DNA barcoding of genus Toxoptera Koch (Hemiptera: Aphididae): Identification and molecular phylogeny inferred from mitochondrial COI sequences

Abstract Identification of aphid species is always difficult due to the shortage of easily distinguishable morphological characters. Aphid genus Toxoptera consists of species with similar morphology and similar to Aphis in most morphological characters except the stridulatory apparatus. DNA barcodes with 1 145 bp sequences of partial mitochondrial cytochrome‐coxidase I (COI) genes were used for accurate identification of Toxoptera. Results indicated mean intraspecific sequence divergences were 1.33%, whereas mean interspecific divergences were greater at 8.29% (0.13% and 7.79% if T. aurantii 3 and T. aurantii 4 are cryptic species). Sixteen samples were distinguished to four species correctly by COI barcodes, which implied that DNA barcoding was successful in discrimination of aphid species with similar morphology. Phylogenetic relationships among species of this genus were tested based on this portion of COI sequences. Four species of Toxoptera assembled a clade with low support in maximum‐parsimony (MP) analysis, maximum‐likelihood (ML) analysis and Bayesian phylogenetic trees, the genus Toxoptera was not monophyletic, and there were two sister groups, such as T. citricidus and T. victoriae, and two clades of T. aurantii which probably presented cryptic species in the genus.     

The complete mitochondrial genome of <Emphasis Type="Italic">Thrinchus schrenkii</Emphasis> (Orthoptera: Caelifera,Acridoidea, Pamphagidae)

Complete nucleotide sequences of mitochondrial genome (mitogenome) of Thrinchus schrenkii (Orthoptera: Acridoidea: Pamphagidae) were determined. It is 15672 bp in length and contains 71.2% A + T. All T. schrenkii protein-coding sequences except for the cytochrome oxidase subunit I (COI) start with a typical ATN codon. Instead, CCG, which is a rare but possible initiation codon, is located at the initiation context of COI. The usual termination codons (TAA and TAG) were found from 12 PCGs. However, the ND5 had incomplete termination codon (T). All tRNA genes could be folded into the typical cloverleaf secondary structure, excluding tRNA ^Ser(AGN) which forms another structure according to the Steinberg–Cedergren tertiary structure. The sizes of the large and small ribosomal RNA genes are 1319 and 848 bp, respectively. The A + T content of the A + T-rich region is 78.7%, which is the lowest among the known mitogenome of Acridoidea.     

Recent emergence and worldwide spread of the red tomato spider mite, <Emphasis Type="Italic">Tetranychus evansi</Emphasis>: genetic variation and multiple cryptic invasions

Plant biosecurity is increasingly challenged by emerging crop pests. The spider mite Tetranychus evansi has recently emerged as a new threat to solanaceous crops in Africa and the Mediterranean basin, with invasions characterized by a high reproductive output and an ability to withstand a wide range of temperatures. Mitochondrial (868 bp of COI) and nuclear (1,137 bp of ITS) loci were analyzed in T. evansi samples spanning the current geographical distribution to study the earliest stages of the invasive process. The two sets of markers separate the samples into two main clades that are only present together in South America and Southern Europe. The highest COI diversity was found in South America, consistent with the hypothesis of a South American origin of T. evansi. Among the invaded areas, the Mediterranean region displayed a high level of genetic diversity similar to that present in South America, that is likely the result of multiple colonization events. The invasions of Africa and Asia by T. evansi are characterized by a low genetic variation associated with distinct introductions. Genetic data demonstrate two different patterns of invasions: (1) populations in the Mediterranean basin that are a result of multiple cryptic introductions and (2) emerging invasions of Africa and Asia, each likely the result of propagules from one or limited sources. The recent invasions of T. evansi illustrate not only the importance of human activities in the spread of agricultural pests, but also the limits of international quarantine procedures, particularly for cryptic invasions.     

Genetic diversity and phylogeny of the Kanzawa spider mite, Tetranychus kanzawai, in Japan

Two types are known in the Kanzawa spider mite, Tetranychus kanzawai (K and T; see Gotoh et al., 1999), which differ in host range and have a unidirectional incompatibility. Prior to DNA analyzes, crossing between females of a known K type and males of each of 17 strains collected in Japan showed that six of the strains were of the K type, five were the T type and the rest consisted of a mixture of the two types. In order to elucidate the genetic diversity and phylogenetic relationship of T. kanzawai in Japan, we analyzed the DNA sequences of two regions – the internal transcribed spacer 1 (ITS1) of the nuclear ribosomal DNA (rDNA) and a fragment of the cytochrome oxidase subunit I gene (COI) of mitochondrial DNA – using 11 strains (six K-type strains and five T-type strains). Base substitutions were detected on 25 sites of COI (375bp) and 19 sites of ITS1 (486bp), resulting in eight and 17 haplotypes, respectively. The phylogenetic trees constructed using the DNA sequences failed to clearly distinguish between the two types. The results suggested that the T type was derived from the K type.     

Analysis of mitochondrial COI sequences of the Diachasmimorpha longicaudata (Hymenoptera: Braconidae) species complex in Thailand

Diachasmimorpha longicaudata is an Opiinae parasitoid used to control tephritid fruit flies, which cause tremendous economic losses of fruits worldwide. In Thailand, D. longicaudata is classified as three sibling species, DLA, DLB and DLBB, based on the morphological and biological species concepts but their genetic variation has not been studied. Therefore, we investigated the genetic differentiation of the mitochondrial COI gene to clarify the ambiguous taxonomy of this species complex. The 603‐bp COI region was sequenced from laboratory‐bred colonies and field‐collected specimens from seven locations representing five geographical regions in Thailand. DLA was associated with the host Bactrocera correcta while DLB and DLBB were associated with Bactrocera dorsalis. The interspecific nucleotide differences of COI sequences among the three groups ranged from 6.70% to 7.62% (Kimura 2‐parameter distance), which adequately separates species complexes within the order Hymenoptera and supports the current sibling species classification. The neighbor joining, maximum likelihood and consensus Bayesian phylogenetic trees constructed from COI sequences revealed that the three sibling species of laboratory and field‐collected D. longicaudata are monophyletic with 100% support. The high genetic variation and molecular phylogeny of the COI sequences were shown to discriminate between the D. longicaudata species examined in this study.     

A critical review on some closely related species of <Emphasis Type="Italic">Tetranychus</Emphasis> sensu stricto (Acari: Tetranychidae) in the public DNA sequences databases

Taxonomic misidentification of the specimens used to obtain DNA sequences is a growing problem reported for different groups of organisms, which threatens the utility of the deposited sequences in public DNA databases. This paper provides new evidence of misidentifications in molecular DNA public databases in phytophagous mites of the Tetranychidae family belonging to the group Tetranychus (Tetranychus). Several species in this group are of economic and quarantine importance in agriculture and among them Tetranychus urticae, a highly polyphagous mite causing outbreaks in many crops worldwide, is certainly the most studied. We analyzed and evaluated the identity of 105 GenBank accessions of ITS2 rDNA and 138 COI mtDNA sequences which were deposited as T. urticae and those of 14 other taxa morphologically closely related to Tetranychus sensu stricto. In addition, ITS2 and COI sequences of 18 T. urticae samples collected for this study and identified by morphological criteria, were generated and included in the analyzed dataset. Among the deposited sequences in the GenBank database, numerous cases of apparently mistaken identities were identified in the group Tetranychus s. str., especially between T. urticae, T. cinnabarinus, T. kanzawai and T. truncatus. Unreliable sequences (misidentified or dubious) were estimated at nearly 30%. In particular the analysis supports the invalidity of the controversial species status of T. cinnabarinus. More generally, it highlights the need of using combined morphological and molecular approaches to guarantee solid species diagnostics for reliable sequence accessions in public databases.     

Exploring phylogenetic informativeness and nuclear copies of mitochondrial DNA (numts) in three commonly used mitochondrial genes: mitochondrial phylogeny of peppermint,cleaner, and semi‐terrestrial shrimps (Caridea: Lysmata,Exhippolysmata, and Merguia)

Of paramount importance to studies that profit from molecular trees is the accuracy and robustness of the reconstructed phylogenies. Causes of systematic error that can mislead phylogenetic methods include nuclear copies of mitochondrial DNA (numts) and low phylogenetic informativeness (PI). Herein, numts and PI were explored in three mitochondrial genes commonly used for phylogenetic reconstruction: 16S, 12S, and cytochrome c oxidase I (COI). Shrimps from the genera Lysmata, Exhippolysmata, and Merguia were used as a model system. The existence of: (1) multiple bands on gels of COI and 12S polymerase chain reaction (PCR) products from various species; (2) double peaks, background noise, and ambiguity in sequence chromatograms of COI and 12S PCR products that produced a single clear band in other species; and (3) indels, stop codons, and considerable composition bias in COI‐like cloned sequences of one problematic species (Lysmata seticaudata), was interpreted as evidence of pervasive non‐functional nuclear copies of mitochondrial DNA (numts) of the targeted COI (and probably 12S) mtDNA fragment. The information content of the three mtDNA markers studied was investigated using PI profiling, spectral analysis, and neighbour‐nets. Marker‐specific PI profiles suggested that the COI marker has the highest information content and greatest power for resolving both shallow and deep nodes in trees depicting the phylogenetic relationship among the species studied. Nonetheless, spectral analysis of splits and neighbour‐nets suggested that the 16S and 12S markers were equally or even more powerful than the COI marker for resolving nodes at all phylogenetic levels. Altogether, these analyses suggest that all three mtDNA markers are equally useful for resolving phylogenetic relationships in the shrimps studied, and that PI profiling is not necessarily useful to estimate overall gene utility. A ‘total‐evidence’ phylogenetic analysis that included 34 species and used a concatenated data set of 1403 characters (from reliable 16S, 12S and COI sequences), demonstrated that the genus Lysmata is paraphyletic, and that the monophyletic clade comprising species of Lysmata and Exhippolysmata can be divided into four well‐supported subclades (Neotropical, Cleaner, Cosmopolitan, and Morphovariable). © 2013 The Linnean Society of London     

Reliable molecular identification of nine tropical whitefly species

The identification of whitefly species in adult stage is problematic. Morphological differentiation of pupae is one of the better methods for determining identity of species, but it may vary depending on the host plant on which they develop which can lead to misidentifications and erroneous naming of new species. Polymerase chain reaction (PCR) fragment amplified from the mitochondrial cytochrome oxidase I (COI) gene is often used for mitochondrial haplotype identification that can be associated with specific species. Our objective was to compare morphometric traits against DNA barcode sequences to develop and implement a diagnostic molecular kit based on a RFLP‐PCR method using the COI gene for the rapid identification of whiteflies. This study will allow for the rapid diagnosis of the diverse community of whiteflies attacking plants of economic interest in Colombia. It also provides access to the COI sequence that can be used to develop predator conservation techniques by establishing which predators have a trophic linkage with the focal whitefly pest species.     

The complete mitogenome and phylogenetic analysis of <Emphasis Type="Italic">Bombyx mandarina</Emphasis> strain Qingzhou

The complete nucleotide sequence of the mitogenome of Bombyx mandarina strain Qingzhou was determined. The circular genome is 15,717 bp long and has the typical gene organization and order of lepidopteran mitogenomes. All protein-coding sequences are initiated with a typical ATN codon, except the COI gene, which has a 4-bp TTAG putative initiator codon. Eleven of the 13 protein-coding gene have a complete termination codon (all TAA), but the remaining two genes terminate with incomplete codons. All transfer RNAs (tRNAs) have a clover-leaf structure typical of the mitochondrial tRNAs, and some of them have a mismatch in the four-stem-and-loop structure. The length of the A + T rich region of B. mandarina strain Qingzhou is 495 bp, shorter than that of B. mandarina strain Tsukuba (747 bp) but similar to that of Bombyx mori. Phylogenetic analysis based on the whole mitochondrial genome sequences of the available sequenced species (B. mori strains C-108, Aojuku, Backokjam, and Xiafang, B. mandarina strains Tsukuba, Ankang, and Qingzhou, and Antheraea pernyi) shows the origin of the domesticated silkmoth B. mori to be the Chinese B. mandarina. Nuclear mitochondrial pseudogene sequences were detected in the nuclear genome of B. mori with the MEGA BLAST search program. A phylogenetic analysis of these nuclear mitochondrial pseudogene sequences suggests that B. mori was domesticated independently in different areas and periods.     

Theileria ovis discovered in China

A polymerase chain reaction (PCR) using 989/990 primers was conducted to identify a newly isolated Theileria sp. in Xinjiang Province of China. The target DNA fragments of the complete 18S rRNA gene were cloned and sequenced. The phylogenetic relationship of newly isolated Theileria spp. was inferred based on the 18S rRNA gene. The results showed that the new Theileria sp. belonged to the cluster of Theileria ovis. Moreover, the findings were confirmed by T. ovis species-specific PCR. An expected 520 bp fragment of T. ovis DNA was obtained from 25 out of 320 (8%) field blood samples, and blood of an experimental sheep infested by Hyalomma anatolicum anatolicum collected in Xinjiang. The infection rate of T. ovis was 78% (25/32) in Xinjiang province. The investigation did not find T. ovis positive samples from the field samples collected from the other twelve provinces. This study indicates that T. ovis is prevalent in Xinjiang province of China and its transmission vector is H. anatolicum anatolicum.     

Species-specific COI primers for rapid identification of Bemisia tabaci Mediterranean (MED) species

Bemisia tabaci (Gennadius) is a rapidly evolving species complex, and is small in size and difficult to identify quickly and accurately. For the accurate identification and effective prevention of this species, the specific PCR method based on the mitochondrial DNA cytochrome oxidase subunit I (mt DNA COI) gene was used in the present study to evaluate rapid molecular detection technological applications for Mediterranean (MED) species. The MED was targeted and whitefly species from different regions were used as references. Fragments of the mt DNA COI gene of the MED and other closely related species were amplified with universal primers. Species-specific mitochondrial DNA cytochrome oxidase subunit I (SS-COI) primers BQLF/BQLR and BQJF/BQJR were designed from variable sites of MED and other whitefly species partial COI gene sequences. Subsequently, the lengths of target fragments were amplified by two pairs of SS-COI primers. Meanwhile, the accuracy, specificity and sensitivity of SS-COI primers were determined using various life stages of the MED and other related species collected from different locations. The primer pairs BQLF/BQLR and BQJF/BQJR generated 334 bp and 483 bp amplified fragment length respectively. Accuracy test results showed that primers can detect the MED single-head adults and also accurately detect single-egg and first instar, second instar and third instar nymphs, MED pupae, etc. Specific detection results demonstrated that the primers were able to amplify the MED but not the following species/populations: Middle East-Asia Minor 1 (MEAM1), Asia I, Asia II 1, Asia II 6 and Asia II 7, Aleurocanthus spiniferus (Quaintanca), A. camelliae, Siphoninus phillyreae, Aleuroclava rhododendri, A. thysanospermi, Aleurolobus taonabae, Dialeurodes citri and Trialeurodes vaporariorum (Westwood) in different areas. Sensitivity detection results showed that primers can detect the minimum threshold of 2,160 pg/μl and 1.38 pg/μl, respectively (equivalent to 1/1280 and 1/2000000 adult). This technique solves the problem that MED cannot be identified based on morphology. This method simultaneously adopted SS-COI PCR technological applications that improved detection accuracy and saved detection time.     

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 g–1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.     

Hagfish phylogeny and taxonomy,with description of the new genus Rubicundus (Craniata,Myxinidae)

A recent phylogenetic analysis of the Myxinidae based on the 16S rRNA gene resulted in synonymization of Paramyxine with Eptatretus. This created homonymy of Paramyxine fernholmi with Eptatretus fernholmi and Paramyxine wisneri with Eptatretus wisneri. In order to resolve this nomenclatural dilemma, we made a more extensive phylogenetic assessment of the Myxinidae and examined the nomenclature of the family. We used 75 sequences (37 of which new for this study) of a 561 bp fragment of the 16S rRNA gene, representing 33 species, and 72 sequences (37 of which new for this study) of a 687 bp fragment of the cytochrome c oxidase subunit I (COI) gene, representing 23 species, to reconstruct the phylogeny of Myxinidae. The monophyly of the subfamily Myxininae, traditionally characterized by having a single pair of external gill openings, was rejected (0.50 Bayesian posterior probability) by the 16S analysis, but supported by the COI and combined COI+16S analyses (0.99 and 0.81 Bpp, respectively). The monophyly of the subfamily Eptatretinae, characterized by having several pairs of external gill openings, was not supported by the 16S analysis and rejected by the COI and combined COI+16S analysis due to the placement of Eptatretus lopheliae as the earliest branch of Myxinidae (0.71 and 0.57 Bpp, respectively). Eptatretus lopheliae and Eptatretus rubicundus formed a monophyletic group and were allocated to a new genus, Rubicundus, characterized by the presence of an elongated tubular nostril and reddish coloration. A new monotypic subfamily, Rubicundinae, was proposed for Rubicundus. The synonymy of the genera Paramyxine and Quadratus with Eptatretus was confirmed. E. fernholmi is renamed Eptatretus luzonicus. Eptatretus wisneri was renamed Eptatretus bobwisneri. Petromyzon cirrhatus Forster, 1801, Homea banksii Fleming, 1822, and Bdellostoma forsteri Müller, 1836 are synonyms, but no type specimens are known to exist. Petromyzon cirrhatus was designated as type species of Eptatretus, conserving present usage. Gastrobranchus dombeyi Shaw, 1804 has priority over other names for Chilean myxinids. Bdellostoma stoutii was designated as type species of Polistotrema Gill. The validity of the Western Atlantic Myxine limosa as distinct from the Eastern Atlantic Myxine glutinosa was confirmed.     

Female dimorphism in Japanese diving beetle Dytiscus marginalis czerskii (Coleoptera: Dytiscidae) evidenced by mitochondrial gene sequence analysis

Three species of the Japanese diving beetle Dytiscus have been identified: D. dauricus Gebler, 1832; D. marginalis czerskii Zeitzev, 1953; and D. sharpi. At present, the latter consists of the subspecies D. sharpi sharpi Wehncke, 1875 and D. sharpi validus Régimbart, 1899 based on the comparative data of mitochondrial DNA cytochrome‐c oxidase I (COI) sequences. Many Dytiscus species have smooth and grooved elytra, which are female dimorphic traits. For many years it has been thought that Japanese D. marginalis czerskii has a single morph, that is, only grooved females, although there were some collecting reports of smooth females occurring at the foot of Mt. Chokaisan in Yamagata and Akita Prefectures. However, the population of smooth females (smooth population) has not yet been identified by DNA markers. To understand the species status of the smooth population, we sequenced 769 bp of COI of a male derived from a smooth mother insect and compared it with the sequence from a known grooved female. The sequences of 769 bp of the COI gene in the smooth population were identical to that in the grooved female, indicating that Japanese D. marginalis czerskii has female dimorphic traits.     

Genetic Variation and Population Structure of Hair Crab (<Emphasis Type="Italic">Erimacrus isenbeckii</Emphasis> ) in Japan Inferred from Mitochondrial DNA Sequence Analysis

Genetic variation and population structure of hair crab (Erimacrus isenbeckii) were examined using nucleotide sequence analysis of 580 base pairs (bp) in the 3′ portion of the mitochondrial cytochrome c oxidase subunit I gene (COI) of 20 samples collected from 16 locales in Japan (the Hokkaido and Honshu Islands) and one in Korea. A total of 27 haplotypes was defined by 23 variable nucleotide sites in the examined COI region. Pairwise population F _ST estimates and neighbor-joining tree inferred distinct genetic differentiation between the representative samples from the Pacific Ocean off the Eastern Hokkaido Island and the Sea of Japan, while others were intermediate between these two groups. AMOVA also showed a weak but significant differentiation among these three groups. The present results suggest a moderate population structure of hair crab, probably influenced by high gene flow between regional populations due to sea current dependent larval dispersal of this species.     

DNA barcoding supports reclassification of Japanese Chironomus species (Diptera: Chironomidae)

Non‐biting midges (Diptera: Chironomidae) adapt to species‐specific environmental conditions and hence are promising bioindicators for aquatic and ecotoxicological monitoring. Although their utility for these purposes was historically limited by difficulties in their morphological identification, DNA barcoding offers a possible solution. Here, eight Japanese species of the genus Chironomus, which is characterized by its worldwide distribution and abundance among Chironomidae, were subjected to DNA barcoding using cytochromec oxidase subunit I (COI). To examine whether this DNA barcode is a useful indicator for Japanese species of Chironomus, we calculated genetic distances within and between the COI sequences of Chironomus species both from this study and worldwide and constructed phylogenetic trees. Based on 415 bp COI sequences and the Kimura two‐parameter model, the average genetic distances within 37 species and between 72 species were 2.6% and 17.2%, respectively. Although the ranges of genetic distances within and between species overlapped from 0.8% to 17.3%, 99.7% of average genetic distances between species were >3.0%. Some of this overlap is attributable to distances within species that were “too large” as well as those between species that were “too small”. Of eight Japanese species examined, two showed genetic distances between species that were below a 3.0% threshold, and four had distances within species that were greater than 3.0%. These results suggest a possible reclassification of these species and the need for further sampling to unveil biogeographic variations among different countries and regions.     

Comparative and evolutionary analysis of new variants of ω-gliadin genes from three A-genome diploid wheats

A genomic polymerase chain reaction (PCR) cloning strategy was applied to isolate ω-gliadin sequences from three A-genome diploid wheats (Triticum monococcum, T. boeoticum and T. urartu). Amplicon lengths varied from 744 and 1,044 bp, and those of the corresponding deduced mature proteins from 248 to 348 residues. The primary structure of the deduced polypeptides comprised a short N- and C-terminal conserved domain, and a long, variable repetitive domain. A phylogenetic analysis recognised several clades: the first consisted of three T. aestivum sequences; the second and the third two T. boeoticum and six T. monococcum sequences; and the rest four T. urartu and three T. aestivum sequences. Among the functional (non-pseudogene) ARQ/E-type ω-gliadin sequences, two were derived from T. boeoticum and three from T. monococcum; one of the latter sequences appeared to be a chimera originating via illegitimate recombination between the other two T. monococcum sequences. None of the 12 intact ω-gliadin sequences contained any cysteine or methionine residues. We discussed the variation and evolution of A-genome ω-gliadin genes.     

Biological and Molecular Characterization of Alfalfa Mosaic Virus Infecting Trumpet Creeper (Campsis radicans) in Iran

Samples of trumpet creeper (Campsis radicans) leaves showing mottling and mosaic were collected from plants growing in a private garden in Tehran province, Iran, in 2012. Symptomatic leaf samples were tested for Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV) and Peanut stunt virus (PSV) infection in enzyme‐linked immunosorbent assay (ELISA), using specific antibodies. None of the samples were positive for CMV and PSV; however, all reacted positively with that of AMV antiserum. In biological assay, systemic infection was found on Datura stramonium, Nicotiana tabacum cvs., White Burley, and Xanthi, 21 days postinoculation (DPI), while necrotic local lesions were obtained following inoculation of Phaseolus vulgaris and Vigna unguiculata within three to four DPI. Using a pair of primers specific for AMV, a DNA fragment of 880 bp was RT‐PCR‐amplified. Analysis of the sequences revealed the presence of 657 nucleotides of AMV complete coat protein (CP) gene (translating 218 amino acid residues). Phylogenetic analysis using neighbour‐joining (NJ) method clustered AMV isolates into two main types and the IRN‐Tru (GenBank Accession No. JX865593 ) isolate fell into type I. Pairwise nucleotide distances also confirmed two main types with the highest and lowest similarities for type I and II, respectively. The association of AMV with mosaic disease of C. radicans represents the first record from the world.     

Haemosporidian infection in passerine birds from Lower Saxony

Blood samples from 94 coal tits (Parus ater), 56 great tits (Parus major) and 219 pied flycatchers (Ficedula hypoleuca), caught between 1993 and 2002 at two localities in Lower Saxony, Germany, were examined for haemosporidian infection by parasite-specific polymerase chain reaction (PCR). A simple PCR targeting the 18 SSU rRNA gene of the parasites was used for rapid screening of the samples and generated a total infection prevalence of 20.6% (76/369): 6.8% (n = 15) of the pied flycatchers, 19.1% (n = 18) of the coal tits and 76.8% (n = 43) of the great tits were infected. The positive specimens were re-examined by a cytochrome b gene-directed nested PCR producing significantly longer DNA fragments (approx. 520 bp) that were sequenced and analysed against GenBank-deposited nucleotide sequences. In various numbers (once to 30 times), a total of 13 parasitic DNA sequences differing from 2.9 to 8.5% (13–45 nucleotides) were demonstrated in the three bird species. Due to similarities of 98–100% with GenBank entries, 11 sequences could be assigned to Plasmodium sp. and two to the genus Haemoproteus. In summary, 57 birds were infected with Plasmodium and 19 with Haemoproteus, corresponding to 15.4 and 5.1% of all birds examined, and to 75 and 25% of all birds tested positive. As the only defined species, Haemoproteus majoris was identified in 17 great tits.     

Old and new DNA unweave the phylogenetic position of the eastern Atlantic gall crab Detocarcinus balssi (Monod, 1956) (Decapoda: Cryptochiridae)

Phylogeny reconstructions based on mtDNA and nDNA have become the standard in studies on relationships between taxa. Difficulties in obtaining material, for example because of small endemic distributions, often lead to gaps in datasets. Collections in natural history museums can provide us with material to fill these gaps, but extracting DNA from historical specimens can be challenging. We used a PCR protocol for small amounts of sample material and high PCR yield on eggs of specimens of the coral‐dwelling gall crab family Cryptochiridae collected in 1984, including material from the eastern Atlantic species Detocarcinus balssi. We obtained DNA sequences from seven older museum specimens using newly developed primers, which we combined with COI sequences from recently collected material. Results show that D. balssi is closest to the Indo‐Pacific species Utinomiella dimorpha and not closely related to one of the other three Atlantic Cryptochiridae species. The remaining newly acquired DNA sequences from museum material cluster with the respective sequences from recently collected specimens.