene loci. Twenty-three isolates of H. nana were sequenced at the ITS1 locus and this confirmed the existence of spacers which, although similar in length (approximately 646 bp), differed in their primary sequences which led to the separation of the isolates into 2 clusters when analysed phylogenetically. This sequence variation was not, however, related to the host of origin of the isolate, thus was not a marker of genetic distinction between H. nana from rodents and humans. Sequencing of a 444 bp fragment of the mitochondrial cytochrome c oxidase 1 gene (C01) in 9 isolates of H. nana from rodents and 6 from humans identified a phylogenetically supported genetic divergence of approximately 5% between some mouse and human isolates. This suggests that H. nana is a species complex, or 'cryptic' species (=morphologically identical yet genetically distinct). A small segment of the nuclear gene, paramyosin, (625 bp or 840 bp) was sequenced in 4 mouse and 3 human isolates of H. nana. However, this gene did not provide the level of heterogeneity required to distinguish between isolates from rodent and human hosts. From the results obtained from faster evolving genes, and the epidemiological evidence, we believe that the life-cycle of H. nana that exists in the north-west of Western Australia is likely to involve mainly 'human to human' transmission.

Animals↗

Polymerase chain reaction species diagnostic assay for Anopheles quadrimaculatus cryptic species (Diptera: Culicidae) based on ribosomal DNA ITS2 sequences.

Species-specific differences in the nucleotide sequences of the 2nd internal transcribed spacer (ITS2) of nuclear ribosomal DNA (rDNA) were used to develop a diagnostic assay based on the polymerase chain reaction (PCR) that can distinguish 4 of the 5 cryptic sibling species in the common malaria mosquito, Anopheles quadrimaculatus Say, complex. The assay requires only a small amount of tissue from an individual mosquito and a mixture of 5 PCR primers. The plus strand universal primer is derived from a sequence in the 5.8S coding region that is identical in all members of the complex. The 4 minus strand primers were selected from species-unique sequences within the ITS2 region. PCR amplification produces a different sized fragment for each of the 4 species which can be visualized readily under ultraviolet light after electrophoresis through an ethidium bromide-containing agarose gel. The assay has been developed and tested only with An. quadrimaculatus complex specimens from Florida populations.

Animals↗

Cryptic species as a window on diversity and conservation.

The taxonomic challenge posed by cryptic species (two or more distinct species classified as a single species) has been recognized for nearly 300 years, but the advent of relatively inexpensive and rapid DNA sequencing has given biologists a new tool for detecting and differentiating morphologically similar species. Here, we synthesize the literature on cryptic and sibling species and discuss trends in their discovery. However, a lack of systematic studies leaves many questions open, such as whether cryptic species are more common in particular habitats, latitudes or taxonomic groups. The discovery of cryptic species is likely to be non-random with regard to taxon and biome and, hence, could have profound implications for evolutionary theory, biogeography and conservation planning.

Adaptation, Biological↗

Genetic and behavioral components of the cryptic species boundary between Laupala cerasina and L. kohalensis (Orthoptera: Gryllidae).

Cryptic species are often hypothesized on the basis of differences in courtship signals. These signal differences suggest that mate recognition systems, which include both courtship signals and responses to those signals, have diverged between genetically isolated populations. Cryptic species are therefore thought to represent distinct genetic units, the boundaries of which are maintained by premating incompatibilities, specifically by receiver preferences for conspecific signals. Laupala cerasina and L. kohalensis are sympatric species of swordtail crickets endemic to the big island of Hawaii, that are distinguishable by differences in male courtship song. We first tested whether groupings hypothesized by acoustic similarity reflect genetic groupings, using AFLP data to estimate genetic relationships. Second, we tested whether genetic boundaries are maintained by female preferences for conspecific song characteristics. Phonotaxis trials were used to determine the extent of female preferences for conspecific male song. Results generally support both hypotheses, but suggest the presence of porous species boundaries.

Acoustic Stimulation↗

A comparison between mitochondrial DNA and the ribosomal internal transcribed regions in prospecting for cryptic species of platyhelminth parasites.

We examined the relative merits of mitochondrial DNA loci and ribosomal DNA internal transcribed spacers for their use in prospecting for cryptic species of platyhelminth parasites. Sequence divergence at ITS1 and ITS2 was compared with divergence at 2 mtDNA loci (NADH dehydrogenase-1 and cytochrome c oxidase I) between closely related species of trematodes and cestodes. Both spacers accumulated substitutions substantially more slowly than mtDNA, which clearly shows a higher level of divergence among species relative to intra-specific variation. Besides a slow rate of substitution, other caveats that may be encountered when using ITS sequences as a prospecting marker are discussed. In particular, we note recent studies that suggest the existence of substantial levels of intra-individual variation in ITS sequences of flatworms. Because it is likely that closely related species share this phenomenon, it may confound the detection of cryptic species, especially if small sample sizes are studied. Although potential limitations of mtDNA are also recognized, the higher rate of evolution and smaller effective population size of this marker increases the probability of detecting diagnostic characters between cryptic species.

Animals↗

[Differentiation of cryptic species A and D of Anopheles dirus complex by polymerase chain reaction].

AIM: To distinguish cryptic species A and D of Anopheles dirus complex using polymerase chain reaction (PCR). METHODS: A diagnostic PCR assay of species was developed by use of three primers, one derived from highly conservative 5.8 S coding sequences and two from different interspecies sequence in the second internal transcribed spacer (ITS2) of ribosomal DNA. RESULTS: Using the PCR method, specific fragments were amplified in both species, the size of fragments is 374 bp for species A and 663 bp for species D. Thirty samples of species A from AFRIMS and HN laboratory colony and seven samples of the species D from Yunnan Province were correctly identified by PCR. Satisfactory results were obtained from the amount of DNA as little as 1/1,600 of extracted DNA of a single mosquito or 1/5 of DNA derived from one leg of a mosquito triturated in water. A total of 148 field-collected specimens of Anopheles dirus from Heping(HP), Baisha(BS), Loukui(LK), and Maoyang (MY) in Hainan Province revealed fragment characteristic of species A, while 30 specimens from Mengla (ML) in Yunnan Province showed the specific fragment of species D. CONCLUSION: A simple and reliable method was developed to identify cryptic species A and D of Anopheles dirus complex and it was further verified that Anopheles dirus from Hainan and Yunnan Provinces is the species A and the species D, respectively.

Animals↗

Morphological and genetic variation indicate cryptic species within Lamarck's little sea star, Parvulastra (=Patiriella) exigua.

The asterinid sea star Parvulastra exigua (Lamarck) is a common member of temperate intertidal marine communities from geographically widespread sites around the southern hemisphere. Individuals from Australian populations lay benthic egg masses (through orally directed gonopores) from which nonplanktonic offspring hatch and metamorphose without a dispersing planktonic larval phase. Scattered reports in the taxonomic literature refer to a similar form in southern Africa with aborally directed gonopores (and possibly broadcast spawning of planktonic eggs and larvae); such differences would be consistent with cryptic species variation. Surveys of morphology and mtDNA sequences have revealed cryptic species diversity in other asterinid genera. Here we summarize the taxonomic history of Lamarck's "Astérie exiguë" and survey morphological variation (the location of the gonopores) for evidence that some P. exigua populations include cryptic species with a different mode of reproduction. We found strong evidence for multiple species in the form of two phenotypes and modes of reproduction (oral and aboral gonopore locations) in populations from southern Africa and islands in the Atlantic and Indian oceans. Both modes of reproduction have broad geographic ranges. These results are consistent with previously published genetic data that indicate multiple species in African and island (but not Australian) populations.

Animals↗

Identification of two cryptic species in the Anopheles (Cellia) annularis complex using ribosomal DNA PCR-RFLP.

Anopheles (Cellia) annularis Van der Wulp is a complex of two sibling species provisionally designated as species A and B and can only be differentiated on the basis of the paracentric inversion in the ovarian polytene chromosomes. To analyze the distribution of these two species and to develop a molecular method for the identification of these two cryptic species, we sequenced the ribosomal DNA internal transcribed spacer 2 (ITS2) and domain 3 (D3) of A. annularis specimens collected from Sonapur (Assam), Jabalpur (Madhya Pradesh), Ranchi (Jharkhand), and Ghaziabad (Uttar Pradesh). We did not find any sequence variation among the specimens collected from Assam, Madhya Pradesh, and Jharkhand states, whereas two types of sequences were obtained from the specimens collected from the state of Uttar Pradesh, which correspond to species A and B of the A. annularis complex. Species A was more prevalent among the all four regions studied. The ITS2 sequence of species A showed unique restriction sites for MvaI and Eco24I, while species B displayed HinfI and NruI sites. Similarly, the D3 sequence of species A showed unique restriction site for Alw26I, while species B showed a unique KpnI site. In this study, we report for the first time the development of ribosomal DNA polymerase chain reaction-restriction fragment length polymorphism methods for identifying these two cryptic species of the Annularis complex.

Animals↗