On the conspecificity ofAnopheles fluviatilis species S withAnopheles minimus species C

Anopheles fluviatilis andAn. minimus complexes, each comprising of at least three sibling species, are closely related and important malaria vectors in Oriental Region. RecentlyAn. fluviatilis species S, which is a highly efficient malaria vector in India, has been made conspecific withAn. minimus species C (senior synonym) on the basis of homology in 335 base pair nucleotide sequence of D3 domain of 28S ribosomal DNA(rDNA). We examined the conspecificity of these two nominal species by obtaining and analysing the DNA sequences of nuclear ribosomal loci internal transcribed spacer 2 (ITS2) and D2-D3 domain of 28S rDNA (28S-D2/D3) from those ofAn. fluviatilis S andAn. minimus C. We found that the sequences ofAn. fluviatilis S are appreciably different from those ofAn. minimus C with pair-wise distance (Kimura-2-parametre model) of 3.6 and 0.7% for loci ITS2 and 28S-D2/D3, respectively. Pair-wise distance and phylogenetic analyses using ITS2 sequences of members of Minimus and Fluviatilis Complexes revealedthat An. fluviatilis S is distantly related toAn. minimus C as compared to any other members of the Fluviatilis Complex. These findings suggest that the two nominal species,An. fluviatilis S andAn. minimus C, do not merit synonymy. The study also confirms that the reported speciesAn. fluviatilis X is synonym with species S.     

Molecular phylogenetics of the Oriental members of the Myzomyia Series of Anopheles subgenus Cellia (Diptera: Culicidae) inferred from nuclear and mitochondrial DNA sequences

Abstract. The phylogenetic relationships of fifteen Oriental and two Afrotropical taxa of the Myzomyia Series of Anopheles subgenus Cellia and two outgroup species, An. maculatus (Neocellia Series) and An. dirus A (Neomyzomyia Series), were inferred from nucleotide sequences of the entire 685 bp of the mitochondrial cytochrome oxidase subunit II locus (COII) and 372 bp of the third domain (D3) of the 28S rDNA locus, both separately and together. Alignment of the D3 sequences was achieved with the aid of secondary structure comparisons, and the pattern of nucleotide substitution was best explained by the GTR + I + G model for either separate or combined datasets. Maximum likelihood and maximum parsimony analyses robustly identified five monophylies: An. fluviatilis U and T; An. fluviatilis U and T + An. minimus A, C, E and #157 + An. leesoni; An. filipinae + An. mangyanus; An. filipinae + An. mangyanus + An. aconitus; and An. culicifacies A and B. The results confirm the specific status of An. flavirostris, the close relationship of An. leesoni with the Minimus Complex, and the exclusion of An. jeyporiensis, An. culicifacies s.l and An. funestus from the Minimus Group. All of the species classified as members of the Minimus Group on morphological grounds formed a single clade, which comprised two subgroups: the Minimus Subgroup, including An. minimus s.l., An. fluviatilis s.l., An. leesoni and An. flavirostris, and the Aconitus Subgroup, including An. filipinae, An. mangyanus, An. aconitus, An. pampanai and An. varuna. However, these clades are only weakly supported by the present dataset.     

Multilocus nuclear DNA markers reveal population structure and demography of Anopheles minimus

Utilization of multiple putatively neutral DNA markers for inferring evolutionary history of species population is considered to be the most robust approach. Molecular population genetic studies have been conducted in many species of Anopheles genus, but studies based on single nucleotide polymorphism (SNP) data are still very scarce. Anopheles minimus is one of the principal malaria vectors of Southeast (SE) Asia including the Northeastern (NE) India. Although population genetic studies with mitochondrial genetic variation data have been utilized to infer phylogeography of the SE Asian populations of this species, limited information on the population structure and demography of Indian An. minimus is available. We herewith have developed multilocus nuclear genetic approach with SNP markers located in X chromosome of An. minimus in eight Indian and two SE Asian population samples (121 individual mosquitoes in total) to infer population history and test several hypotheses on the phylogeography of this species. While the Thai population sample of An. minimus presented the highest nucleotide diversity, majority of the Indian samples were also fairly diverse. In general, An. minimus populations were moderately substructured in the distribution range covering SE Asia and NE India, largely falling under three distinct genetic clusters. Moreover, demographic expansion events could be detected in the majority of the presently studied populations of An. minimus. Additional DNA sequencing of the mitochondrial COII region in a subset of the samples (40 individual mosquitoes) corroborated the existing hypothesis of Indian An. minimus falling under the earlier reported mitochondrial lineage B.     

Diversity of Anopheles species and trophic behavior of putative malaria vectors in two malaria endemic areas of northwestern Thailand

We determined the species diversity, blood‐feeding behavior, and host preference of Anopheles mosquitoes in two malaria endemic areas of Tak (Mae Sot District) and Mae Hong Son (Sop Moei District) Provinces, located along the Thai border with Myanmar, during a consecutive two‐year period. Anopheline mosquitoes were collected using indoor and outdoor human‐landing captures and outdoor cow‐baited collections. Mosquitoes were initially identified using morphological characters, followed by the appropriate multiplex AS‐PCR assay for the identification of sibling species within Anopheles (Cellia) complexes and groups present. Real‐time PCR was performed for parasite‐specific detection in mosquitoes (Plasmodium spp. and Wuchereria bancrofti). A total of 7,129 Anopheles females were captured, 3,939 from Mae Sot and 3,190 from Sop Moei, with 58.6% and 37% of all anophelines identified as An. minimus, respectively. All three malaria vector complexes were detected in both areas. One species within the Minimus Complex (An. minimus) was present along with two related species in the Funestus Group, (An. aconitus, An. varuna), two species within the Dirus Complex (An. dirus, An. baimaii), and four species within the Maculatus Group (An. maculatus, An. sawadwongporni, An. pseudowillmori, and An. dravidicus). The trophic behavior of An. minimus, An. dirus, An. baimaii, An. maculatus, and An. sawadwongporni are described herein. The highest An. minimus densities were detected from February through April of both years. One specimen of An. minimus from Mae Sot was found positive for Plasmodium vivax.     

Molecular and morphological studies on the Anopheles minimus group of mosquitoes in southern China: taxonomic review,distribution and malaria vector status

Abstract. Mosquitoes of the Anopheles minimus group (Diptera: Culicidae) from nine Provinces of southern China were identified morphologically and by molecular characterization, using single‐strand conformation polymorphisms (SSCPs) and sequence data for the D3 region of the 28S ribosomal DNA and the mitochondrial COII locus. Species A and C (sensu Green et al., 1990 ) of the An. minimus complex were found to be sympatric in Yunnan Province. Species A occurs eastward from Yunnan through southern Guangxi, Hainan, Guangdong and Taiwan Provinces, whereas species C occurs northward to northern Guangxi, Guizhou and Sichuan Provinces. Morphological and molecular evidence (based on specimens from the field and four isofemale lines) shows that An. minimus forms A and B (sensu Yu & Li, 1984 ) are morphological variants of species A, which is accepted as An. minimus Theobald sensu stricto (type‐locality: Pokfulam, Hong Kong). The so‐called subspecies x of An. minimus (sensu Baba, 1950 ) is reinterpreted as An. aconitus Dönitz. The distribution and vector status of members of the An. minimus group are discussed in relation to the historical and current transmission of malaria and filariasis in China. Both An. minimus A and C have been implicated as widespread vectors of malaria, whereas only species A has been found in Hainan, where An. minimus s.l. was a vector of Bancroftian filariasis. The presence of An. aconitus in Hainan and Yunnan Provinces is confirmed, but the occurrence of An. varuna Iyengar and An. fluviatilis James, which were previously recorded in China, could not be verified.     

Vector bionomics and malaria transmission along the Thailand‐Myanmar border: a baseline entomological survey

Baseline entomological surveys were conducted in four sentinel sites along the Thailand‐Myanmar border to address vector bionomics and malaria transmission in the context of a study on malaria elimination. Adult Anopheles mosquitoes were collected using human‐landing catch and cow‐bait collection in four villages during the rainy season from May‐June, 2013. Mosquitoes were identified to species level by morphological characters and by AS‐PCR. Sporozoite indexes were determined on head/thoraces of primary and secondary malaria vectors using real‐time PCR. A total of 4,301 anopheles belonging to 12 anopheline taxa were identified. Anopheles minimus represented >98% of the Minimus Complex members (n=1,683), whereas the An. maculatus group was composed of two dominant species, An. sawadwongporni and An. maculatus. Overall, 25 Plasmodium‐positive mosquitoes (of 2,323) were found, representing a sporozoite index of 1.1% [95%CI 0.66–1.50]. The transmission intensity as measured by the EIR strongly varied according to the village (ANOVA, F=17.67, df=3, P<0.0001). Our findings highlight the diversity and complexity of the biting pattern of malaria vectors along the Thailand‐Myanmar border that represent a formidable challenge for malaria control and elimination.     

Multiplex PCR assay for malaria vector Anopheles minimus and four related species in the Myzomyia Series from Southeast Asia

Abstract. Mosquitoes (Diptera: Culicidae) of the Anopheles (Cellia) Myzomyia Series are important malaria vectors in Africa, India and Southeast Asia. Among 10 named species of Myzomyia known from the Oriental Region, seven form the An. minimus group. Even for expert taxonomists, the adults of these species remain difficult to identify morphologically. For technical staff of malaria control programmes, confusion may extend to misidentification of species that are not formally within the minimus group. For identification of specimens from Indochina (Cambodia, Laos, Vietnam), we describe a multiplex polymerase chain reaction (PCR) assay, based on rDNA internal transcribed spacer 2 (ITS2) sequences, that employs a cocktail of primers to identify An. minimus Theobald sibling species A and C (sensu; Green et al., 1990) and three other species in the An. minimus group (An. aconitus Dönitz, An. pampanai Büttiker & Beales, An. varuna Iyengar), as well as An. jeyporiensis James, also belonging to the Myzomyia Series. As the test is DNA‐based, it can be applied to all life stages of these mosquitoes for ecological investigations and vector incrimination studies. This PCR assay is simpler, quicker, cheaper and more readily interpreted than previous assays.     

Presence of Anopheles culicifacies B in Cambodia established by the PCR-RFLP assay developed for the identification of Anopheles minimus species A and C and four related species

Abstract A polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north‐eastern Cambodia. In addition to finding An. aconitus Dönitz, An. minimus species A and An. pampanai Büttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR‐RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980 ), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR‐RFLP assay can detect species not included in the initial set‐up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non‐vector anophelines in South‐east Asia.     

EVOLUTIONARY IMPLICATIONS OF DNA DIVERGENCE IN THE DROSOPHILA OBSCURA GROUP

Using DNA–DNA hybridization, we have determined the degree of single-copy DNA (scDNA) divergence among eight species of the Drosophila obscura group. These include Old World and New World species as well as members of two subgroups. Contrary to classical systematics, members of the affinis subgroup are more closely related to American members of the obscura subgroup than are Old World species. The Old World species are not a monophyletic group. The degree of scDNA divergence among species is not necessarily correlated with morphology, chromosomal divergence, or ability to form hybrids. A unique pattern of hybrid formation was found: species separated by a ΔT_m of 6.5°C can form hybrids whereas species separated by a ΔT_m of 2.5°C cannot. As with other groups of Drosophila, the obscura group has discrete parts of the genome evolving at very different rates. The slow evolving fraction of the nuclear genome is evolving at about the same rate as mitochondrial DNA. The additional scDNA divergence accompanying the step from partial reproductive isolation (between North American pseudoobscura and the isolated Bogotà population) to full isolation is very small. The resolution of the technique was challenged by five closely related taxa with a maximum ΔT_m of 2.5°C separating them; the taxa were unambiguously resolved and the “correct” phylogeny recovered. Finally, there is some indication that scDNA in the obscura group may be evolving considerably slower than in the melanogaster subgroup.     

NUCLEAR AND MITOCHONDRIAL SEQUENCE DATA REVEAL AND CONCEAL DIFFERENT DEMOGRAPHIC HISTORIES AND POPULATION GENETIC PROCESSES IN CARIBBEAN REEF FISHES

Mitochondrial and nuclear sequence data should recover historical demographic events at different temporal scales due to differences in their effective population sizes and substitution rates. This expectation was tested for two closely related coral reef fish, the tube blennies Acanthemblemaria aspera and A. spinosa. These two have similar life histories and dispersal potentials, and co‐occur throughout the Caribbean. Sequence data for one mitochondrial and two nuclear markers were collected for 168 individuals across the species’ Caribbean ranges. Although both species shared a similar pattern of genetic subdivision, A. spinosa had 20–25 times greater nucleotide sequence divergence among populations than A. aspera at all three markers. Substitution rates estimated using a relaxed clock approach revealed that mitochondrial COI is evolving at 11.2% pairwise sequence divergence per million years. This rapid mitochondrial rate had obscured the signal of old population expansions for both species, which were only recovered using the more slowly evolving nuclear markers. However, the rapid COI rate allowed the recovery of a recent expansion in A. aspera corresponding to a period of increased habitat availability. Only by combining both nuclear and mitochondrial data were we able to recover the complex demographic history of these fish.     

Z chromosome divergence,polymorphism and relative effective population size in a genus of lekking birds

Sex chromosomes contribute disproportionately to species boundaries as they diverge faster than autosomes and often have reduced diversity. Their hemizygous nature contributes to faster divergence and reduced diversity, as do some types of selection. In birds, other factors (mating system and bottlenecks) can further decrease the effective population size of Z-linked loci and accelerate divergence (Fast-Z). We assessed Z-linked divergence and effective population sizes for two polygynous sage-grouse species and compared them to estimates from birds with various mating systems. We found lower diversity and higher F_ST for Z-linked loci than for autosomes, as expected. The π_Z/π_A ratio was 0.38 in Centrocercus minimus, 0.48 in Centrocercus urophasianus and 0.59 in a diverged, parapatric population of C. urophasianus, a broad range given the mating system among these groups is presumably equivalent. The full data set had unequal males and females across groups, so we compared an equally balanced reduced set of C. minimus and individuals pooled from both C. urophasianus subgroups recovering similar estimates: 0.54 for C. urophasianus and 0.38 for C. minimus. We provide further evidence that N_eZ/N_eA in birds is often lower than expected under random mating or monogamy. The lower ratio in C. minimus could be a consequence of stronger selection or drift acting on Z loci during speciation, as this species differs strongly from C. urophasianus in sexually selected characters with minimal mitochondrial divergence. As C. minimus also exhibited lower genomic diversity, it is possible that a more severe demographic history may contribute to its lower ratio.     

Systematics,biogeography, and evolution of Pristurus minimus (Squamata,Sphaerodactylidae) with the discovery of the smallest Arabian vertebrate

Almost 20% of Oman’s terrestrial reptiles are found on Masirah Island. Despite its ancient geological history and its long isolation, Masirah Island only harbours one endemic reptile species, Hemidactylus masirahensis. In this study, we use an integrative approach to explore the variation in Pristurus minimus, to revise its systematics and to assess its phylogeography by using molecular (mitochondrial and nuclear DNA sequences) and morphological data. Our results uncovered a deep divergence within P. minimus that dates back to ～4?Ma, during the Pliocene Epoch. The old divergence separated P. minimus into two allopatric species: one from mainland Arabia, P. minimus, and one endemic to Masirah Island, described as a new species herein. Despite the general similarity between the two sister species, there are morphological differences related mainly to body size. The new Pristurus species endemic to Masirah Island is significantly smaller than its mainland sister taxon, becoming the smallest known vertebrate species in Arabia and one of the smallest lizard species in the world. The phylogenetic analyses also uncovered a low level of genetic diversity within the newly described Pristurus species endemic to Masirah Island and a relatively deep genetic divergence within P. minimus that dates back to the Pleistocene. Once more, the present study highlights the relatively high levels of reptile diversity and endemicity in south-eastern Arabia despite its harsh, arid climate and stresses its relevance from a conservation point of view.

The LSIDs for this publication is: urn:lsid:zoobank.org:pub:DB0658D5-7F68-4E66-885F-75E27F9CD512.     

Photoreceptor Guanylate Cyclases: A Review

Almost three decades of research in the field of photoreceptor guanylate cyclases are discussed in this review. Primarily, it focuses on the members of membrane-bound guanylate cyclases found in the outer segments of vertebrate rods. These cyclases represent a new guanylate cyclase subfamily, termed ROS-GC, which distinguishes itself from the peptide receptor guanylate cyclase family that it is not extracellularly regulated. It is regulated, instead, by the intracellularly-generated Ca²⁺ signals. A remarkable feature of this regulation is that ROS-GC is a transduction switch for both the low and high Ca²⁺ signals. The low Ca²⁺ signal transduction pathway is linked to phototransduction, but the physiological relevance of the high Ca²⁺ signal transduction pathway is not yet clear; it may be linked to neuronal synaptic activity. The review is divided into eight sections. In Section I, the field of guanylate cyclase is introduced and the scope of the review is briefly explained; Section II covers a brief history of the investigations and ideas surrounding the discovery of rod guanylate cyclase. The first five subsections of Section III review the experimental efforts to quantify the guanylate cyclase activity of rods, including in vitro and in situ biochemistry, and also the work done since 1988 in which guanylate cyclase activity has been determined. In the remaining three subsections an analytical evaluation of the Ca²⁺ modulation of the rod guanylate cyclase activity related to phototransduction is presented. Section IV deals with the issues of a biochemical nature: isolation and purification, subcellular localization and functional properties of rod guanylate cyclase. Section V summarizes work on the cloning of the guanylate cyclases, analysis of their primary structures, and determination of their location with in situ hybridization. Section VI summarizes studies on the regulation of guanylate cyclases, with a focus on guanylate cyclases activating proteins. In Section VII, the evidence about the localization and functional role of guanylate cyclases in other retinal cells, especially in on-bipolar cells, in which guanylate cyclase most likely plays a critical role in electrical signaling, is discussed. The review concludes with Section VIII, with remarks about the future directions of research on retinal guanylate cyclases.     

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 morphologic variation of the Pecos assiminea,an endangered mollusk of the Rio Grande region,United States and Mexico (Caenogastropoda: Rissooidea: Assimineidae)

Assiminea pecos is an endangered species of amphibious gastropod that occupies four widely separated portions of the Rio Grande region in the southwestern United States (Pecos River basin) and northeastern Mexico (Cuatro Cienegas basin). Our statistical and discriminant function analyses of shell variation among the disjunct populations of this species indicate that Mexican specimens differ in their morphometry from those of the United States and can be diagnosed by several characters. We also analyzed variation in the mitochondrial genome by sequencing 658 bp of mitochondrial COI from populations of A. pecos, representatives of the other three North American species of Assiminea, and several outgroups. Our results indicated substantial divergence of the Mexican population of A. pecos, which was consistently depicted as a monophyletic unit nested within or sister to the shallowly structured group comprised of American members of this species. Consistent with our findings, we describe the Mexican population as a new species, which is provisionally placed in the large, worldwide genus Assiminea pending further study of the phylogentic relationships of the North American assimineids. Our molecular data suggest that the Rio Grande region assimineids, which are among the few inland members of the otherwise estuarine subfamily Assimineinae, diverged from coastal progenitors in the late Miocene, with subsequent Pleistocene vicariance of Mexican and American species perhaps associated with development of the modern, lower course of the Rio Grande. Handling editor: K. Martens     

Mitochondrial phylogeny and biogeography of the genus Gobio (Teleostei: Cyprinidae) in Turkey

Species of the cyprinid genus Gobio Cuvier, 1816 are widely distributed in freshwater lakes and rivers in Turkey, which is a hotspot for freshwater fish diversity and endemism. The mitochondrial 16S, coxI and cytb genes were sequenced for 217 individuals representing 15 species of Gobio from Turkey. A total of 23 haplotypes were identified for each mitochondrial gene. The genetic distance matrices show that Turkish Gobio species are clustered into three groups (northwestern, central and northeastern Anatolia). Phylogenetic trees constructed with combined dataset by using maximum parsimony, maximum likelihood and Bayesian inference methods revealed that the Turkish Gobio species belongs to three well-supported groups in accordance with their geographic distribution: Group I comprises species found in Central Anatolia, Group II species ranging from eastern Thrace to western Anatolia (northwestern Group) and Group III contains only one species from Western Transcaucasia (northeastern Group). The estimated divergence times between the three Gobio groups, calculated using a conventional 1% rate of mutation for a fish mitochondrial cytb gene per million years, coincide with the late Miocene period in which the tectonic uplift of Anatolia and global climate fluctuations occurred. The relatively low genetic distance between Gobio species in the Turkish Lake District indicate that they are not good species.     

Maintenance of mosquito vectors: effects of blood source on feeding,survival, fecundity,and egg hatching rates

Artificial membrane‐feeding techniques have replaced direct feeding on animals for the maintenance of malaria and arbovirus vectors in many laboratories. Membrane feeding facilitates controlled experimentation of pathogen transmission during mosquito feeding. Sheep blood is commonly used due to its availability and low cost. We evaluated the impact of blood source (human, guinea pig, sheep, and hamster via direct feeding) on feeding rates, adult survival, fecundity, hatching rates, and developmental times for five species of laboratory‐colonized mosquitoes (Anopheles dirus, An. cracens, An. minimus, An. sawadwongporni, and Ae. aegypti). We found that feeding rates differ among blood sources within mosquito species. Survival, fecundity, and hatching rates were lower in all Anopheles species and Ae. aegypti after membrane feeding on sheep blood. Survival rates seven days post‐feeding on sheep blood were significantly lower (P<0.05) for An. dirus (84.2%), An. minimus (67.2%), An. sawadwongporni (51.5%), and An. cracens (35.5%) relative to other blood sources. An. minimus and An. sawadwongporni laid no eggs by seven days post‐feeding with sheep blood, while An. dirus and An. cracens produced significantly fewer numbers of eggs and demonstrated significantly lower hatching rates relative to what was observed with the other blood sources. These findings support the conclusion that sheep blood is not a suitable blood source for laboratory rearing of Anopheles spp.     

Phylogeography of Pinus tabulaeformis Carr. (Pinaceae), a dominant species of coniferous forest in northern China

How coniferous trees in northern China changed their distribution ranges in response to Quaternary climatic oscillations remains largely unknown. Here we report a study of the phylogeography of Pinus tabulaeformis, an endemic and dominant species of coniferous forest in northern China. We examined sequence variation of maternally inherited, seed‐dispersed mitochondrial DNA (mtDNA) (nad5 intron 1 and nad4/3–4) and paternally inherited, pollen‐ and seed‐dispersed chloroplast DNA (cpDNA) (rpl16 and trnS‐trnG) within and among 30 natural populations across the entire range of the species. Six mitotypes and five chlorotypes were recovered among 291 trees surveyed. Population divergence was high for mtDNA variation (G_ST = 0.738, N_ST = 0.771) indicating low levels of seed‐based gene flow and significant phylogeographical structure (N_ST > G_ST, P < 0.05). The spatial distribution of mitotypes suggests that five distinct population groups exist in the species: one in the west comprising seven populations, a second with a north–central distribution comprising 15 populations, a third with a southern and easterly distribution comprising five populations, a fourth comprising one central and one western population, and a fifth comprising a single population located in the north‐central part of the species’ range. Each group apart from the fourth group is characterized by a distinct mitotype, with other mitotypes, if present, occurring at low frequency. It is suggested, therefore, that most members of each group apart from Group 4 are derived from ancestors that occupied different isolated refugia in a previous period of range fragmentation of the species, possibly at the time of the Last Glacial Maximum. Possible locations for these refugia are suggested. A comparison of mitotype diversity between northern and southern subgroups within the north‐central group of populations (Group 2) showed much greater uniformity in the northern part of the range both within and between populations. This could indicate a northward migration of the species from a southern refugium in this region during the postglacial period, although alternative explanations cannot be ruled out. Two chlorotypes were distributed across the geographical range of the species, resulting in lower levels of among‐population chlorotype variation. The geographical pattern of variation for all five chlorotypes provided some indication of the species surviving past glaciations in more than one refugium, although differentiation was much less marked, presumably due to the greater dispersal of cpDNA via pollen.     

Identification of bacterial guanylate cyclases

The ability of bacteria to use cGMP as a second messenger has been controversial for decades. Recently, nucleotide cyclases from Rhodospirillum centenum, GcyA, and Xanthomonas campestris, GuaX, have been shown to possess guanylate cyclase activities. Enzymatic activities of these guanylate cyclases measured in vitro were low, which makes interpretation of the assays ambiguous. Protein sequence analysis at present is insufficient to distinguish between bacterial adenylate and guanylate cyclases, both of which belong to nucleotide cyclases of type III. We developed a simple method for discriminating between guanylate and adenylate cyclase activities in a physiologically relevant bacterial system. The method relies on the use of a mutant cAMP receptor protein, CRP_G, constructed here. While wild‐type CRP is activated exclusively by cAMP, CRP_G can be activated by either cAMP or cGMP. Using CRP‐ and CRP_G‐dependent lacZ expression in two E. coli strains, we verified that R. centenum GcyA and X. campestris GuaX have primarily guanylate cyclase activities. Among two other bacterial nucleotide cyclases tested, one, GuaA from Azospillrillum sp. B510, proved to have guanylate cyclase activity, while the other one, Bradyrhizobium japonicum CyaA, turned out to function as an adenylate cyclase. The results obtained with this reporter system were in excellent agreement with direct measurements of cyclic nucleotides secreted by E. coli expressing nucleotide cyclase genes. The simple genetic screen developed here is expected to facilitate identification of bacterial guanylate cyclases and engineering of guanylate cyclases with desired properties. Proteins 2015; 83:799–804. © 2015 Wiley Periodicals, Inc.     

Conservation and genetics of the frosted flatwoods salamander (Ambystoma cingulatum) on the Atlantic coastal plain

The federally threatened frosted flatwoods salamander, Ambystoma cingulatum, occurs in isolated populations on the coastal plain of northern Florida, Georgia and South Carolina. An earlier phylogeographic study based primarily on mitochondrial DNA (mtDNA) and morphometrics demonstrated that the previously recognized species A. cingulatum contained two cryptic species, and that two distinct mtDNA clades were contained within the newly restricted A. cingulatum. However, salamanders from the northern extent of the species range in South Carolina were not previously available for analysis. Here, we used individuals from a newly discovered South Carolina breeding site to determine whether A. cingulatum from South Carolina are genetically distinct from their more southerly conspecifics. These analyses included the mitochondrial gene studied previously as well as broad geographic sampling of three rapidly evolving nuclear genes that allowed us to further investigate lineage diversification of flatwoods salamanders. The mitochondrial and nuclear results are largely congruent, yielding strong support for two distinct species of flatwoods salamanders and also two lineages within the eastern species. Further, the South Carolina specimens are closely related to other haplotypes found in eastern Georgia and Florida. Our summary of field surveys over the past 20 years indicates that this South Carolina population may be one of only three remaining in the entire Atlantic coastal plain distribution of this rare and declining amphibian species.