Seven new species within western Atlantic Starksia atlantica, S. lepicoelia, and S. sluiteri (Teleostei, Labrisomidae), with comments on congruence of DNA barcodes and species

Specimens of Starksia were collected throughout the western Atlantic, and a 650-bp portion of the mitochondrial gene cytochrome oxidase-c subunit I (COl) was sequenced as part of a re-analysis of species diversity of western Central Atlantic shorefishes. A neighbor-joining tree constructed from the sequence data suggests the existence of several cryptic species. Voucher specimens from each genetically distinct lineage and color photographs of vouchers taken prior to dissection and preservation were examined for diagnostic morphological characters. The results suggest that Starksia atlantica, Starksia lepicoelia, and Starksia sluiteri are species complexes, and each comprises three or more species. Seven new species are described. DNA data usually support morphological features, but some incongruence between genetic and morphological data exists. Genetic lineages are only recognized as species if supported by morphology. Genetic lineages within western Atlantic Starksia generally correspond to geography, such that members of each species complex have a very restricted geographical distribution. Increasing geographical coverage of sampling locations will almost certainly increase the number of Starksia species and species complexes recognized in the western Atlantic. Combining molecular and morphological investigations is bringing clarity to the taxonomy of many genera of morphologically similar fishes and increasing the number of currently recognized species. Future phylogenetic studies should help resolve species relationships and shed light on patterns of speciation in western Atlantic Starksia.     

New molecular data for tardigrade phylogeny, with the erection of Paramacrobiotus gen. nov.

Up to few years ago, the phylogenies of tardigrade taxa have been investigated using morphological data, but relationships within and between many taxa are still unresolved. Our aim has been to verify those relationships adding molecular analysis to morphological analysis, using nearly complete 18S ribosomal DNA gene sequences (five new) of 19 species, as well as cytochrome oxidase subunit 1 (COI) mitochondrial DNA gene sequences (15 new) from 20 species, from a total of seven families. The 18S rDNA tree was calculated by minimum evolution, maximum parsimony (MP) and maximum likelihood (ML) analyses. DNA sequences coding for COI were translated to amino acid sequences and a tree was also calculated by neighbour-joining, MP and ML analyses. For both trees (18S rDNA and COI) posterior probabilities were calculated by MrBayes. Prominent findings are as follows: the molecular data on Echiniscidae (Heterotardigrada) are in line with the phylogenetic relationships identifiable by morphological analysis. Among Eutardigrada, orders Apochela and Parachela are confirmed as sister groups. Ramazzottius (Hypsibiidae) results more related to Macrobiotidae than to the genera here considered of Hypsibiidae. Macrobiotidae and Macrobiotus result not monophyletic and confirm morphological data on the presence of at least two large groups within Macrobiotus. Using 18S rDNA and COI mtDNA genes, a new phylogenetic line has been identified within Macrobiotus , corresponding to the ' richtersi-areolatus group'. Moreover, cryptic species have been identified within the Macrobiotus ' richtersi group' and within Richtersius . Some evolutionary lines of tardigrades are confirmed, but others suggest taxonomic revision. In particular, the new genus Paramacrobiotus gen. n. has been identified, corresponding to the phylogenetic line represented by the ' richtersi-areolatus group'.     

Phylogenetic signal in mitochondrial and nuclear markers in sea anemones (Cnidaria, Actiniaria)

The mitochondrial genome of basal animals is generally more slowly evolving than that of bilaterians. This difference in rate complicates the study of relationships among members of these lineages and the discovery of cryptic species or the testing of morphological species concepts within them. We explore the properties of mitochondrial and nuclear ribosomal genes in the cnidarian order Actiniaria, using both an ordinal- and familial-scale sample of taxa. Although the markers do not show significant incongruence, they differ in their phylogenetic informativeness and the kinds of relationships they resolve. Among the markers studied here, the fragments of 12S rDNA and 18S rDNA most effectively recover well-supported nodes; those of 16S rDNA and 28S rDNA are less effective. The general patterns we observed are similar to those in other hexacorallians, although Actiniaria alone show saturation of transitions for ordinal-scale analyses.     

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.     

Phylogeography and population genetics of the cryptic bonnethead shark Sphyrna aff. tiburo in Brazil and the Caribbean inferred from mtDNA markers

Resolving the identity, phylogeny and distribution of cryptic species within species complexes is an essential precursor to management. The bonnethead shark, Sphyrna tiburo, is a small coastal shark distributed in the Western Atlantic from North Carolina (U.S.A.) to southern Brazil. Genetic analyses based on mitochondrial markers revealed that bonnethead sharks comprise a species complex with at least two lineages in the Northwestern Atlantic and the Caribbean (S. tiburo and Sphyrna aff. tiburo, respectively). The phylogeographic and phylogenetic analysis of two mitochondrial markers [control region (mtCR) and cytochrome oxidase I (COI)] showed that bonnethead sharks from southeastern Brazil correspond to S. aff. tiburo, extending the distribution of this cryptic species >5000 km. Bonnethead shark populations are only managed in the U.S.A. and in the 2000s were considered to be regionally extinct or collapsed in southeast Brazil. The results indicate that there is significant genetic differentiation between S. aff. tiburo from Brazil and other populations from the Caribbean (Φ_ST = 0.9053, P < 0.000), which means that collapsed populations in the former are unlikely to be replenished from Caribbean immigration. The species identity of bonnethead sharks in the Southwest Atlantic and their relationship to North Atlantic and Caribbean populations still remains unresolved. Taxonomic revision and further sampling are required to reevaluate the status of the bonnethead shark complex through its distribution range.     

Dactylorhiza (Orchidaceae) in European Russia: combined molecular and morphological analysis

Four plastid and two nuclear (internal transcribed spacer [ITS] ribosomal DNA) markers were used in this study of the Dactylorhiza maculata and D. incarnata complexes (Orchidaceae: Orchidinae) to determine diversity and taxonomic distribution of haplotypes, hybridization frequencies, and maternal parentage of hybrids in 125 samples from 78 populations from European Russia and the Caucasus. A morphometric study of all populations revealed significant correspondence between morphological and plastid DNA data. Most D. maculata sensu stricto (s.s.) specimens from Russia have D. fuchsii haplotypes; this could be evidence for introgression or widespread hybridization between these species in northern Russia. Heterogeneity within populations is much higher for ITS data and is strongly correlated with latitude. Both plastid and nuclear data are significantly correlated with distribution along a south-north axis. Several haplotypes and ITS alleles uncommon in western Europe are more widely distributed in Russia, whereas some frequent haplotypes from western Europe are absent.     

Taxonomic implications for diaptomid copepods based on contrasting patterns of mitochondrial DNA sequence divergences in four morphospecies

Morphological identification methods do not provide reliable and meaningful species identifications for taxa where morphological differences among distinct species are either absent or overlooked (i.e., cryptic species). For example, due to the minute nature of the morphological characters used to delineate diaptomid copepod species and the apparent potential for copepod speciation to occur with little or no morphological change (i.e., morphological stasis), morphological identifications of diaptomid species may not adequately capture their true species diversity. Here, we present results from a geographic survey of mtDNA sequences from populations across the geographic ranges of four North American diaptomid species—Leptodiaptomus minutus, Skistodiaptomus pallidus, Skistodiaptomus reighardi, and Onychodiaptomus sanguineus. Shallow mitochondrial DNA sequence divergences (maximum of 1.1%) among haplotypes of L. minutus from across its geographic range suggest that current morphological identification techniques reliably identify this species. In contrast, we found large mitochondrial DNA sequence divergences (14–22%) among populations within the currently recognized morphospecies of S. pallidus, S. reighardi, and O. sanguineus. However, pairwise sequence divergences within four distinct S. pallidus clades and within populations of S. reighardi and O. sanguineus were similarly low (maximum of 1.5%) as found within L. minutus as a whole. Thus, the S. pallidus, S. reighardi, and O. sanguineus morphospecies may be considered best as cryptic species complexes. Our study therefore indicates that morphological identifications, while sufficient for some species, likely underestimate the true species diversity of diaptomid copepods. As such, we stress the need for extensive taxonomic revision that integrates genetic, morphological, reproductive, and ecological analyses of this diverse and important group of freshwater zooplankton. Furthermore, we believe an extensive taxonomic revision will shed important insight into major questions regarding the roles of geography, phylogeny, and habitat on the frequency of cryptic species on earth. Handling editor: S. I. Dodson     

Three genetic lineages of the Japanese skink Plestiodon japonicus (Scincidae, Squamata) and the genetic composition of their contact zones

Geographic differentiation of the Japanese skink ( Plestiodon japonicus ) throughout the entire Japanese main islands was surveyed using two DNA markers, mitochondrial DNA (mtDNA) and an internal transcribed spacer of nuclear ribosomal genes (ITS-1). Both of the markers concordantly demonstrated geographically discontinuous differentiation between three genetic lineages: the northeastern, central and western lineages. Little to no gene flow occurs between the western and central lineages despite their parapatric distribution suggesting an existence of reproductive isolation despite no known morphological and ecological dissimilarities. On the other hand, the geographic ranges of the northeastern and central lineages are partially separated by a wide area occupied by genetically intermediate type having northeastern mtDNA and central ITS-1. The current data imply that the intermediate type was established by past secondary hybridization between the northeastern and central lineages. Although current gene flow status between the northeastern and central lineages should be elaborated by further data, at least the divergence between the central and western lineages should be maintained by reproductive isolation and therefore taxonomic revision of the species is desired.     

Ribosomal DNA sequence variation among sympatric strains of the Cyclotella meneghiniana complex (Bacillariophyceae) reveals cryptic diversity

Cyclotella meneghiniana Kützing is one of the most commonly found and intensively studied freshwater diatom species. However, it is considered taxonomically problematic because of its unusually wide ecological range and large frustule ultrastructural variation. As part of a study of morphological and genetic variation in this morphospecies, we surveyed nucleotide variation in the hypervariable D1/D2 regions of the 28S rDNA, in the ribosomal internal transcribed spacer region (containing ITS1, the 5.8S rDNA and ITS2) and in the 18S rDNA in a collection of 20 sympatric strains. High genetic variability and strong indications of genetic structure among the Cyclotella meneghiniana strains were found. Representatives of four genetically distinct--apparently reproductively isolated--groups were revealed among them. The random distribution of ITS variation within these four groups indicated that the genetic structure in Cyclotella meneghiniana can probably be explained by the presence of cryptic sexual species rather than by the lack of allogamous sexual reproduction. The morphological features traditionally used for species identification in this group cannot distinguish these putative cryptic species.     

DNA barcoding of bark and ambrosia beetles reveals excessive NUMTs and consistent east‐west divergence across Palearctic forests

A comprehensive DNA barcoding library is very useful for rapid identification and detection of invasive pest species. We tested the performance of species identification in the economically most damaging group of wood‐boring insects – the bark and ambrosia beetles – with particular focus on broad geographical sampling across the boreal Palearctic forests. Neighbour‐joining and Bayesian analyses of cytochrome oxidase I (COI) sequences from 151 species in 40 genera revealed high congruence between morphology‐based identification and sequence clusters. Inconsistencies with morphological identifications included the discovery of a likely cryptic Nearctic species of Dryocoetes autographus, the possible hybrid origin of shared mitochondrial haplotypes in Pityophthorus micrographus and P. pityographus, and a possible paraphyletic Xyleborinus saxeseni. The first record of Orthotomicus suturalis in North America was confirmed by DNA barcoding. The mitochondrial data also revealed consistent divergence across the Palearctic or Holarctic, confirmed in part by data from the large ribosomal subunit (28S). Some populations had considerable variation in the mitochondrial barcoding marker, but were invariant in the nuclear ribosomal marker. These findings must be viewed in light of the high number of nuclear insertions of mitochondrial DNA (NUMTs) detected in eight bark beetle species, suggesting the possible presence of additional cryptic NUMTs. The occurrence of paralogous COI copies, hybridization or cryptic speciation demands a stronger focus on data quality assessment in the construction of DNA barcoding databases.     

Two deep evolutionary lineages in the circumtropical glasseye Heteropriacanthus cruentatus (Teleostei,Priacanthidae) with admixture in the south‐western Indian Ocean

A phylogeographic study of the circumtropical glasseye Heteropriacanthus cruentatus was conducted. Molecular analyses indicate two mitochondrial cytochrome c oxidase subunit I (coI) lineages that are 10·4% divergent: one in the western Atlantic (Caribbean) and another that was detected across the Indo‐Pacific. A fixed single nucleotide polymorphism (SNP) was detected at a nuclear locus (S7 ribosomal protein) and is consistent with this finding. There is evidence of recent dispersal from the Atlantic to the Indian Ocean with individuals of mixed lineages detected in South Africa and the Mozambique Channel. Using coalescent analyses of the mitochondrial dataset, time of divergence between lineages was estimated to be c. 15·3 million years. The deep divergence between these two lineages indicates distinct evolutionary units, however, due to the lack of morphological differences and evidence of hybridization between lineages, taxonomic revision is not suggested at this time.     

Cryptic species in the cosmopolitan Bugula neritina complex (Bryozoa,Cheilostomata)

Previous analyses of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) and γ‐proteobacterial endosymbiont diversity have suggested that the marine bryozoan Bugula neritina is a complex of three cryptic species, namely Types S, D and N. Types D and N were previously reported to have restricted distributions along California (western USA) and Delaware and Connecticut (eastern USA), respectively, whereas Type S is considered widespread in tropical, subtropical and temperate regions due to anthropogenic transport. Here, Bayesian species delimitation analysis of a data set composed of two mitochondrial (COI and large ribosomal RNA subunit [16S]) and two nuclear genes (dynein light chain roadblock type‐2 protein [DYN] and voltage‐dependent anion‐selective channel protein [VDAC]) demonstrated that Types S, D and N correspond to three biological species. This finding was significantly supported, in spite of the combinations of priors applied for ancestral population size and root age. Furthermore, COI sequences were used to assess the introduction patterns of the cosmopolitan Type S species. Two COI haplotypes of Type S (S1a and S1d) were found occurring at a global scale. Mantel tests showed correlation between these haplotypes and local sea surface temperature tolerance. Accordingly, the distributions of Type S haplotypes may reflect intraspecific temperature tolerance variation, in addition to the role of introduction vectors. Finally, we show that the Type N may also have been introduced widely, as this species was found for the first time in Central California and north‐eastern Australia.     

Discovering Hidden Diversity of Characins (Teleostei: Characiformes) in Ecuador’s Yasuní National Park

Background

Management and conservation of biodiversity requires adequate species inventories. The Yasuní National Park is one of the most diverse regions on Earth and recent studies of terrestrial vertebrates, based on genetic evidence, have shown high levels of cryptic and undescribed diversity. Few genetic studies have been carried out in freshwater fishes from western Amazonia. Thus, in contrast with terrestrial vertebrates, their content of cryptic diversity remains unknown. In this study, we carried out genetic and morphological analyses on characin fishes at Yasuní National Park, in eastern Ecuador. Our goal was to identify cryptic diversity among one of the most speciose fish families in the Amazon region. This is the first time that genetic evidence has been used to assess the species content of the Napo Basin, one of the richest regions in vertebrate diversity.

Results

Phylogenetic analyses of partial mitochondrial 16S ribosomal RNA gene (∼600 pb) DNA sequences from 232 specimens of the family Characidae and its closest groups revealed eight candidate new species among 33 species sampled, representing a 24% increase in species number. Analyses of external morphology allowed us to confirm the species status of six of the candidate species.

Conclusions

Our results show high levels of cryptic diversity in Amazonian characins. If this group is representative of other Amazonian fish, our results would imply that the species richness of the Amazonian ichthyofauna is highly underestimated. Molecular methods are a necessary tool to obtain more realistic inventories of Neotropical freshwater fishes.     

Molecular differentiation of the microgastrine species commonly found in paddy fields from Southeast Asia,with additional data on their phylogeny （Hymenoptera：Braconidae）

Partial DNA sequences of three genes, that is, mitochondrial large ribosomal subunit (16S), nuclear large ribosomal subunit (28S D2) and mitochondrial NADH1 dehydrogenase (NADH1) gene, were sequenced from different microgas trine species(Braconidae: Microgastrinae) collected fresh from paddy fields. The DNA sequences were used to determine the extent of sequence variation among species in order to evaluate the specific status of each species. Cladistic analysis was also used to infer a phylogenetic relationship among these species. The results showed that sequence divergence among species of the same genus Cotesia was much lower than those among different genera, such as Cotesia, Exoryza and Apanteles; the sequence similarity of 16S rDNA and NADH 1 genes between Cotesia sp. and C. chilonis was higher than that between C. sp. and C. ruficrus.Phylogenetic analyses suggested that four species of Cotesia were always grouped in the same clade regardless of using different analysis methods; Cotesia sp. and C. chilonis are more closely related to each other than to C. ruficrus, different from previous morphological results. Additionally, sequence analyses indicated that NADH1 gene has more parsimony informative characters than 28S rDNA D2 and 16S rDNA at the species-level analysis,indicating that NADH1 gene might be a useful marker for species-level analysis.     

An unexpected pattern of migration revealed in the Subalpine Warbler Sylvia cantillans complex by mitochondrial DNA analyses

Genetic markers are often used to trace the geographical origin of migrating birds. Such an approach has been used to attribute individuals of a given species to a given population, but it could also be applied to cryptic species, which are not fully diagnosable on the basis of their morphological appearance alone, despite often being genetically distinct. We sampled migrants of the Sylvia cantillans complex, which include cryptic taxa not readily identified on the basis of their appearance, at spring stopover sites in the central Mediterranean. We identified these individuals taxonomically using mitochondrial DNA sequence data. Molecular analyses enabled us to establish that four different taxa/clades of the Sylvia cantillans complex (two S. c. cantillans clades, S. c. albistriata and S. subalpina) migrate through the central‐western Mediterranean from Africa.     

Molecular resolution of the family Dreissenidae (Mollusca: Bivalvia) with emphasis on Ponto-Caspian species, including first report of Mytilopsis leucophaeata in the Black Sea basin

Considerable uncertainty exists in determination of the phylogeny among extant members of the Dreissenidae, especially those inhabiting the Ponto-Caspian basin, as multiple systematic revisions based on morphological characteristics have failed to resolve relationships within this group of bivalves. In this study we use DNA sequence analyses of two mitochondrial gene fragments, 16S rRNA and cytochrome c oxidase subunit I (COI), to determine phylogenetic relationships among Dreissena rostriformis, D. bugensis, D. polymorpha, D. stankovici, Congeria kusceri, and Mytilopsis leucophaeata. Dreissena stankovici was determined to represent a sister taxa to D. polymorpha and both are more closely related to other extant Dreissena species than Congeria or Mytilopsis. Sequence divergence between D. rostriformis and D. bugensis was relatively low (0.3-0.4%), suggesting that these two taxa constitute a single species. However, environmental differences suggest two races of D. rostriformis, a brackish water race (rostriformis) and a freshwater race (bugensis). Spread of bugensis-type individuals into habitats in the Caspian Sea that are occupied by rostriformis-type individuals may create novel hybridization opportunities. Species-specific molecular markers also were developed in this study since significant intraspecific variation in morphological features complicates dreissenid identification. Using two gene fragments (nuclear 28S and 16S), we identified restriction fragment length polymorphisms (RFLPs) that distinguish among D. rostriformis/bugensis, D. polymorpha, and D. stankovici and revealed the presence of a cryptic invader to the Black Sea basin, Mytilopsis leucophaeata. This is the first report of this North American native in southern Europe.     

Evolution of poecilogony from planktotrophy: cryptic speciation, phylogeography, and larval development in the gastropod genus Alderia

Poecilogony, a rare phenomenon in marine invertebrates, occurs when alternative larval morphs differing in dispersal potential or trophic mode are produced from a single genome. Because both poecilogony and cryptic species are prevalent among sea slugs in the suborder Sacoglossa (Gastropoda: Opisthobranchia), molecular data are needed to confirm cases of variable development and to place them in a phylogenetic context. The nominal species Alderia modesta produces long-lived, feeding larvae throughout the North Atlantic and Pacific, but in California can also produce short-lived larvae that metamorphose without feeding. We collected morphological, developmental, and molecular data for Alderia from 17 sites spanning the eastern and western Pacific and North Atlantic. Estuaries south of Bodega Harbor, California, contained a cryptic species (hereafter Alderia sp.) with variable development, sister to the strictly planktotrophic A. modesta. The smaller Alderia sp. seasonally toggled between planktotrophy and lecithotrophy, with some individuals differing in development but sharing mitochondrial DNA haplotypes. The sibling species overlapped in Tomales Bay, California, but showed no evidence of hybridization; laboratory mating trials suggest postzygotic isolation has arisen. Intra- and interspecific divergence times were estimated using a molecular clock calibrated with geminate sacoglossans. Speciation occurred about 4.1 million years ago during a major marine radiation in the eastern Pacific, when large inland embayments in California may have isolated ancestral populations. Atlantic and Pacific A. modesta diverged about 1.7 million years ago, suggesting trans-Arctic gene flow was interrupted by Pleistocene glaciation. Both Alderia species showed evidence of late Pleistocene population expansion, but the southern Alderia sp. likely experienced a more pronounced bottleneck. Reduced body size may have incurred selection against obligate planktotrophy in Alderia sp. by limiting fecundity in the face of high larval mortality rates in warm months. Alternatively, poecilogony may be an adaptive response to seasonal opening of estuaries, facilitating dispersal by long-lived larvae. An improved understanding of the forces controlling seasonal shifts in development in Alderia sp. may yield insight into the evolutionary forces promoting transitions to nonfeeding larvae.     

From ‘cryptic species’ to integrative taxonomy: an iterative process involving DNA sequences,morphology, and behaviour leads to the resurrection of Sepsis pyrrhosoma (Sepsidae: Diptera)

Tan, D. S. H., Ang, Y., Lim, G. S., Ismail, M. R. B. & Meier, R. (2010). From ‘cryptic species’ to integrative taxonomy: an iterative process involving DNA sequences, morphology, and behaviour leads to the resurrection of Sepsis pyrrhosoma (Sepsidae: Diptera). —Zoologica Scripta, 39, 51–61. The increased availability of DNA sequences has led to a surge of ‘cryptic species’ in the literature. These units are usually proposed based on finding genetically distinct lineages within species that were initially defined based on morphological characters. However, few authors attempt to confirm whether these ‘cryptic’ units are species and even fewer authors are explicit about which species concept is applied. Here, we use an example from Sepsidae (Diptera) to demonstrate how cryptic species can be validated by an iterative process involving several data sources and an evaluation of the data under different species concepts. A phylogeographic analysis based on 50 specimens for five species of the flavimana group revealed deep mitochondrial splits within Sepsis flavimana which was suggestive of a cryptic species. We resolve the initial conflict between DNA sequences and morphology by adding new morphological data as well as behavioural evidence and tests for reproductive isolation. One cryptic species is confirmed and Sepsis pyrrhosoma, a former synonym of S. flavimana, is here shown to be a valid species under most species concepts. We can thus document that the same data can lead to similar conclusions under conflicting concepts once different kinds of data are integrated.