Evolutionary Diversification of New Caledonian Araucaria

New Caledonia is a global biodiversity hotspot. Hypotheses for its biotic richness suggest either that the island is a ‘museum’ for an old Gondwana biota or alternatively it has developed following relatively recent long distance dispersal and in situ radiation. The conifer genus Araucaria (Araucariaceae) comprises 19 species globally with 13 endemic to this island. With a typically Gondwanan distribution, Araucaria is particularly well suited to testing alternative biogeographic hypotheses concerning the origins of New Caledonian biota. We derived phylogenetic estimates using 11 plastid and rDNA ITS2 sequence data for a complete sampling of Araucaria (including multiple accessions of each of the 13 New Caledonian Araucaria species). In addition, we developed a dataset comprising 4 plastid regions for a wider taxon sample to facilitate fossil based molecular dating. Following statistical analyses to identify a credible and internally consistent set of fossil constraints, divergence times estimated using a Bayesian relaxed clock approach were contrasted with geological scenarios to explore the biogeographic history of Araucaria. The phylogenetic data resolve relationships within Araucariaceae and among the main lineages in Araucaria, but provide limited resolution within the monophyletic New Caledonian species group. Divergence time estimates suggest a Late Cretaceous-Cenozoic radiation of extant Araucaria and a Neogene radiation of the New Caledonian lineage. A molecular timescale for the evolution of Araucariaceae supports a relatively recent radiation, and suggests that earlier (pre-Cenozoic) fossil types assigned to Araucaria may have affinities elsewhere in Araucariaceae. While additional data will be required to adequately resolve relationships among the New Caledonian species, their recent origin is consistent with overwater dispersal following Eocene emersion of New Caledonia but is too old to support a single dispersal from Australia to Norfolk Island for the radiation of the Pacific Araucaria sect. Eutacta clade.     

Genome sizes of all 19 Araucaria species are correlated with their geographical distribution

Nuclear genome size was determined to investigate the relationships between all 19 species of Araucaria de Jussieu. Species from the two other genera of Araucariaceae, Wollemia and Agathis, were also studied. The genome size of 17 out of the 19 species of Araucaria are reported here for the first time. All Araucariaceae have the same chromosome number 2n?=?26. However, the nuclear DNA contents (2C value) for Araucaria range from 31.3 to 45.4?pg. There is a good correlation between genome size and division in sections, and geographical distribution. The two species from South America have 44.7 and 45.4?pg, the two species from Australia have 35.7 and 44.4?pg and the two species from New Guinea 34.7 and 40.4?pg. All 13 species of New Caledonia and the one from Norfolk Island have a similar, if not identical, amount of nuclear DNA of, on average, 31.9?pg. This corroborates the identical DNA rbcL sequences found for the New Caledonian araucarias. It suggests that the species from New Caledonia diversified more recently and it questions their status as separate species. Compared with this 31.9?pg a strong increase seems to have occurred in the genome size of the “mainland” araucarias. Genome sizes are evaluated and compared with available taxonomic treatments and extant geographic spreading. The nuclear DNA contents found within the sections are close, making it possible to assign an unknown plant to a section. A difference of 1?pg, which amounts to a difference of 978?Mbp, far exceeds a single character. Nuclear DNA content, as measured by flow cytometry, may conveniently be used to produce systematic data. It is applicable even with young plants or seeds for monitoring the trade in endangered species.     

Characterization of nuclear microsatellite loci in South American Araucariaceae species

The Araucariaceae family has only two species in South America: Araucaria angustifolia and Araucaria araucana. Both species are mainly used for timber and have been overexploited in the past. Currently, they are found as fragmented populations and are classified under the International Union for Conservation of Nature and Natural Resources (IUCN) guidelines as vulnerable species. Population fragmentation may seriously affect the genetic diversity of these two species of Araucariaceae and can consequently lead to decreased survival. To better understand the genetic structure of these South American Araucaria species, eight nuclear microsatellites are reported: six new microsatellites loci developed based on a membrane enrichment procedure and two microsatellites loci transferred from the related species, Araucaria cunninghamii.     

Does complete plastid genome sequencing improve species discrimination and phylogenetic resolution in Araucaria?

Obtaining accurate phylogenies and effective species discrimination using a small standardized set of plastid genes is challenging in evolutionarily young lineages. Complete plastid genome sequencing offers an increasingly easy‐to‐access source of characters that helps address this. The usefulness of this approach, however, depends on the extent to which plastid haplotypes track morphological species boundaries. We have tested the power of complete plastid genomes to discriminate among multiple accessions of 11 of 13 New Caledonian Araucaria species, an evolutionarily young lineage where the standard DNA barcoding approach has so far failed and phylogenetic relationships have remained elusive. Additionally, 11 nuclear gene regions were Sanger sequenced for all accessions to ascertain the success of species discrimination using a moderate number of nuclear genes. Overall, fewer than half of the New Caledonian Araucaria species with multiple accessions were monophyletic in the plastid or nuclear trees. However, the plastid data retrieved a phylogeny with a higher resolution compared to any previously published tree of this clade and supported the monophyly of about twice as many species and nodes compared to the nuclear data set. Modest gains in discrimination thus are possible, but using complete plastid genomes or a small number of nuclear genes in DNA barcoding may not substantially raise species discriminatory power in many evolutionarily young lineages. The big challenge therefore remains to develop techniques that allow routine access to large numbers of nuclear markers scaleable to thousands of individuals from phylogenetically disparate sample sets.     

Selecting barcoding loci for plants: evaluation of seven candidate loci with species-level sampling in three divergent groups of land plants

There has been considerable debate, but little consensus regarding locus choice for DNA barcoding land plants. This is partly attributable to a shortage of comparable data from all proposed candidate loci on a common set of samples. In this study, we evaluated the seven main candidate plastid regions (rpoC1, rpoB, rbcL, matK, trnH‐psbA, atpF‐atpH, psbK‐psbI) in three divergent groups of land plants [Inga (angiosperm); Araucaria (gymnosperm); Asterella s.l. (liverwort)]. Across these groups, no single locus showed high levels of universality and resolvability. Interspecific sharing of sequences from individual loci was common. However, when multiple loci were combined, fewer barcodes were shared among species. Evaluation of the performance of previously published suggestions of particular multilocus barcode combinations showed broadly equivalent performance. Minor improvements on these were obtained by various new three‐locus combinations involving rpoC1, rbcL, matK and trnH‐psbA, but no single combination clearly outperformed all others. In terms of absolute discriminatory power, promising results occurred in liverworts (e.g. c. 90% species discrimination based on rbcL alone). However, Inga (rapid radiation) and Araucaria (slow rates of substitution) represent challenging groups for DNA barcoding, and their corresponding levels of species discrimination reflect this (upper estimate of species discrimination = 69% in Inga and only 32% in Araucaria; mean = 60% averaging all three groups).     

Characterization of highly conserved microsatellite loci in Araucaria cunninghamii and related species

 Ten microsatellite loci are described in Araucaria cunninghamii, the first reported in the Araucariaceae. Eight were tested in sections Eutacta and Bunya, which diverged more than 200 MYA, and to the sister genus Agathis. Specific amplification products within the expected size range were obtained for six to eight loci in section Eutacta (depending on species), five loci in section Bunya and three loci in Agathis. Two of the loci (CRCAc1 and CRCAc2, both GA repeats) produced specific amplification products in all taxa, with orthology confirmed by sequence analysis. The repeats were perfect in all taxa. The flanking sequences were extremely conserved, with sequence divergence of 0% to 2.0% within Araucaria species and 2.9% to 7.5% between Araucaria and Agathis. These microsatellites represent some of the most conserved microsatellite loci reported in plants. This may be due to a low evolutionary rate in Araucariaceae genome or the loci may be closely associated with highly conserved, unreported genes. Received January 14, 2002; accepted June 14, 2002 Published online: February 4, 2003 Current address: The Centre for Identification and Diagnostics, School of Life Sciences, The University of Queensland, Brisbane 4072, Australia.     

Microsatellite DNA markers for the Stable Fly,Stomoxys calcitrans (Diptera: Muscidae)

Eight polymorphic microsatellite markers have been isolated from a microsatellite‐enriched DNA library from the Stable Fly, Stomoxys calcitrans. These loci exhibited four to 15 alleles per locus and an expected heterozygosity ranging from 0.19 to 0.84 in the three populations studied from La Réunion island (Indian Ocean). They should therefore be valuable for studying genetic diversity and population structure. Cross‐species amplification of these eight loci in the closely allied species Stomoxys niger niger was successful for four of these loci, one of which was monomorphic and one of which strongly departed from Hardy–Weinberg expectations.     

Species limits and population differentiation in New Zealand snipes (Scolopacidae: <Emphasis Type="Italic">Coenocorypha</Emphasis>)

At least four species of New Zealand snipes (Coenocorypha) became extinct following the introduction of predatory mammals, and another two species suffered massive range reductions. To investigate species limits and population differentiation in six of the seven remaining offshore populations, we assayed variation in nine microsatellite loci and 1,980 base pairs of four mitochondrial DNA (mtDNA) genes. Genetic diversity in all populations except the largest one on Adams Island in the Auckland Islands was very low in both genomes. Alleles were fixed at many microsatellite loci and for single mtDNA haplotypes, particularly in the populations in the Chathams, Snares, Antipodes and Campbell Islands. Strong population structure has developed, and Chathams and Snares Islands populations are effectively genetically isolated from one another and from the more southern island populations. Based on reciprocal monophyly of lineages and their morphological distinctiveness we recommend that three phylogenetic species should be recognized, C. pusilla in the Chatham Islands, C. huegeli in the Snares Islands and C. aucklandica in the southern islands. The populations of C. aucklandica in the Auckland Islands, Antipodes Island and Campbell Island may warrant recognition as subspecies, and all should be managed as separate conservation units.     

Fire history of Araucaria–Nothofagus forests in Villarrica National Park, Chile

Aim In this study we examine fire history (i.e. c. 500 yr bp to present) of Araucaria–Nothofagus forests in the Andes cordillera of Chile. This is the first fire history developed from tree rings for an Araucaria–Nothofagus forest landscape. Location The fire history was determined for the Quillelhue watershed on the north side of Lanin volcano in Villarrica National Park, Chile. The long‐lived Araucaria araucana was commonly associated with Nothofagus pumilio and N. antarctica in more mesic and drier sites respectively. Methods Based on a combination of fire‐scar proxy records and forest stand ages, we reconstructed fire frequency, severity, and the spatial extent of burned areas for an c. 4000 ha study area. We used a composite fire chronology for the purpose of determining centennial‐scale changes in fire regimes and comparing the pre‐settlement (pre‐1883) and post‐settlement fire regimes. In addition, we contrasted Araucaria and Nothofagus species as fire‐scar recorders. Results In the study area, we dated a total of 144 fire‐scarred trees, representing 46 fire years from ad 1446 to the present. For the period from ad 1696 to 2000, using fire dates from Araucaria and Nothofagus species, the composite mean fire interval varied from 7 years for all fires to 62 years for widespread events (i.e. years in which ≥ 25% of recorder trees were scarred). Sensitivity to fire was different for Araucaria and Nothofagus species. More than 98% of the fires recorded by Nothofagus species occurred during the 1900s. The lack of evidence for older fire dates (pre‐1900) in Nothofagus species was due to their shorter longevity and greater susceptibility to being killed by more severe fires. Whereas the thin‐barked N. pumilio and N. antarctica are often destroyed in catastrophic fire events, large and thick‐barked Araucaria trees typically survive. The spatial extent of fires ranged from small patchy events to those that burned more than 40% of the entire landscape (c. > 1500 ha). Main conclusions Fire is the most important disturbance shaping the Araucaria–Nothofagus landscape in the Araucarian region. The forest landscape has been shaped by a mixed‐severity fire regime that includes surface and crown fires. High‐severity widespread events were relatively infrequent (e.g. 1827, 1909 and 1944) and primarily affected tall Araucaria–N. pumilio forests and woodlands dominated by Araucaria–N. antarctica. Although there is abundant evidence of the impact of Euro‐Chilean settlers on the area, the relative influence of this settlement on the temporal pattern of fire could only be tentatively established due to the relatively small number of pre‐1900 fire dates. An apparent increase in fire occurrence is evident in the fire record during Euro‐Chilean settlement (post‐1880s) compared with the Native American era, but it may also be the result of the destruction of evidence of older fires by more recent stand‐devastating fires (e.g. 1909 and 1944). Overall, the severe and widespread fires that burned in Araucaria–Nothofagus forests of this region in 2002, previously interpreted as an ecological novelty, are within the range of the historic fire regimes that have shaped this forested landscape.     

Isolation and characterization of polymorphic microsatellite DNA markers in the brown sole,Pleuronectes herzensteini

New microsatellite DNA markers from brown sole were developed and characterized. Fourteen primer sets were designed from 40 microsatellite regions. Eight of 14 loci exhibited variations comprising 8–31 alleles. Observed and expected heterozygosities ranged from 0.611 to 0.833 and from 0.647 to 0.968 among 36 individuals, respectively. Phz3, Phz8 and Phz12 significantly deviated from Hardy–Weinberg equilibrium, and there was a significant linkage disequilibrium between Phz2 and Phz12. Seven of eight loci conformed to the Mendelian manner of inheritance in a full‐sib family. Seven to four loci of three related species were cross‐amplified by primers for brown sole.     

Development of new microsatellite primers for green and white sturgeon

Sixty-eight primer sets for microsatellite loci were developed from microsatellite motif enriched genomic libraries of pooled DNA from the polyploid green and white sturgeon (Acipenser medirostris and A. transmontanus). Four individuals from each species were screened for polymorphism at these loci. Forty-eight loci amplified in both species, and some exhibited species-specific amplification for white or green sturgeon (8 and 12 loci, respectively). The number of alleles per locus ranged from one to 12. At least 68% of the green and 65% of the white sturgeon loci we developed are polysomic.     

Microsatellites for tree of heaven (Ailanthus altissima)

Nine (CT)_n microsatellites were developed for tree of heaven, Ailanthus altissima, from invasive populations on the Mediterranean islands. These loci had seven to 12 alleles in 96 trees from five islands. Two loci had significant deficits of heterozygotes within islands while the other loci were in Hardy–Weinberg equilibrium, and four pairs of loci had significant linkage disequilibrium within a single island. These loci were also polymorphic in one to three individuals of the tree of heaven varieties, Ailanthus altissima erythrocarpa, Ailanthus altissima sutchuenensis and Ailanthus altissima tanakai, and the related species Ailanthus giraldii and Ailanthus vilmariniana.     

Population genetic structure of Sorex unguiculatus and Sorex caecutiens (Soricidae, Mammalia) in Hokkaido, based on microsatellite DNA polymorphism

We investigated the genetic structure of Sorex unguiculatus and Sorex caecutiens populations in Hokkaido, Japan, using hypervariable microsatellite DNA markers. We used five microsatellite loci to type 475 S. unguiculatus individuals from 20 localities on the Hokkaido mainland and four localities from each of four offshore islands (and 11 shrews from one locality in southern Sakhalin for a particular analysis). We used six microsatellite loci to type 240 S. caecutiens individuals from 13 localities on the Hokkaido mainland. Genetic variation was high in mainland populations of both species and low in the island populations of S. unguiculatus. Allelic richness and island size were positively correlated for S. unguiculatus, suggesting that genetic drift occurred on those islands due to small population size. In addition, four insular populations of S. unguiculatus were genetically differentiated from the mainland populations, although clear phylogeographic clustering was not confirmed among populations on the Hokkaido mainland for either S. unguiculatus or S. caecutiens. Heterozygosity excess was observed in more than half of the populations including the mainland populations of the two species, suggesting recent bottleneck events in these populations. Population dynamics of the shrews might be explained by a metapopulation scheme. According to autocorrelation analysis, the extent of non-random spatial genetic structure was approximately 100 km. Isolation by distance was observed in S. unguiculatus, but not in S. caecutiens although there is a positive trend. The lack of correlation for S. caecutiens might have been due to small sample size. Thus, no obvious differences in population genetic structure were found between the two species on the Hokkaido mainland in the present study, while previous investigations using mitochondrial DNA sequences inferred that these two species might have rather different biogeographic histories.     

Microsatellite loci development in endangered pamphagid grasshoppers endemic to the Canary Islands (Orthoptera)

The five recognized endemic pamphagid species (Orthoptera) of the Canary Islands have restricted and fragmented ranges due to habitat decline. Seven polymorphic microsatellite markers have been developed for Acrostira tamarani, and the performance of primer pairs amplifying these loci in related taxa has been tested. The number of alleles in A. tamarani samples taken from two distant localities in the island of Gran Canaria ranged from two to eight per locus. Observed heterozygosities were from 0.151 to 0.559. Up to four primer pairs amplified in related species with moderate heterozigosities (maximum of 0.687 and 10 alleles for Ata67 locus in Purpuraria erna). These markers could be useful tools to study the population structure and management of endemic threatened pamphagids of the Canary archipelago.     

Characterization of microsatellite loci in a seed chalcid,Megastigmus spermotrophus (Hymenoptera: Torymidae)

Highly polymorphic microsatellite markers can supply demographic information on founder events and range expansion following initial introduction of invasive insect species. Six microsatellite loci were isolated from a partial DNA library in order to study the invasion patterns of a seed chalcid, Megastigmus spermotrophus, introduced to Europe and New Zealand. Allelic diversity at all described loci was high, ranging from 17 to 30 alleles per locus. All six loci were successfully amplified in 15 congeneric species.     

EVOLUTIONARY GENETICS OF TWO SIBLING SPECIES,DROSOPHILA SIMULANS AND D. SECHELLIA

Drosophila simulans and D. sechellia are sibling species, the former cosmopolitan and the latter restricted to the Seychelles Islands. We used classical genetic analysis to measure the numbers and effects of genes responsible for reproductive isolation and morphological differences in male genitalia between these species. At least five loci are responsible for male sterility in hybrids, with the strongest effects produced by at least two genes on the X chromosome. At least three (and probably four) loci are responsible for the interspecific difference in the size of the posterior process of the male genital arch. These genetic results, as well as the pattern of morphological divergence between the species, show several parallels with the divergence between D. simulans and its other island relative, D. mauritiana. We also present the DNA sequence of a 4.5 kilobase region containing the alcohol dehydrogenase (Adh) locus of D. sechellia, and combine this with previous data to reconstruct the phylogenies of the three species and their more distant relative D. melanogaster. Both D. mauritiana and D. sechellia are very closely related to D. simulans. Although most phylogenies show the two island species to be independent offshoots of the D. simulans lineage (with D. sechellia the more recent), the branch points are too close to make this conclusion unambiguous. The genetic and evolutionary parallels between the simulans/mauritiana and the simulans/sechellia divergences may therefore represent either a striking evolutionary convergence or a close common ancestry of the island species. A comparison of Adh alleles within species shows that the divergence among them may be almost as large as among alleles from different species. We conclude that many of the nucleotide differences among these species actually represent polymorphisms within common ancestors. It may be difficult to build accurate phylogenies using only a single DNA sequence from each species.     

Six microsatellite loci from the deep‐sea coral Corallium lauuense (Octocorallia: Coralliidae) from the islands and seamounts of the Hawaiian archipelago

Gorgonian octocorals are among the dominant deep‐sea benthic taxa of many seamounts. Seamount fauna are threatened by destructive fisheries practices, yet little is known about the physical and biological processes that maintain species on seamounts. Few informative molecular markers have been found in deep‐water corals or in gorgonian octocorals. Here we report the characterization of six highly polymorphic microsatellite loci for the deep‐sea precious coral Corallium lauuense from Hawaii using enriched genomic DNA libraries. These loci are being used to examine gene flow and stock structure among seamount and island populations to better understand dispersal and connectivity of seamount species.     

Invasion of two exotic terrestrial flatworms to subantarctic Macquarie Island

Invasive species are a serious threat to biodiversity worldwide. The relatively simple ecological systems of the subantarctic have the potential to be significantly damaged by predatory species that invade. Two species of exotic, predatory, terrestrial flatworms were first collected in 1997 from two localities only 2 km apart, in the southeast of subantarctic Macquarie Island. The species were later identified as Kontikia andersoni and Arthurdendyus vegrandis. We report here the results of fieldwork in 2004 that established that both species now occupy about a seventh of the southeast of the island which has a total area of only 170 km² and that there seem to be no barriers to further expansion. The island was first discovered in 1810 and so it is likely the species were introduced by means of human intervention within the last 200 years. We provide evidence to show that both species originated in New Zealand and have probably been on the island for ∼100 years giving an average rate of spread of about 10 m per year. Other species of Arthurdendyus have been introduced from New Zealand to the United Kingdom where they prey on earthworms. The quarantine significance of A. vegrandis for Australia is discussed and recommendations made to reduce the probability of it entering Tasmania where it has the potential to become an agricultural pest.     

Characterization of polymorphic microsatellite loci for the Tawny Pipit Anthus campestris and their utility in other steppe birds

New microsatellite loci for Tawny Pipit Anthus campestris were isolated from a genomic library. We were able to unambiguously score six loci: two were dinucleotide, one trinucleotide, two tetranucleotide and one pentanucleotide that turned out to be sex-linked. Four out of six loci were polymorphic with 7–23 alleles in our population and an observed heterozygosity ranging between 0.286 and 0.936. Cross-utility of these markers was tested in other 17 steppe-bird species of six families. In addition, 16 microsatellite loci developed for other species were tested for cross-species amplification in A. campestris. Eight microsatellite markers were successfully amplified; seven of them were polymorphic with 2–43 alleles and an observed heterozygosity of 0.040–0.863. Overall, 14 functional locus markers have been characterized for A. campestris that could be useful for future studies of paternity, genetic variability and population structure.     

Taxonomy of theAlouatta seniculus group: Biochemical and chromosome data

Wild populations ofAlouatta belzebul andA. seniculus from Brazil were surveyed in relation to 20 protein loci. Estimates of genetic variability demonstrated thatA. seniculus presents the highest level of heterozygosity among several New World monkey species studied for the same loci. Additional information from DNA and chromosomes suggest thatA. seniculus andA. belzebul are not the closest species in theHershkovitz’sAlouatta seniculus group.