A multiplex PCR test for determination of mating type applied to the plant pathogens Tapesia yallundae and Tapesia acuformis

A multiplex PCR test for determining mating type of the pathogens Tapesia yallundae and Tapesia acuformis is described. The test involves three primers: a "common" primer annealing to DNA sequence conserved in the flanking region of both mating-type idiomorphs and two specific primers annealing to sequence in either the MAT-1 or the MAT-2 idiomorphs. Locating the specific primers in different positions relative to the common primer yielded PCR products of 812 or 418 bp from MAT-1 and MAT-2 isolates, respectively. The test was used successfully to determine the mating type of 118 isolates of T. yallundae and T. acuformis from Europe, North America, and New Zealand. Isolates of both mating types were found on all continents for both species despite the rarely observed occurrence of sexual reproduction of T. acuformis. The multiplex test design should be applicable to other ascomycete species, of use in studies of MAT distribution and facilitating sexual crossing by identifying compatible isolates.     

Both mating types in the heterothallic fungus Ophiostoma quercus contain MAT1-1 and MAT1-2 genes

In heterothallic Ascomycota, two opposite but distinct mating types control all sexual processes. Using mating crosses, mating types were assigned to ten isolates of the heterothallic fungal species Ophiostoma quercus. Primers were subsequently designed to target the MAT1-1-1, MAT1-1-3 (of the mating type 1 idiomorph), and MAT1-2-1 (of the mating type 2 idiomorph) genes in these isolates. Results showed that all isolates contained the full gene sequence for the MAT1-2-1 gene. In addition, fragments of the MAT1-1-1 and MAT1-1-3 genes were sequenced from all isolates. These results were unexpected, as each isolate from a heterothallic species would typically contain only one of the two possible MAT idiomorphs.     

Enhanced niche opportunities: can they explain the success of New Zealand's introduced bird species?

Aim  The niche hypothesis could explain why some species introduced to new locations reach higher densities than in their native range: it posits that the new environment provides more abundant or higher quality resources or habitat, a more suitable physical environment or both. We investigate whether 11 bird species occur at higher densities in their introduced range than in their native range and whether the differences can be explained by the availability of preferred habitat or the suitability of climatic conditions in their introduced range relative to their native range.
Location  South Island, New Zealand (the introduced range); UK (the native range).
Methods  We first develop a series of models that accurately predict the density of 11 bird species at 54 UK farmland sites, which are closely matched to our New Zealand sites, from habitat and climatic variables. We then use these models to predict the density of the 11 species at 54 New Zealand farmland sites and compare the predicted and observed values.
Results  Actual densities at New Zealand sites were on average (median) 22 times (range: 1–6361) higher than predicted from the UK models and similarly higher than actually observed at comparable UK sites. Habitat and climatic variables can accurately predict bird densities in the UK but grossly underestimate densities for all species except Turdus merula in New Zealand.
Main conclusions  These findings indicate that factors other than the measured habitat and climatic variables must differ between the two regions and explain the much higher densities of New Zealand birds. We suggest that introduced birds, other than T. merula , in New Zealand may still experience enhanced niche opportunities due to greater availability of higher quality resources within habitats, release from natural enemy regulation, less exposure to extreme weather events, particularly during winter, or some combination of these processes.     

Using testis size to predict the mating systems of New Zealand geckos

Abstract

The mating system is unknown for the majority of New Zealand geckos (Family Gekkonidae). I investigated interspecific variation in relative testis volume across 19 New Zealand gecko taxa and 63 additional species of squamate reptiles from other countries to make predictions about the social mating system of the New Zealand geckos. Relative testis size varied greatly between species of New Zealand gecko, with testes ranging from 119% larger than expected to 75% smaller than expected. This variation was even greater across other squamates, with testes ranging from 55 to 152% of expected size. This variation could not be explained by seasonality of breeding, clutch size or the sex ratio of populations. As species subject to more intense sperm competition sport relatively larger testes in other vertebrate groups, it is likely that similar variation in gecko testis size is due to differences in mating systems between species. Based on testis volume, it is predicted that at least half of the New Zealand gecko taxa studied are likely to have polygynandrous mating systems.     

Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility

A diverse collection of isolates identified as Colletotrichum acutatum, including a range of fruit-rot and foliar pathogens, was examined for mtDNA RFLPs and RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase (GS) gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene. RFLPs of mtDNA, RFLPs of the 900-bp GS intron and sequence analysis of each intron identified the same seven distinct molecular groups, or clades, within C. acutatum sensu lato. Sequence analysis produced highly concordant tree topologies with definitive phylogenetic relationships within and between the clades. The clades might represent phylogenetically distinct species within C. acutatum sensu lato. Mating tests also were conducted to assess sexual compatibility with tester isolates known to outcross to form the teleomorph Glomerella acutata. Mating compatibility was identified within one clade, C, and between two phylogenetically distinct clades, C and J4. The C clade represented isolates from a wide range of hosts and geographic origins. J4 clade contained isolates from Australia or New Zealand recovered from fruit rot and pine seedlings with terminal crook disease. That isolates in two phylogenetically distinct clades were capable of mating suggests that genetic isolation occurred before reproductive isolation. No other isolates were sexually compatible with the mating testers, which also were in groups C and J4. Certain clades identified by mtDNA and intron analysis (D1, J3 and J6) appeared to represent relatively host-limited populations. Other clades (C1, F1 and J4) contained isolates from a wide range of hosts. Isolates described as C. acutatum f. sp. pineum were clearly polyphyletic.     

Phylogenetic Analysis of Phaeosphaeria Species Using Mating Type Genes and Distribution of Mating Types in Iran

Phaeosphaeria species are pathogenic on wheat, barley and a wide range of wild grasses. To analyze mating type loci of the Phaeosphaeria species and investigate mating type distribution in Iran, we sequenced mating type loci of 273 Phaeosphaeria isolates including 67 isolates obtained from symptomatic leaves and ears of wheat, barley, and wild grasses from two wheat-growing region in Iran as well as 206 isolates from our collection from other regions in Iran which were isolated in our previous studies. Mating type genes phylogeny was successfully used to determine the species identity and relationships among isolates within the Phaeosphaeria spp. complex. In this study, we reported seven new host records for Phaeosphaeria species and the Phaeosphaeria avenaria f. sp. tritici 3 group was first reported from Iran in this study. Mating type distribution among Phaeosphaeria species was determined. Both mating types were present in all sampling regions from Iran. We observed skewed distribution of mating types in one region (Kohgiluyeh va Boyer-Ahmad) and equal distribution in the other region (Bushehr). However, when considering our entire dataset of 273 Iranian Phaeosphaeria isolates, the ratio of mating types was not deviated significantly from 1:1 suggesting possibilities for isolates of opposite mating type to interact and reproduce sexually, although the sexual cycle may infrequently occur in some regions especially when the climatic conditions are unfavorable for teleomorph development.     

Biogeographic and phylogenetic diversity of thermoacidophilic cyanidiales in Yellowstone National Park, Japan, and New Zealand

Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extreme environments that combine low pH levels ( approximately 0.2 to 4.0) and moderately high temperatures of 40 to 56 degrees C. These unicellular algae occur in far-flung volcanic areas throughout the earth. Three genera (Cyanidium, Galdieria, and Cyanidioschyzon) are recognized. The phylogenetic diversity of culture isolates of the Cyanidiales from habitats throughout Yellowstone National Park (YNP), three areas in Japan, and seven regions in New Zealand was examined by using the chloroplast RuBisCO large subunit gene (rbcL) and the 18S rRNA gene. Based on the nucleotide sequences of both genes, the YNP isolates fall into two groups, one with high identity to Galdieria sulphuraria (type II) and another that is by far the most common and extensively distributed Yellowstone type (type IA). The latter is a spherical, walled cell that reproduces by internal divisions, with a subsequent release of smaller daughter cells. This type, nevertheless, shows a 99 to 100% identity to Cyanidioschyzon merolae (type IB), which lacks a wall, divides by "fission"-like cytokinesis into two daughter cells, and has less than 5% of the cell volume of type IA. The evolutionary and taxonomic ramifications of this disparity are discussed. Although the 18S rRNA and rbcL genes did not reveal diversity among the numerous isolates of type IA, chloroplast short sequence repeats did show some variation by location within YNP. In contrast, Japanese and New Zealand strains showed considerable diversity when we examined only the sequences of 18S and rbcL genes. Most exhibited identities closer to Galdieria maxima than to other strains, but these identities were commonly as low as 91 to 93%. Some of these Japanese and New Zealand strains probably represent undescribed species that diverged after long-term geographic isolation.     

Genotype and mating type analysis of Cryptococcus neoformans and Cryptococcus gattii isolates from China that mainly originated from non-HIV-infected patients

Cryptococcosis has been reported to be mostly associated with non-HIV-related patients in China. However, little is known about the molecular characteristics of clinical isolates from the Cryptococcus neoformans species complex in this country. In this study, 115 clinical isolates were included. Molecular type VNI was the most representative ( n =103), followed by VGI ( n =8), VNIII ( n =2), VNIV ( n =1), and VGII ( n =1). With the exception of a serotype D mating type a isolate, all possessed the MAT α locus. Multilocus sequence typing (MLST) revealed that most Cryptococcus gattii isolates from China shared identical MLST profiles with the most common MLST genotype reported in the VGI group, and the only one VGII isolate resembled the Vancouver Island outbreak minor genotype. The C. gattii strains involved in this study were successfully grouped according to their molecular type and mating types by PCR-restriction fragment length polymorphism (RFLP) analysis of the GEF1 gene. Our results suggest that (1) in China, cryptococcosis is mostly caused by C. neoformans var. grubii (molecular type VNI), and mating type α; (2) The most common causative agents of C. gattii infection in China are closely related to a widely distributed MLST genotype; and (3) The PCR-RFLP analysis of the GEF1 gene has the potential to identify the molecular and mating types of C. gattii simultaneously.     

Two new species of Kazachstania that form ascospores connected by a belt-like intersporal body: Kazachstania zonata and Kazachstania gamospora

Four strains of ascomycetous yeasts were isolated from samples collected at two locations in southern Japan. The strains formed two warty ascospores that were joined together by an intersporal body appearing as a belt. Phylogenetic analysis of rRNA gene nucleotide sequences indicated that the strains represented two new and closely related species of the genus Kazachstania. Isolates of one of the species were from Miyazaki Prefecture and those of the other species were from the Iriomote Islands. Genetic separation of the two species was further confirmed by DNA-DNA reassociation, which gave values of 63.3-78.1%, and from interspecific crosses, which gave nonviable ascospores. On the basis of these data, the isolates from Miyazaki Prefecture are described as Kazachstania zonata sp. nov. [type strain NBRC 100504=CBS 10326, mating types NBRC 101821 (+), NBRC 101822 (-)], and the isolates from the Iriomote Islands are described as Kazachstania gamospora sp. nov. [type strain NBRC 11056=CBS 10328, mating types NBRC 101825 (+), NBRC 101826 (-)].     

<Emphasis Type="Italic">Cylindrocladium</Emphasis> species and the related fungi isolated in the Ogasawara (Bonin) Islands and the Ryukyu Islands,Japan

A total of 28 deuteromycetous isolates obtained from forest environments in the Ogasawara (Bonin) Islands and the Ryukyu Islands, Japan, were identified to five Cylindrocladium and related fungal species (Calonectria kyotoensis (anamorph: Cylindrocladium floridanum), Cylindrocladiella lageniformis, Cylindrocladium camelliae, Cylindrocladium citri, and Cylindrocladium tenue), excluding two unknowns. Cylindrocladiella lageniformis is a new record, and the others are rarely reported in Japan.     

Beauveria caledonica is a naturally occurring pathogen of forest beetles

In New Zealand, two introduced scolytid beetles, Hylastes ater and Hylurgus ligniperda (Curculionidae: Scolytinae) are pests in pine plantations. Investigation of the naturally occurring pathogens of these exotic pests revealed that both are attacked by Beauveria caledonica, a species originally isolated and described from soil in Scotland. The isolates in New Zealand were identical in morphology and conserved DNA region (rDNA, elongation factor α) sequence to isolates held in the USDA-ARS insect pathogens culture collection. In bioassay, the B. caledonica isolates were highly pathogenic to adults of H. ligniperda and larvae of Tenebrio molitor. Sporulation was observed on cadavers, confirming the species can utilise the cadavers. As both species were likely to have been introduced to New Zealand from Europe, a search was made for B. caledonica in the northern UK and Ireland. The fungus was found as a naturally-occurring pathogen of the weevil pest, Hylobius abietis (Curculionidae: Scolytinae), developing in spruce and other beetles in forests in both regions.     

ITS rDNA variation of the Coprinopsis phlyctidospora (syn.: Coprinus phlyctidosporus) complex in the Northern and Southern Hemispheres

 Coprinopsis phlyctidospora (syn: Coprinus phlyctidosporus) from the Netherlands, Japan, New Zealand, and Australia can be segregated into two groups, northern and southern, based on the nucleotide sequences of their ITS regions. The mating type of a C. phlyctidospora isolate was tetrapolar. Mating reactions were compatible between monokaryotic testers derived from basidiospores of a Japanese isolate and dikaryotic isolates obtained from a wide geographic area in Japan. In contrast, mating between the Japanese monokaryotic and dikaryotic isolates from Australia and New Zealand were incompatible. These results indicated that C. phlyctidospora was complex and individuals currently recognized as C. phlyctidospora in the Northern Hemisphere and those in the Southern Hemisphere are distinct taxa. The relationship between the clades and the biogeography of the C. phlyctidospora complex are also discussed. Received: January 22, 2002 / Accepted: March 15, 2002     

Isolation and characterization of microsatellite loci in Cylindrocladium pauciramosum

Ten polymorphic microsatellite markers were developed for Cylindrocladium pauciramosum, a plant pathogen with a wide host range, which poses a serious problem in South African Eucalyptus nurseries. Polymorphism was evaluated on 43 isolates collected from Colombia and South Africa. Each locus had between three and six alleles. Testing for random mating showed multilocus equilibrium for a population of 40 isolates from a South African forestry nursery. Cross‐species transferability tested for 19 other Cylindrocladium species found amplification only in C. spathulatum, which is phylogenetically closely related to C. pauciramosum.     

Mating Genes of the Trichophyton mentagrophytes Complex

The mating type (-)-specific gene of the alpha-box and the mating type (+)-specific gene of the high-mobility group (HMG) DNA-binding domain were confirmed in zoophilic dematophytes of Arthroderma simii and A. vanbreuseghemii. The sequence of the alpha-box gene was 1,375 bp, containing 2 exons (from 172 to 463 bp and from 513 to 1,375 bp) in the A. simii (-) mating type strain and 1,380 bp, containing 2 exons (from 177 to 468 bp and from 518 to 1,380 bp) in the A. vanbreuseghemii (-) mating type strain. The sequence of the HMG gene was 1,871 bp, containing 2 exons (from 181 to 362 bp and from 426 to 1,440 bp, coding a protein of 398 amino acids) in the A. simii (+) mating type strain and 1,811 bp containing 2 exons (from 158 to 339 bp and from 403 to 1,381 bp, coding a protein of 386 amino acids) in the A. vanbreuseghemii (+) mating type strain. Of 15 animal isolates and 72 human isolates examined, the alpha-box gene was detected in five of the animal isolates and in none of the human isolates, while the HMG gene was detected in the other 10 of the animal isolates and in all of the human isolates. Phylogenetic analysis of the alpha-box and HMG genes of Trichophyton mentagrophytes complex strains and the Microsporum gypseum strain revealed that these strains were divided into 4 clusters; the first cluster consisting of A. vanbreuseghemii and the isolates from animals and humans, the second cluster consisting of A. simii, the third cluster consisting of A. benhamiae and the fourth cluster consisting of M. gypseum. These results indicate that anthropophilic T. mentagrophytes evolved from the A. vanbreuseghemii (+) mating strain.     

Multiple mating strategies explain unexpected genetic mixing of New Zealand fur seals with two congenerics in a recently recolonized population

Human impacts on natural systems can cause local population extinctions, which may promote redistribution of taxa and secondary contact between divergent lineages. In mammalian populations that have mating systems shaped by polygyny and sexual selection, the potential for hybridization to ensue and persist depends on individual and demographic factors. At Macquarie Island, a recently formed fur seal population is comprised of both sexes of breeding Antarctic (Arctocephalus gazella) and subantarctic (A. tropicalis) fur seals, and an itinerant collection of male New Zealand fur seals (A. forsteri), presumed to be non-breeders due to their absence from principle breeding areas. The mating system of the three species is described as resource-defence polygyny: males defend beach territories containing breeding females for exclusive mating rights. A recent genetic study identified a high level of hybridization in the population (17-30%), unexpectedly involving all three species. This study examined the source of involvement in breeding by A. forsteri with respect to mating strategies operating in the population. Ninety-five (10%) pups born from 1992 to 2003 were genetically identified as New Zealand hybrids. Most resulted from reproduction within territories by New Zealand hybrids of both sexes, although some were conceived extra-territorially, indicating that males successfully utilize strategies other than territory holding to achieve paternities. Female reproductive status influenced mating partner and mating location, and females without pups were more likely to conceive extra-territorially and with A. forsteri males. This study illustrates an important consequence of low heterospecific discrimination in a sympatric population of long-lived mammals.     

Unexpectedly diverse Mesorhizobium strains and Rhizobium leguminosarum nodulate native legume genera of New Zealand, while introduced legume weeds are nodulated by Bradyrhizobium species

The New Zealand native legume flora are represented by four genera, Sophora, Carmichaelia, Clianthus, and Montigena. The adventive flora of New Zealand contains several legume species introduced in the 19th century and now established as serious invasive weeds. Until now, nothing has been reported on the identification of the associated rhizobia of native or introduced legumes in New Zealand. The success of the introduced species may be due, at least in part, to the nature of their rhizobial symbioses. This study set out to address this issue by identifying rhizobial strains isolated from species of the four native legume genera and from the introduced weeds: Acacia spp. (wattles), Cytisus scoparius (broom), and Ulex europaeus (gorse). The identities of the isolates and their relationship to known rhizobia were established by comparative analysis of 16S ribosomal DNA, atpD, glnII, and recA gene sequences. Maximum-likelihood analysis of the resultant data partitioned the bacteria into three genera. Most isolates from native legumes aligned with the genus Mesorhizobium, either as members of named species or as putative novel species. The widespread distribution of strains from individual native legume genera across Mesorhizobium spp. contrasts with previous reports implying that bacterial species are specific to limited numbers of legume genera. In addition, four isolates were identified as Rhizobium leguminosarum. In contrast, all sequences from isolates from introduced weeds aligned with Bradyrhizobium species but formed clusters distinct from existing named species. These results show that native legume genera and these introduced legume genera do not have the same rhizobial populations.     

Population genetic analysis of the ovine parasitic nematode Teladorsagia circumcincta and evidence for a cryptic species

An understanding of genetic variation in parasite populations, and how it is partitioned, is required to underpin many areas of basic and applied research. Population genetic studies on parasitic nematode populations are still in their infancy and have been dominated by the use of single locus markers. We have used a panel of five microsatellite markers to undertake a genetic study of a number of field and laboratory populations of Teladorsagia circumcincta. High levels of polymorphism were seen in all the populations examined with the majority of diversity being within rather than between populations. There was no detectable genetic differentiation between the UK populations examined although they included both laboratory passaged and field isolates derived from different geographical regions and host species. This broadly supports previous mtDNA sequence diversity studies of this parasite in the UK and USA. However, some between-population genetic differentiation was apparent when several populations from French goats and a laboratory population from New Zealand were examined. Most notably, a population from a French goat farm, which has previously been suggested to contain a cryptic species, showed very high levels of genetic differentiation from all the other populations. Analysis of multi-locus genotypes suggested the presence of two sympatric parasite populations on this farm with little or no gene flow between them. This supports the hypothesis that parasites currently defined as T. circumcincta by routine morphological criteria comprise more than a single species.     

Change of Mating Type in an EU1 Lineage Isolate of Phytophthora ramorum

All Phytophthora ramorum EU1 lineage isolates tested are of A1 mating type, except for three rare isolates from 2002 to 2003 from Belgium, which were originally assigned the A2 mating type. In one of these isolates (2338), a switch from A2 to A1 mating type was observed in 2006. This observation initiated a larger study in which all cultures and subcultures of the original three EU1 A2 isolates, maintained in three laboratories under different storage conditions, were checked for mating type change. The A2 to A1 mating type switch was observed in four of seven independently maintained isolates that were derived from isolate 2338 in two laboratories, using different transfer regimes and storage conditions. Following the mating type switch to A1 in these four derived isolates, no reversion back to A2 mating was observed, even after up to 5 years of additional isolate maintenance and several more subculturing events. The three other isolates that were derived from isolate 2338 as well as the other EU1 A2 isolates collected in 2002 and 2003 and stored in the same conditions did not display such mating type change. The potential causes of the mating type conversions as well as their epidemiological implications are discussed.     

New Zealand geckos (Diplodactylidae): Cryptic diversity in a post-Gondwanan lineage with trans-Tasman affinities

We used a multi-gene approach to assess the phylogenetic relationships of New Zealand diplodactylid geckos to their Australian and New Caledonian relatives and to one another. Data from nuclear (RAG-1, PDC) and mitochondrial (ND2, 16S) genes from >180 specimens representing all 19 recognized New Zealand taxa and all but two of 20 putatively new species suggested by previous studies were analyzed using Maximum Parsimony, Maximum Likelihood and Bayesian inference. All analyses retrieved a monophyletic New Zealand clade, most closely related to the Australian Diplodactylidae exclusive of Pseudothecadactylus. Hoplodactylus is paraphyletic and composed of two morphological groups: a broad-toed clade, consisting of the island-restricted, largest extant species, Hoplodactylus duvaucelii, and the species-rich, wide-ranging Hoplodactylus maculatus clade; and a narrow-toed clade, comprising five monophyletic subgroups: Naultinus, the Hoplodactylus pacificus and Hoplodactylus granulatus clades, and the distinctive species Hoplodactylus rakiurae and Hoplodactylus stephensi. Each of these lineages is here recognized at the generic level. Our data support recognition of 16 new species (36 total), and five new or resurrected genera (seven total). The New Zealand diplodactylid radiation split from its Australian relatives 40.2mya (95% highest posterior density estimate 28.9-53.5), after the opening of the Tasman Sea. Their distribution cannot, therefore, be regarded as derived as a result of Gondwanana vicariance. The age of the New Zealand crown group, 24.4mya (95% highest posterior density estimate 15.5-33.8), encompasses the period of the 'Oligocene drowning' of New Zealand and is consistent with the hypothesis that New Zealand was not completely inundated during this period. Major lineages within New Zealand geckos diverged chiefly during the mid- to late Miocene, probably in association with a suite of geological and climatological factors that have characterized the region's complex history.     

Quantifying functional heterothallism in the pseudohomothallic ascomycete Neurospora tetrasperma

Neurospora tetrasperma is a pseudohomothallic filamentous ascomycete that has evolved from heterothallic ancestors. Throughout its life cycle, it is predominantly heterokaryotic for mating type, and thereby self-fertile. However, studies of N. tetrasperma have revealed the occasional production of self-sterile asexual and sexual spores of a single-mating type, indicating that it can be functionally heterothallic. Here, we report the extensive sampling and isolation of natural, heterokaryotic, strains of N. tetrasperma from the United Kingdom (UK): 99 strains were collected from Surrey, England, and four from Edinburgh, Scotland. We verified by phylogenetic analyses that these strains belong to N. tetrasperma. We isolated cultures from single germinated asexual spores (conidia) from 17 of these newly sampled UK strains from Surrey, and 16 previously sampled strains of N. tetrasperma from New Zealand (NZ). Our results show that the N. tetrasperma strains from the UK population produced a significantly greater proportion of self-sterile, homokaryotic conidia than the NZ population: the proportion of homokaryotic conidia was 42.6 % (133/312 spores) and 15.3 % (59/386) from the UK and the NZ populations, respectively. Although homokaryons recovered from several strains show a bias for one of the mating types, the total ratio of mat A to mat a mating type in homokaryons (UK: 72/61, NZ 28/31) did not deviate significantly from the expected 1:1 ratio for either of these populations. These results indicate that different populations exhibit differences in their life cycle characteristics, and that a higher degree of outcrossing might be expected from the UK population. This study points to the importance of studying multiple strains and populations when investigating life history traits of an organism with a complex life cycle, as previously undetected differences between populations may be revealed.