Genetic divergence between morphological forms of brown trout Salmo trutta L. in the Balkan region of Macedonia

The objective of this study was to characterize the genetic structure of two Balkan brown trout morphotypes, Salmo macedonicus and Salmo pelagonicus, and to test whether molecular traits support the species' status proposed by traditional morphological identification. The mitochondrial DNA 12S‐rDNA, cyt b and control region genes were sequenced in 15 specimens collected from three localities in the Former Yugoslav Republic of Macedonia. The results of these markers did not support the taxonomic category of species but confirmed the existence of two morphotypes, Salmo trutta macedonicus and Salmo trutta pelagonicus, in the Aegean–Adriatic lineages of the Salmo trutta species complex.     

Molecular and morphological boundaries of the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae)

Neoseiulus californicus (McGregor) is a natural enemy of pest mites used worldwide in many crops. Its correct identification is thus essential to ensure biological control success. The present study aimed to characterize molecular and morphological intraspecific variations for assisting in the diagnosis of the species and to build baseline information about expected variations within a commercially important phytoseiid species. Morphological and molecular [12S rRNA, cytochrome b mitochondrial (mt)DNA, and internal transcribed spacer] analyses were carried out on fourteen populations collected worldwide and on one mass‐reared strain. The genetic distances between the specimens of N. californicus and another related species were high and no overlap was observed, sustaining the reliability of such molecular methods for assisting a specific diagnosis. Furthermore, the genetic distances between populations of N. californicus were very low and overlap between intra‐ and interpopulations distances was observed, except for two populations collected in France (Marsillargues and Midi‐Pyrénées). The high mitochondrial differentiation between these two latter populations and the others questions their specific status: do they belong to the species N. californicus or to another cryptic species? However, using nuclear DNA marker analyses, no distinct differences were observed. Furthermore, even if significant morphological differences were observed between the populations, these differences were very small and the standard errors within each population were very low. We thus concluded that all the populations studied belong to the species N. californicus, despite unexpected high mitochondrial variations. The present study thus displays the importance of an integrative taxonomic approach for avoiding misidentifications. A discussion on morphological and mtDNA variations in relation to diagnostic reliability is developped. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 393–406.     

Is Neoseiulus wearnei the Neoseiulus californicus of Australia?

Species of the family Phytoseiidae are known as predatory mites, some of them being used in crops to control mite pests, all around the world. Neoseiulus (=Cydnodromus) californicus is among the most commonly used Phytoseiidae species in biological control programs, especially in vineyards, orchards and vegetable fields. This species is distributed world-wide but has never been reported from Australia. On the other hand, specimens morphologically close to N. californicus have been assigned to a species called Neoseiulus wearnei, only reported from Australia. Investigations based on morphological and molecular comparisons were carried out to investigate whether these two taxa are conspecific. Morphological analyses showed no significant difference between specimens identified as N. wearnei and N. californicus. Similarly, genetic distances between these taxa were null, showing that all these specimens belong to the same species. Although it is not yet possible to conclude that all the specimens identified as N. wearnei are N. californicus, we can conclude that N. californicus is present in Australia. The information about the biology of N. californicus can thus now be applied to the Australian population of this species for biological control purposes.     

Morphological and molecular diagnostics of <Emphasis Type="Italic">Phytoseiulus persimilis</Emphasis> and <Emphasis Type="Italic">Phytoseiulus macropilis</Emphasis> (Acari: Phytoseiidae)

This study focuses on the diagnostics of two natural enemy species, belonging to the genus Phytoseiulus in the family Phytoseiidae (sub-family Amblyseiinae): P. macropilis and P. persimilis. These two species are of primary importance in biological control all over the world. However, they are morphologically very similar and specific diagnostics is difficult. This study utilizes mitochondrial molecular markers (12S rRNA and Cytb mtDNA) to differentiate these two species. Morphological analyses showed significant differences between P. persimilis and P. macropilis for 17 morphological characters of the 32 considered. However, despite these significant differences, the ranges of all characters overlap. Only the serration of the macroseta on the basitarsus (StIV) allows the differentiation between P. persimilis and P. macropilis. Despite these small morphological differences, molecular results, for both mitochondrial DNA fragments considered (rRNA and Cytb mtDNA), showed a clear delineation between the specimens of P. macropilis and P. persimilis. This study emphasizes (i) that only one morphological character (serration of the seta StIV) clearly separates these two species, and (ii) the usefulness of an automatical molecular and simple diagnostic tool for accurate differentiation of the two species and ensure the morphological diagnostics. Further studies are proposed, including more DNA sequences especially for P. macropilis.     

DNA Barcodes indicate members of the Anopheles fluviatilis (Diptera: Culicidae) species complex to be conspecific in India

Anopheles fluviatilis, a major vector of malaria in India has been described as a complex of three sibling species members, named as S, T and U, based on variations in chromosomal inversions. Also, ribosomal DNA markers (repetitive Internal Transcribed Spacer 2 (ITS2) and 28S D3 region) were described to differentiate these three sibling species members. However, controversies prevail on the genetic isolation status of these cryptic species. Hence, we evaluated this taxonomic incongruence employing DNA barcoding, the well established methodology for species identification, using 60 An. fluviatilis sensu lato specimens, collected from two malaria endemic eastern states of India. These specimens were also subjected to sibling species characterization by ITS2 and D3 DNA markers. The former marker identified 31 specimens among these as An. fluviatilis S and 21 as An. fluviatilis T. Eight specimens amplified DNA fragments specific for both S and T. The D3 marker characterized 39 specimens belonging to species S and 21 to species T. Neither marker identified species U. Neighbor Joining analysis of mitochondrial cytochrome c oxidase gene 1 sequences (the DNA barcode) categorized all the 60 specimens into a single operational taxonomic unit, their Kimura 2 parameter (K2P) genetic variability being only 0.8%. The genetic differentiation (F_ST) and gene flow (N_m) estimates were 0.00799 and 62.07, respectively, indicating these two ‘species’ (S & T) as genetically con‐specific intermixing populations with negligible genetic differentiation. Earlier investigations have refuted the existence of species U. Also, this study demonstrated that An. fluviatilis and the closely related An. minimus could be taxonomically differentiated by the DNA Barcode approach (K2P = 5.0%).     

Genetic and morphological differentiation of Porphyra and Pyropia species (Bangiales,Rhodophyta) coexisting in a rocky intertidal in Central Chile

A recent molecular taxonomic study along the Chilean coast (18° S–53° S) described 18 candidate species of bladed Bangiales of which only two were formally described. Few studies focused on local genetic and morphological diversity of bladed Bangiales and attempted to determine their intertidal distribution in contrasting habitats, and none were performed in Chile. To delimit intertidal distributions of genetic species, 66 samples of bladed Bangiales were collected at Maitencillo (32° S) in four zones: a rocky platform, a rocky wall, and two boulders zones surrounded by sandy and rocky bottoms, respectively. These samples were identified based on sequences of the mitochondrial COI and chloroplast rbcL markers. We also collected 87 specimens for morphological characterization of the most common species, rapidly assessing their putative species identity using newly developed species‐diagnostic (PCR‐RFLP) markers. Eight microscopic and two macroscopic morphological traits were measured. We described and named three of four species that predominate in Maitencillo (including Pyropia orbicularis): Pyropia variabilis Zapata, Meynard, Ramírez, Contreras‐Porcia, sp. nov., Porphyra luchea Meynard, Ramírez, Contreras‐Porcia sp. nov., and Porphyra longissima Meynard, Ramírez, Contreras‐Porcia, sp. nov. With the exception of Po. longissima restricted to boulders surrounded by sandy bottom, and a morphotype of Py. variabilis restricted to rocky walls, the other species/morphotypes have overlapping intertidal distributions. Except for Po. longissima, which is clearly differentiated morphologically (longest and thinnest blades), we conclude that morphology is not sufficient to differentiate bladed Bangiales. Our findings underscore the importance of refining our knowledge of intrinsic and environmental determinants on the distribution of bladed Bangiales.     

Plant DNA‐barcode library and community phylogeny for a semi‐arid East African savanna

Applications of DNA barcoding include identifying species, inferring ecological and evolutionary relationships between species, and DNA metabarcoding. These applications require reference libraries that are not yet available for many taxa and geographic regions. We collected, identified, and vouchered plant specimens from Mpala Research Center in Laikipia, Kenya, to develop an extensive DNA‐barcode library for a savanna ecosystem in equatorial East Africa. We amassed up to five DNA barcode markers (rbcL, matK, trnL‐F, trnH–psbA, and ITS) for 1,781 specimens representing up to 460 species (~92% of the known flora), increasing the number of plant DNA barcode records for Africa by ~9%. We evaluated the ability of these markers, singly and in combination, to delimit species by calculating intra‐ and interspecific genetic distances. We further estimated a plant community phylogeny and demonstrated its utility by testing if evolutionary relatedness could predict the tendency of members of the Mpala plant community to have or lack “barcode gaps”, defined as disparities between the maximum intra‐ and minimum interspecific genetic distances. We found barcode gaps for 72%–89% of taxa depending on the marker or markers used. With the exception of the markers rbcL and ITS, we found that evolutionary relatedness was an important predictor of barcode‐gap presence or absence for all of the markers in combination and for matK, trnL‐F, and trnH–psbA individually. This plant DNA barcode library and community phylogeny will be a valuable resource for future investigations.     

Genetic diversity of Philaenus spumarius and P. tesselatus (Hemiptera,Aphrophoridae): implications for evolution and taxonomy

The meadow spittlebugs Philaenus spumarius and P. tesselatus are closely related taxa with uncertain taxonomic position in the light of previous morphological, ecological, cytological and molecular research. Despite morphological homogeneity of P. spumarius (with the exception of high colour and pattern polymorphism) across its wide Holarctic range, it is possible that additional taxa (species or subspecies) exist. Philaenus spumarius is a potentially important pest in parts of its range where it was introduced. We used DNA markers to describe the genetic diversity of P. spumarius and P. tesselatus and to verify the taxonomic status of P. tesselatus and remote populations of the former species. The mitochondrial (cytochrome B) data showed that there are two main groups encompassing a northeastern (Asia and north‐central Europe) and a southwestern (Mediterranean area and western Europe, including North American specimens) clade. According to the elongation factor‐1α gene, there are three main clades: northeastern (Eurasiatic clade, E1), southeastern (east Mediterranean – Caucasus clade, E2) and southwestern (Iberian clade, E3). These two or three mitochondrial and nuclear clades could be considered as separate taxonomic units. On the other hand, all studied individuals of both species possessed the same internal transcribed spacer 2 haplotype. American specimens most probably originated from some western European populations. All studied specimens of P. tesselatus belong to the southwestern clade and western Mediterranean cluster. Therefore, together with cytological data, its species status is doubtful. To definitively solve the taxonomic status of P. tesselatus and populations of P. spumarius, further research using more samples and more genetic markers are needed.     

A molecular characterization of the charismatic Faroe house mouse

Faroe house mice are a ‘classic’ system of rapid and dramatic morphological divergence highlighted by J. S. Huxley during the development of the Modern Synthesis. In the present study, we characterize these charismatic mice using modern molecular techniques, examining specimens from all Faroe islands occupied by mice. The aims were to classify the mice within the modern house mouse taxonomy (i.e. as either Mus musculus domesticus or Mus musculus musculus) using four molecular markers and a morphological feature, and to examine the genetic diversity and possible routes of colonization using mitochondrial (mt) control region DNA sequences and microsatellite data (15 loci). Mice on the most remote islands were characterized as M. m. domesticus and exhibited exceptionally low genetic diversity, whereas those on better connected islands were more genetically diverse and had both M. m. musculus and M. m. domesticus genetic elements, including one population which was morphologically M. m. musculus‐like. The mtDNA data indicate that the majority of the mice had their origins in south‐western Norway (or possibly southern Denmark/northern Germany), and probably arrived with the Vikings, earlier than suggested by Huxley. The M. m. musculus genetic component appears to derive from recent mouse immigration from Denmark. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 471–482.     

Mitochondrial DNA analysis of Rhipicephalus sanguineus s.l. from the western Iberian peninsula

Rhipicephalus sanguineus Latreille (1806) (Ixodida: Ixodidae) is considered to be the most widely distributed tick and to have a vast range of habitats and hosts, including livestock, pets and wildlife. In addition to morphological differences, recent investigations using approaches based on molecular genetic markers have revealed the existence of different R. sanguineus lineages in different geographic regions. In this study, 475 ticks collected from dogs in the western Iberian peninsula were studied both morphologically and genetically, using 12S and 16S rDNA and COI gene markers in order to clarify the controversy over the systematic status of R. sanguineus sensu lato in Western Europe, and to compare the present data with those sourced from studies conducted in other regions of the world. Despite the high morphometric variability, particularly on spiracles in both genders and in female genitalia, data obtained with different genetic molecular markers show very low variability, suggesting the existence of a unique species. In addition, the phylogenetic analysis showed genetic uniformity, supporting the existence of a well‐defined clade consisting of R. sanguineus s.l. specimens from Western Europe that are distinct from R. sanguineus s.l. from Africa. Furthermore, these data corroborate the existence of a polymorphic species in Western Europe, which requires to be consensually redescribed in view of its medical and veterinary importance in pathogen transmission.     

RECOGNIZING DINOFLAGELLATE SPECIES USING ITS rDNA SEQUENCES1

Dinoflagellate taxonomy is based primarily on morphology and morphometric data that can be difficult to obtain. In contrast, molecular data can be rapidly and cost‐effectively acquired, which has led to a rapid accumulation of sequence data in GenBank. Currently there are no systematic criteria for utilizing taxonomically unassigned sequence data to identify putative species that could in turn serve as a basis for testable hypotheses concerning the taxonomy, diversity, distribution, and toxicity of these organisms. The goal of this research was to evaluate whether simple, uncorrected genetic distances (p) calculated using ITS1/5.8S/ITS2 (ITS region) rDNA sequences could be used to develop criteria for recognizing putative species before formal morphological evaluation and classification. The current analysis used sequences from 81 dinoflagellate species belonging to 14 genera. For this diverse assemblage of dinoflagellate species, the within‐species genetic distances between ITS region copies (p=0.000–0.021 substitutions per site) were consistently less than those observed between species (p=0.042–0.580). Our results indicate that a between‐species uncorrected genetic distance of p≥0.04 could be used to delineate most free‐living dinoflagellate species. Recently evolved species, however, may have ITS p values <0.04 and would require more extensive morphological and genetic analyses to resolve. For most species, the sequence of the dominant ITS region allele has the potential to serve as a unique species‐specific “DNA barcode” that could be used for the rapid identification of dinoflagellates in field and laboratory studies.     

Statistical approaches for morphological continuous characters: a conceptual model applied to Phytoseiidae (Acari: Mesostigmata)

Marie‐Stephane, T. (2012). Statistical approaches for morphological continuous characters: a conceptual model applied to Phytoseiidae (Acari: Mesostigmata). —Zoologica Scripta, 42, 327–334. Species discrimination is certainly the most current and essential taxonomic task. Despite molecular development, species continue to be delimited using morphological characters. This study provides statistical approaches to assess decision rules, using continuous morphological characters, to determine whether specimens examined belong or not to a same species. As species discrimination is usually based on no overlapping between intraspecific distributions, a general statistical approach has been developed to assess, for a character x, the relation between intraspecific overlapping and (i) the differences between the means of specimen lots corresponding to two species and (ii) the differences between the values borne by two specimens belonging to two species. Then, this conceptual approach was applied to the predatory mite family Phytoseiidae, highlighting that the minimal difference between means of two specimen lots belonging to two species should be of 10.58 μm (for setae <65 μm) and 33.99 μm (for setae >65 μm). When no specimen sets are available but only two specimens compared, the model shows that a difference of 13.24 μm (for setae <65 μm) and 31.74 μm (for setae >65 μm) would be sufficient to conclude that these specimens belong to two species. The presently proposed decision rules are assumed to improve species discrimination and to limit synonymies. Further developments will consist in applying this approach to databases containing species features in order to automatically extract the putative synonyms. Furthermore, such decision rules would also be useful to determine whether a species newly described is really new for science.     

Resurrection of Mesoplodon hotaula Deraniyagala 1963: A new species of beaked whale in the tropical Indo‐Pacific

We present genetic and morphological evidence supporting the recognition of a previously synonymized species of Mesoplodon beaked whale in the tropical Indo‐Pacific, Mesoplodon hotaula. Although the new species is closely‐related to the rare ginkgo‐toothed beaked whale M. ginkgodens, we show that these two lineages can be differentiated by maternally (mitochondrial DNA), biparentally (autosomal), and paternally (Y chromosome) inherited DNA sequences, as well as by morphological features. The reciprocal monophyly of the mtDNA genealogies and the largely parapatric distribution of these lineages is consistent with reproductive isolation. The new lineage is currently known from at least seven specimens: Sri Lanka (1), Gilbert Islands, Republic of Kiribati (1+), Palmyra Atoll, Northern Line Islands, U.S.A. (3), Maldives (1), and Seychelles (1). The type specimen (Sri Lanka) was described as a new species, M. hotaula, in 1963, but later synonymized with M. ginkgodens. This discovery brings the total number of Mesoplodon species to 15, making it, by far, the most speciose yet least known genus of cetaceans.     

Two synonymy cases within the genus Neoseiulella (Acari: Phytoseiidae): is the molecular evidence so evident?

Synonymy is considered as a great problem for diversity characterization and further applications, especially for biological control success with regard to the accurate identification of natural enemies. The present study focuses on two synonymy cases of natural enemies, belonging to the family Phytoseiidae, the genus Neoseiulella, specifically five species of this genus: Neoseiulella tiliarum, Neoseiulella formosa, Neoseiulella aceri, Neoseiulella squamiger, and Neoseiulella aceris. Morphological and molecular analyses [12S rRNA, cytochrome b (Cytb) mitochondrial (mt)DNA, internal transcribed spacer DNA] were applied. First, the results obtained support the synonymy of N. tiliarum and N. formosa. Second, because morphological differences (solenostomes occurrence) were observed for the first time between the type material of N. aceris and N. squamiger, the present study does not provide sufficient evidence to synomymize these two species and further analyses are required. Lastly, we assume that N. squamiger and N. aceri are synonyms. However, three groups of specimens, including N. aceri and N. squamiger, were identified by two mitochondrial DNA genes (12S rRNA and Cytb mtDNA). Therefore, the present study highlights the problems encountered when using only mitochondrial genes to diagnose species. The great mtDNA variations observed appear to reflect population differentiation (linked to plant support). This is the first time that such high intraspecific differentiation is be observed within the family Phytoseiidae. Further experiments, such as cross‐breeding and microsatellite DNA marker analyses, are planned to characterize gene flow and reproductive isolation levels within this species. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 323–344.     

Evolutionary relationships within European Monochamus (Coleoptera: Cerambycidae) highlight the role of altitude in species delineation

Phylogenetic relationships within the European Monochamus (Coleoptera: Cerambycidae) remain understudied despite their increasing importance in the Pine Wood Nematode spread in Europe. To clarify the delimitation and the evolutionary history of the two main European Monochamus species, Monochamus galloprovincialis and Monochamus sutor, as well as their sub‐species, a comparative study using morphological, molecular, and biogeographical criterions was conducted. Four morphological characters, including a newly‐described morphological character on the male genitalia, separated the two species. Additionally, molecular data revealed twelve and two single nucleotide polymorphisms in cytochrome oxidase c subunit I and 28S, respectively, supporting species segregation. By contrast, incongruence between morphological and genetic results did not allow discriminating the sub‐species of M. galloprovincialis and M. sutor, even though mitochondrial DNA revealed intraspecific differentiation, mostly consenting to a multiple refugia origin. Within‐species variability was explained to a large extent by biogeography (i.e. altitude, climate). These different ecological adaptations within beetle species, together with potential climate change impact, increase the risk of spreading the nematode across Europe to novel conifer hosts and challenge the European biosecurity. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 354–376.     

Phylogeny and Genetic/Morphological Variation of Strombidinopsis minima‐like Species (Ciliophora: Choreotrichia)

Six isolates of mineral‐enveloped Strombidinopsis minima‐like species were collected from the coastal waters across several regions in Korea. Morphological observations and molecular analyses were performed. The ribosomal DNA sequences (including small subunit ribosomal DNA, internal transcriber spacer 1‐5.8S ribosomal DNA‐internal transcriber spacer 2; and part of large subunit ribosomal DNA) of these six isolates were compared. Their morphological characteristics were also compared with those of S. minima populations reported. The marked genetic differences (with a similarity range of 96.85–98.48%) in SSU rDNA among these S. minima‐like entities suggest the existence of multiple species. This finding is also supported by morphological variations detected in this study and reported in the literature (e.g. 15–32 collar membranelles in different populations). In addition, S. minima‐like species are clustered with S. batos and S. sinicum, and therefore, our SSU rDNA results support previous results suggesting that the genus Strombidinopsis is not monophyletic in origin. Further collection of morphological and molecular data may facilitate the determination of a new genus carrying mineral‐enveloped Strombidinopsis species.     

Morphological and genetic relationship of two closely‐related giant water bugs: Appasus japonicus Vuillefroy and Appasus major Esaki (Heteroptera: Belostomatidae)

The East Asian giant water bug species Appasus japonicus Vuillefroy and Appasus major Esaki are aquatic hemipteran insects whose ranges overlap, particularly in the Japanese Archipelago and on the Korean Peninsula. In rare cases, the two species co‐occur. Furthermore, they are very similar ecologically and also morphologically, making their identification extremely difficult, and the possibility of hybridization has also been suggested. In the present study, we re‐examined their taxonomic validity, and the characteristics useful for identifying them. To re‐examine the morphological traits useful for distinguishing these two species, 222 specimens of A. japonicus collected from Japan, Korea, and China, and 132 specimens of A. major from Japan and Korea, were examined. We also performed molecular phylogenetic analyses based on the mitochondrial DNA 16S rRNA and cytochrome oxidase subunit I (COI) regions and the nuclear DNA Histone 3 region. Although the two species are very similar ecologically and also morphologically, they showed significant genetic differentiation. Thus, there is likely some form of reproductive isolation acting between them. Major morphological characteristics overlap extensively between A. japonicus and A. major, and no particular trait was identified as being effective for differentiating these species. All the morphological characteristics examined overlapped between A. japonicus and A. major. However, a principal component analysis based on all of the morphological characteristics revealed that, despite the overlap between these species, it was possible to morphologically distinguish them. Therefore, a more accurate identification becomes possible using multiple characteristics rather than a single characteristic. The male genital paralobes, evaluated as the most useful morphological characteristic, was effective with 100% probability for the Japanese Appasus species. However, for the Asian (i.e. Korean) specimens, this characteristic was not useful. On the other hand, the results of molecular phylogenetic analyses based on the mitochondrial DNA 16S rRNA and COI regions and the nuclear DNA Histone 3 region clearly showed significant genetic differentiation between the two species. Notably, the results for the mitochondrial COI region strongly supported the independence of each monophyletic group (i.e. validity of each species). Therefore, DNA barcoding based on the mitochondrial DNA COI region is also considered useful for the identification of A. japonicus and A. major. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 615–643.     

Identification of a uniquely southern African clade of coastal pipefishes Syngnathus spp

The taxonomic status of two southern African coastal pipefish species, Syngnathus temminckii and Syngnathus watermeyeri, was investigated using a combination of morphological and genetic data. Morphological data showed that S. temminckii is distinct from the broadly distributed European pipefish Syngnathus acus, and a molecular phylogeny reconstructed using mitochondrial DNA recovered S. temminckii and S. watermeyeri as sister taxa. The southern African species share an evolutionary origin with north‐eastern Atlantic Ocean and Mediterranean Sea species, including S. acus. These data support the existence of a distinct southern African clade of Syngnathus pipefishes that has diverged in situ to form the two species present in the region today.