Genetic diversity of two mitochondrial DNA genes in Spirometra erinaceieuropaei (Cestoda: Diphyllobothridae) from Poland

The tapeworm species Spirometra erinaceieuropaei was documented mainly in Asia and Europe. In recent years, plerocercoid larvae (spargana) of this parasite have been found in different hosts in north‐eastern Poland. The evolutionary history and way of S. erinaceieuropaei spreading across Eurasia have been not described yet. However, this phenomenon could be closely related to the evolutionary history and migration routes of studied tapeworm host species. We investigated the genetic variability and divergence pattern among S. erinaceieuropaei populations in intermediate and paratenic hosts from north‐eastern Poland based on complete mitochondrial sequences of cytochrome b (cytb) and cytochrome c oxidase subunit I (cox1) genes. Analysis of 319 consolidated sequences of these two genes showed no genetic structure across study area. Comparison of sequences from Poland and China showed distinct separation of S. erinaceieuropaei populations from these two regions. They split from their common ancestor approximately 28.6 million years ago. Demographic expansion of Polish population of S. erinaceieuropaei started from glacial refugia approximately 12.5 thousand years ago, and recent population expansion has been observed in the tapeworm population from north‐eastern Poland.     

Cryptic diversity in Chortís Highland moss salamanders (Caudata: Plethodontidae: Nototriton) revealed using mtDNA barcodes and phylogenetics,with a new species from eastern Honduras

The systematic study of Central American moss salamanders has been a challenge to researchers due to their cryptic nature and subsequent difficulty in sampling. In an effort to elucidate relationships among moss salamanders from the Chortís Highlands, we quantified intra- and interspecific variation using data from the mitochondrial genes 16S (the amphibian barcoding gene), cytochrome b (cyt b; widely used in phylogenetic studies of neotropical salamanders), and cytochrome oxidase subunit I (COI, the universal metazoan barcoding gene). All three loci exhibit a bimodal ‘barcoding gap’ between ranges of intra- and interspecific variation, demonstrating unambiguous species boundaries with respect to the mitochondrial loci. A barcoding approach and Bayesian and maximum likelihood analyses of the mtDNA dataset revealed that the nominal species N. barbouri represents a species complex, with two of three populations assigned to this taxon being paraphyletic with respect to a sample from the type locality. These data also revealed an undescribed species from eastern Honduras, and confirmed the identity of allopatric populations of N. lignicola and N. limnospectator. Sequence data from cyt b presented an analytical challenge due to a high level of substitution saturation at the third codon position for some samples. Phylogenetic analyses of the entire dataset including and omitting the third position of cyt b resulted in conflicting results with respect to the position of the new species and N. tomamorum. Based on the molecular data, supplemented with data from external morphology and osteology, we formally describe a new species from Sierra de Agalta.     

Hidden diversity and cryptic speciation refute cosmopolitan distribution in Caprella penantis (Crustacea: Amphipoda: Caprellidae)

Caprella penantis is considered a cosmopolitan species and one of the most challenging caprellids in taxonomic terms because of its remarkable intraspecific morphological variation. This study examined DNA sequences from mitochondrial (COI) and nuclear (18S) markers together with morphological data from 25 localities of C. penantis, and closely related species Caprella dilatata and Caprella andreae, all traditionally considered part of the old ‘acutifrons’ complex. The large genetic divergence and reciprocally allopatric distributions point to the existence of a species complex of at least four species, of which one is reported as a cryptic species. This study provides the first evidence of cryptic speciation in the family Caprellidae, and questions the validity of some traditional morphological characters used to delimit species in the genus Caprella. Our results are consistent with the idea that main factors were probably isolation by distance and ecological traits, promoting diversification in C. penantis. The strong genetic structure reported for this species in the Iberian Peninsula and Moroccan coasts also suggests restriction to dispersal as well as the presence of refugial areas. These results highlight the utility of the COI and 18S genes in combination with morphological characters for shedding light on systematic questions in caprellids, and patterns of genetic connectivity.     

Failed species,innominate forms,and the vain search for species limits: cryptic diversity in dusky salamanders (Desmognathus) of eastern Tennessee

Cytochrome B sequences and allozymes reveal complex patterns of molecular variation in dusky salamander (Desmognathus) populations in eastern Tennessee. One group of allozymically distinctive populations, which we refer to as the Sinking Creek form (SCF), combines morphological attributes of Desmognathus fuscus with cytB sequences characteristic of Desmognathus carolinensis. This form is abruptly replaced by D. fuscus just north of Johnson City, TN with no evidence of either sympatry or gene exchange. To the south, allozymic markers indicate a broad zone of admixture with populations characterized by distinct cytB sequences and that may or may not be ultimately referable to Desmognathus conanti. A third distinctive group of populations, which we refer to as the Lemon Gap form (LGF), occurs in the foothills of the Great Smoky and southern Bald Mountains and exchanges genes with Desmognathus santeetlah along the escarpment of the Great Smokies, D. carolinensis in the southern Bald Mountains, and populations of a different haplotype clade in the Ridge and Valley. We treat all these as innominate forms that may represent “failed species,” recognizing that it may never be possible to reconcile species limits with patterns of phylogeny, morphology, and gene exchange in these salamanders.     

Deep‐sea anglerfishes (Lophiiformes: Ceratioidei) from the western North Atlantic: Testing the efficacy of DNA barcodes

The taxonomy of ceratioid fishes is challenging and frequently based on a small number of female specimens described for each species. Twenty ceratioid specimens caught on Flemish Cap and Grand Bank (western North Atlantic), representing 12 species and six families: Ceratias holboelli and Cryptopsaras couesii (Ceratiidae); Himantolophus albinares (Himantolophidae); Melanocetus johnsonii (Melanocetidae); Lophodolos acanthognathus, Oneirodes eschrichtii, Dolopichthys karsteni, and Leptacanthichthys gracilispinis (Oneirodidae); Caulophryne polynema (Caulophrynidae); and Haplophryne mollis, Linophryne brevibarbata, and L. bicornis (Linophrynidae) were identified by examination of morphological characters. DNA barcode sequences, from the 5′ end of the COI mitochondrial gene, were developed for 18 specimens and compared with all ceratioid barcode sequences available in public repositories. The analyses extended the ranges of some quantitative traits for certain species, highlighted the possible existence of cryptic species in C. couesii with distinct ranges in the Atlantic and Pacific oceans, and indicated a close relationship between Bertella and Dolopichthys meriting further attention. The authoritative identification of the 18 voucher specimens made possible detection of erroneous identifications of some sequences extracted from the repositories and highlighting of taxonomic conflicts that should be the subject of future studies.     

Phylogeography of the cold‐water barnacle Chthamalus challengeri in the north‐western Pacific: effect of past population expansion and contemporary gene flow

Aim Phylogeographical patterns of marine organisms in the north‐western Pacific are shaped by the interaction of past sea‐level fluctuations during glacial maxima and present‐day gene flow. This study examines whether observed population differentiation in the barnacle Chthamalus challengeri, which is endemic to the north‐western Pacific, can be explained by the interactions between historical glacial events and patterns of contemporary gene flow. Location Eleven locations in the north‐western Pacific. Methods Partial sequences of mitochondrial cytochrome c oxidase subunit I (COI), 12S, 16S and nuclear internal transcribed spacer 1 (ITS1) were obtained from 312 individuals. Parsimony haplotype networks and analysis of molecular variance (AMOVA) were used to determine whether the observed genetic structure corresponds to marine provinces (Kuroshio Current and China Sea Coastal Provinces), zoogeographical zones (oriental and Japan warm‐temperate zones) and/or potential refugial areas (Sea of Japan and East China Sea) in the north‐western Pacific. Neutrality tests, mismatch distribution analysis and Bayesian skyline plots were used to infer the demographic history of C. challengeri. Results In total, 312, 117, 182 and 250 sequences were obtained for COI, 12S, 16S and ITS1, respectively. A panmictic population was revealed, which did not conform to the ‘isolation by distance’ model. None of the a priori population groupings based on marine provinces, zoogeographical zones or potential refugial areas was associated with observed genetic patterns. Significant negative values from neutrality tests and the unimodal mismatch distribution and expansion patterns in Bayesian skyline plots for the COI and 16S data sets indicate a population expansion in the mid‐Pleistocene (c. 200 ka). Information from the fossil record suggests that there has been a northward range shift of this species from the East China Sea or the Palaeo‐Pacific coast of Japan to the Sea of Japan since the mid‐Pleistocene. Main conclusions Chthamalus challengeri has experienced a population expansion and range shift since the mid‐Pleistocene. The observed lack of population differentiation can be explained by this past population expansion and present‐day wide‐scale larval dispersal (owing to the long planktonic larval duration) across marine provinces, which have led to the successful establishment of the species in different zoogeographical zones and habitats.     

Phylogeny and evolutionary history of birch mice Sicista Griffith, 1827 (Sminthidae,Rodentia): Implications from a multigene study

Phylogeny of birch mice is estimated using sequences of ten nuclear genes and one mitochondrial gene. Based on the results of tree reconstructions and molecular dating, five major lineages are recognized: “tianschanica,” “concolor,” “caudata,” “betulina,” and “caucasica.” It is established that the three latter lineages constitute a clade and that the long‐tailed birch mouse Sicista caudata is the sister group of the “caucasica” lineage. The “tianschanica” lineage is placed as the sister branch to all other species, however, with insufficient support. The cytochrome b tree is generally concordant with the nuclear topology. The molecular clock results suggest that the radiation among the main lineages occurred in the Late Miocene–Early Pliocene (6.0–4.7 Mya). The correspondence between molecular dating and the fossil record is discussed. Based on nuclear data, a high level of divergence between cryptic species in the “tianschanica” lineage is confirmed. Mitochondrial and nuclear data suggest the existence of a potential cryptic species within Sicista strandi.     

Phylogenetic and mixed Yule‐coalescent analyses reveal cryptic lineages within two South American marine snails of the genus Crepipatella (Gastropoda: Calyptraeidae)

The presence of sibling species within the marine gastropod genus Crepipatella has complicated the taxonomy of members of the group. Since the establishment of the genus, 15 species have been described, but recent studies have indicated that there are only five valid species, two of which inhabit the coasts of Chile, namely C. dilatata and C. fecunda. The two species are morphologically indistinguishable as adults, but can be differentiated on the basis of their encapsulated developmental stages. The primary aim of this study was to reconstruct phylogeny within the genus, and to establish species limits of C. dilatata and C. fecunda, using mitochondrial DNA data. To this end, we used maximum parsimony, maximum likelihood, and Bayesian inference to reconstruct phylogenies using 589 bp of the cytochrome oxidase I (COI) gene. The mtDNA phylogenies were then used as input in a general mixed Yule‐coalescent (GMYC) analysis to estimate species boundaries. In addition, quarter likelihood mapping was used to test a posteriori the confidence of inner branch patterns in the phylogenetic tree. Both DNA tree‐based and GMYC methods provide support for five isolated lineages within this species complex. Our data also suggest that Late Pleistocene and Holocene fragmentation and subsequent range expansion events may have shaped contemporary genetic patterns of Crepipatella in South America.     

Validation of a cheap and simple nondestructive method for obtaining AFLPs and DNA sequences (mitochondrial and nuclear) in amphibians

The use of nondestructive methods for obtaining DNA from amphibians (e.g. buccal swabs) allows genetic studies to be performed without affecting the survival of the studied individuals. In this study, we compared two methods of nondestructive DNA sampling, buccal swabs and interdigital membrane or toe‐clipping, in several amphibian species of different size: Rhinella spinulosa, R. atacamensis, six species of the genus Telmatobius and Pleurodema thaul. We evaluated the integrity of the DNA extracted by sequencing fragments of mitochondrial and nuclear genes and by generating amplified fragment length polymorphisms markers (AFLPs). In all cases, we obtained an adequate amount of DNA (mean range 55–298 ng/μL). We obtained identical DNA sequences from buccal swab and interdigital membrane/toe‐clip for all individuals. The differences in the coding of AFLP markers between the tissues were similar to those reported for replicas of the same type of sample in similar analyses in other species of amphibians. In conclusion, the use of buccal swabs is a trustworthy and inexpensive method to obtain DNA for mitochondrial and nuclear sequencing and AFLP analyses. Given the types of markers evaluated, buccal swabs may be used for phylogenetic, phylogeographic and population genetic studies, even in small amphibians (<33 mm).     

Deceptive single‐locus taxonomy and phylogeography: Wolbachia‐associated divergence in mitochondrial DNA is not reflected in morphology and nuclear markers in a butterfly species

The satyrine butterfly Coenonympha tullia (Nymphalidae: Satyrinae) displays a deep split between two mitochondrial clades, one restricted to northern Alberta, Canada, and the other found throughout Alberta and across North America. We confirm this deep divide and test hypotheses explaining its phylogeographic structure. Neither genitalia morphology nor nuclear gene sequence supports cryptic species as an explanation, instead indicating differences between nuclear and mitochondrial genome histories. Sex‐biased dispersal is unlikely to cause such mito‐nuclear differences; however, selective sweeps by reproductive parasites could have led to this conflict. About half of the tested samples were infected by Wolbachia bacteria. Using multilocus strain typing for three Wolbachia genes, we show that the divergent mitochondrial clades are associated with two different Wolbachia strains, supporting the hypothesis that the mito‐nuclear differences resulted from selection on the mitochondrial genome due to selective sweeps by Wolbachia strains.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Habitat‐driven diversification,hybridization and cryptic diversity in the Fork‐tailed Drongo (Passeriformes: Dicruridae: Dicrurus adsimilis)

Species complexes of widespread African vertebrates that include taxa distributed across different habitats are poorly understood in terms of their phylogenetic relationships, levels of genetic differentiation and diversification dynamics. The Fork‐tailed Drongo (Dicrurus adsimilis) species complex includes seven Afrotropical taxa with parapatric distributions, each inhabiting a particular bioregion. Various taxonomic hypotheses concerning the species limits of the Fork‐tailed Drongo have been suggested, based largely on mantle and upperpart coloration, but our understanding of diversity and diversification patterns remains incomplete. Especially given our lack of knowledge about how well these characters reflect taxonomy in a morphologically conservative group. Using a thorough sampling across Afrotropical bioregions, we suggest that the number of recognized species within the D. adsimilis superspecies complex has likely been underestimated and that mantle and upperpart coloration reflects local adaptation to different habitat structure, rather than phylogenetic relationships. Our results are consistent with recent phylogeographic studies of sub‐Saharan African vertebrates, indicating that widespread and often morphologically uniform species comprise several paraphyletic lineages, often with one or more of the lineages being closely related to phenotypically distinct forms inhabiting a different, yet geographically close, biome.     

When recent and evolutionary histories meet: deciphering temporal events from contemporary patterns of mtDNA from fishers (Martes pennanti) in north‐eastern North America

The current spatial distribution of genetic lineages across a region should reflect the complex interplay of both historical and contemporary processes. Postglacial expansion and recolonization in the distant past, in combination with more recent events with anthropogenic effects such as habitat fragmentation and overexploitation, can help shape the pattern of genetic structure observed in contemporary populations. In this study, we characterize the spatial distribution of mtDNA lineages for fisher (Martes pennanti) in north‐eastern North America. The history of fishers in this region is well understood and thus provides an opportunity to interpret patterns of genetic structure in the light of known historical (e.g. recolonization from glacial refugia) and contemporary events (e.g. reintroductions, fragmentation and natural recolonization). Our results indicate that fishers likely recolonized north‐eastern North America from a single Pleistocene refugium. Three genetically distinct remnant populations persisted through the population declines of the 1800s and served as sources for multiple reintroductions and natural recolonizations that have restored the fisher throughout north‐eastern North America. However, the spatial genetic structure of genetic lineages across the region still reflects the three remnant populations.     

Larval development and post‐larval growth of Branchiomma bairdi (Annelida: Sabellidae) from a Mediterranean population

Branchiomma bairdi is a Caribbean fan worm introduced in several localities worldwide, including the Mediterranean Sea, where the species’ range has rapidly expanded. Reproduction in B. bairdi was previously investigated in both extra‐Mediterranean and Mediterranean areas, but no information is available on larval development and post‐larval growth. In the present article, we examined these features for a population from the Mar Grande of Taranto (Ionian Sea). The species is hermaphrodite, and fertilization occurs in situ. Mucus seems to play an important role in fertilization, and also in preserving eggs before fertilization. The trochophore stage develops within the mucus and after hatching, larvae swim for about 3 d before settlement. The trochophore showed a distinct prototroch and two red dorsolateral larval eyes. The pelagic stage takes only 96 h even though prototroch is maintained after settlement, disappearing at 5 d, when larvae showed three chaetigers and branchial crown consisted of four radioles. Some interesting observations concerning changes in the morphology of chaetae and in the number of uncini during growth are also reported, together with discussion of the development of stylodes, an important diagnostic feature in Branchiomma species identification.     

Molecular and morphometric variation in two sibling species of the genus Praomys (Rodentia: Muridae): implications for biogeography

The rodent genus Praomys is widely distributed in the African tropics. The species are cryptic, rendering the species taxonomy unclear. There are differences of opinion concerning the specific status of Praomys misonnei and Praomys tullbergi, and their geographical distribution. We sequenced the cytochrome b and/or the 16S gene of 221 specimens from 12 countries in order to evaluate the genetic variability within these two species, and to precisely determine their geographical distribution. Morphological and morphometrical analyses on the sequenced specimens were also performed to find criteria useful for the identification of museum specimens. Our results confirm that P. misonnei and P. tullbergi are two valid species that can be separated by molecular data. However, no single discrete morphological character or simple metric measurement can be used to discriminate them. The percentage of misclassified individuals in multivariate discriminant analysis is relatively high (10%). The two species have allopatric distributions: P. tullbergi occurs in West Africa, from eastern Guinea to western Ghana, and P. misonnei is widely distributed from eastern Ghana to western Kenya. Within P. misonnei we identified three or four major geographical clades: a West Central African clade, an East African clade, a Nigerian clade, and a possible West African clade. Within P. misonnei, high geographical morphometrical variability was also identified. The role of both rivers and Pleistocene forest refugia in promoting speciation within the genus Praomys is discussed. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160 , 397–419.