5S-IGS rDNA in wind-pollinated trees (Fagus L.) encapsulates 55 million years of reticulate evolution and hybrid origins of modern species.

Standard models of plant speciation assume strictly dichotomous genealogies in which a species, the ancestor, is replaced by two offspring species. The reality in wind-pollinated trees with long evolutionary histories is more complex: species evolve from other species through isolation when genetic drift exceeds gene flow; lineage mixing can give rise to new species (hybrid taxa such as nothospecies and allopolyploids). The multi-copy, potentially multi-locus 5S rDNA is one of few gene regions conserving signal from dichotomous and reticulate evolutionary processes down to the level of intra-genomic recombination. Therefore, it can provide unique insights into the dynamic speciation processes of lineages that diversified tens of millions of years ago. Here, we provide the first high-throughput sequencing (HTS) of the 5S intergenic spacers (5S-IGS) for a lineage of wind-pollinated subtropical to temperate trees, the Fagus crenata - F. sylvatica s.l. lineage, and its distant relative F. japonica. The observed 4963 unique 5S-IGS variants reflect a complex history of hybrid origins, lineage sorting, mixing via secondary gene flow, and intra-genomic competition between two or more paralogous-homoeologous 5S rDNA lineages. We show that modern species are genetic mosaics and represent a striking case of ongoing reticulate evolution during the past 55 million years.

Phylogenetic origin of two Japanese Torreya taxa found in two regions with strongly contrasting snow depth.

The Japanese archipelago exhibits a notable difference in snow depth in winter, deep snow on the Sea of Japan side and low snow cover on the Pacific Ocean side. This contrasting pattern has shaped the distribution of infraspecific taxon pairs in a range of woody plants, with taxa found on the Sea of Japan side typically exhibiting a stunted shrub form with multiple decumbent stems. The phylogenetic origin of these taxon pairs is unknown, i.e., whether the two taxa diverged from the same species or if they have different origins. This study aimed to reveal the phylogenetic origin of two varieties of Torreya nucifera (Taxaceae); var. nucifera is a tree found on the Pacific Ocean side, whereas var. radicans is a shrub found on the Sea of Japan side. We examined the phylogenetic relationships of the two varieties and worldwide Torreya taxa using whole chloroplast genomes, chloroplast DNA fragments, and the nuclear ribosomal internal transcribed spacer (ITS). The whole chloroplast genome phylogeny indicated that T. nucifera var. radicans was a sister taxon to Chinese T. grandis, rather than to var. nucifera. In contrast, the nuclear ITS phylogeny indicated that while several haplotypes of T. nucifera var. radicans were closely related to T. grandis, most haplotypes of T. nucifera var. radicans formed a single clade with those of var. nucifera. This implies that the homogenization of the ITS has occurred between the two taxa, while taxon-specific chloroplast DNA haplotypes were retained. These discordant phylogenies suggested that the two taxa have different phylogenetic origins, but have an intricate evolutionary history, involving inter-taxa hybridization and gene flow, possibly when their distributions were confined to sympatric refugia. Given the genetic evidence and distinct difference in growth form, we propose that T. nucifera var. radicans should be taxonomically treated as a distinct species, T. fruticosa.

Transient hybridization, not homoploid hybrid speciation, between ancient and deeply divergent conifers.

PREMISE OF THE STUDY: Homoploid hybrid speciation is receiving growing attention due the increasing recognition of its role in speciation. We investigate if individuals intermediate in morphology between the two species of the conifer genus Athrotaxis represent a homoploid hybrid species, A. laxifolia, or are spontaneous F1 hybrids. METHODS: A total of 1055 individuals of Athrotaxis cupressoides and A. selaginoides, morphologically intermediate individuals, and two putative hybrid swarms were sampled across the range of the genus and genotyped with 13 microsatellites. We used simulations to test the power of our data to identify the pure species, F1s, F2s, and backcross generations. KEY RESULTS: We found that Athrotaxis cupressoides and A. selaginoides are likely the most divergent congeneric conifers known, but the intermediates are F1 hybrids, sharing one allele each from A. cupressoides and A. selaginoides at six loci with completely species specific alleles. The hybrid swarms contain wide genetic variation with stronger affinities to the locally dominant species, A. selaginoides and A. selaginoides backcrosses outnumbering A. cupressoides backcrosses. In addition, we observed evidence for isolated advanced generation backcrosses within the range of the pure species. CONCLUSIONS: We conclude that, even though they can be large and long-lived, Athrotaxis hybrid swarms are on a trajectory of decline and will eventually be reabsorbed by the parental species. However, this process may take millennia and fossil evidence suggests that such events have occurred repeatedly since the early Quaternary. Given this timeline, our study highlights the many obstacles to homoploid hybrid speciation.

Outcrossing rates and organelle inheritance estimated from two natural populations of the Japanese endemic conifer Sciadopitys verticillata.

The Japanese endemic conifer Sciadopitys verticillata is one of the most phylogenetically isolated species of all plants. Occurring in small and scattered populations, the species is currently classified as Near Threatened by the International Union for Conservation of Nature and Natural Resources (IUCN) and as Vulnerable in three prefectures of Japan. This study investigated two major factors that should impact the genetic structure of the species at both the nuclear and organelle DNA level, the mating system and the inheritance of both the chloroplast and mitochondrial genomes. The mating system is crucial to determining the degree of outcrossing of plant species and thus should have a key role in shaping the species' population level genetic diversity and gene flow between populations but as yet has not been studied in S. verticillata. Nine mother trees and their seedling progeny from two natural populations were genotyped using genetic markers from three plant genomes (eight nuclear microsatellites and DNA sequence for the chloroplast and mitochondria). Using a maximum likelihood method implemented in the software MLTR, the study found an outcrossing rate in the seedling stage of 0.49 and 0.79 for Aburazaka and Mount Shirotori populations, respectively, and an average of 0.66 at the species level. These outcrossing rates were low for conifers and therefore may have potential deleterious implications for the conservation of the species. The test of organelle inheritance supported paternal transmission of both the chloroplast and mitochondria consistent with previous microscopic evidence.

Climate oscillation during the Quaternary associated with landscape heterogeneity promoted allopatric lineage divergence of a temperate tree Kalopanax septemlobus (Araliaceae) in East Asia.

We investigated the biogeographic history of Kalopanax septemlobus, one of the most widespread temperate tree species in East Asia, using a combined phylogeographic and palaeodistribution modelling approach. Range-wide genetic differentiation at nuclear microsatellites (G'(ST) = 0.709; 2205 samples genotyped at five loci) and chloroplast DNA (G(ST) = 0.697; 576 samples sequenced for 2055 bp at three fragments) was high. A major phylogeographic break in Central China corresponded with those of other temperate species and the spatial delineation of the two temperate forest subkingdoms of East Asia, consistent with the forests having been isolated within both East and West China for multiple glacial-interglacial cycles. Evidence for multiple glacial refugia was found in most of its current range in China, South Japan and the southernmost part of the Korean Peninsula. In contrast, lineage admixture and absence of private alleles and haplotypes in Hokkaido and the northern Korean Peninsula support a postglacial origin of northernmost populations. Although palaeodistribution modelling predicted suitable climate across a land-bridge extending from South Japan to East China during the Last Glacial Maximum, the genetic differentiation of regional populations indicated a limited role of the exposed sea floor as a dispersal corridor at that time. Overall, this study provides evidence that differential impacts of Quaternary climate oscillation associated with landscape heterogeneity have shaped the genetic structure of a wide-ranging temperate tree in East Asia.

Low but structured chloroplast diversity in Atherosperma moschatum (Atherospermataceae) suggests bottlenecks in response to the Pleistocene glacials.

BACKGROUND AND AIMS: The cool temperate rainforests of Australia were much reduced in range during the cold and dry glacial periods, although genetic evidence indicates that two key rainforest species, Nothofagus cunninghamii and Tasmannia lanceolata, survived within multiple locations and underwent only local range expansions at the end of the Last Glacial. To better understand the glacial response of a co-occurring but wind-dispersed and less cold-tolerant rainforest tree species, Atherosperma moschatum, a chloroplast phylogeographic study was undertaken. METHODS: A total of 3294 bp of chloroplast DNA sequence was obtained for 155 samples collected from across the species' range. KEY RESULTS: The distribution of six haplotypes observed in A. moschatum was geographically structured with an inferred ancestral haplotype restricted to Tasmania, while three non-overlapping and endemic haplotypes were found on the mainland of south-eastern Australia. Last glacial refugia for A. moschatum are likely to have occurred in at least one location in western Tasmania and in Victoria and within at least two locations in the Great Dividing Range of New South Wales. Nucleotide diversity of A. moschatum was lower (π = 0·00021) than either N. cunninghamii (0·00101) or T. lanceolata (0·00073), and was amongst the lowest recorded for any tree species. CONCLUSIONS: This study provides evidence for past bottlenecks having impacted the chloroplast diversity of A. moschatum as a result of the species narrower climatic niche during glacials. This hypothesis is supported by the star-like haplotype network and similar estimated rates of chloroplast DNA substitution for A. moschatum and the two more cold tolerant and co-occurring species that have higher chloroplast diversity, N. cunninghamii and T. lanceolata.

The major Australian cool temperate rainforest tree Nothofagus cunninghamii withstood Pleistocene glacial aridity within multiple regions: evidence from the chloroplast.

Glacial aridity of the Pleistocene was inhospitable for the cool temperate rainforest tree Nothofagus cunninghamii over most of its current range in southeastern Australia, particularly in eastern Tasmania. A chloroplast DNA phylogeographic study was undertaken to investigate whether this species was likely to have survived in situ or conforms to a dispersal model of postglacial recovery. Twenty-three chloroplast haplotypes were identified by PCR-RFLP and direct sequencing of 2164 base pairs from 213 N. cunninghamii individuals collected in a range-wide survey. Fine-scale haplotype distribution was investigated using PCR-RFLP in eastern Tasmania. Deep chloroplast divergence occurred in N. cunninghamii. The single haplotype of the sister species, N. moorei, was nested among N. cunninghamii haplotypes. The distribution of N. cunninghamii haplotypes supports: multiple glacial refugia in coastal and inland western Tasmania, the centre of haplotype diversity; glacial survival in the central highlands of Victoria, corroborating pollen data; and the long-term occupation of eastern Tasmania because of the presence of a unique deeply diverged chloroplast lineage. Nothofagus cunninghamii withstood glacial aridity within multiple regions in apparently nonequable climates. This finding contributes to a growing understanding of how the resilience of temperate species during glacial periods has shaped modern biota.

Development of microsatellite markers for the Japanese endemic conifer Thuja standishii and transfer to other East Asian species.

OBJECTIVE: Design polymorphic microsatellite loci that will be useful for studies of the genetic diversity, gene-flow and reproduction in the Japanese endemic conifer Thuja standishii and test the transferability of these loci to the two other East Asian species, T. sutchuenensis and T. koraiensis. RESULTS: Fifteen loci were developed which displayed 3 to 21 alleles per locus (average = 9.2) among 97 samples from three populations of T. standishii. Observed heterozygosity for all samples varied between 0.33 and 0.75 (average = 0.54) while expected heterozygosity values were higher with an average over the 15 loci of 0.62 (0.37-0.91). Low multi-locus probability of identity values (< 0.00002) indicate that these markers will be effective for identifying individuals derived from clonal reproduction. All 15 loci amplified in 13 samples of T. sutchuenensis, the sister species of T. standishii, with 1 to 11 alleles per locus (average = 4.33) while 13 loci amplified in four samples of the more distantly related T. koraiensis with 1 to 5 alleles per locus (average = 2.15).

The complete chloroplast genome of Fagus crenata (subgenus Fagus) and comparison with F. engleriana (subgenus Engleriana).

This study reports the whole chloroplast genome of Fagus crenata (subgenus Fagus), a foundation tree species of Japanese temperate forests. The genome has a total of 158,227 bp containing 111 genes, including 76 protein-coding genes, 31 tRNA genes and 4 ribosomal RNA genes. Comparison with the only other published Fagus chloroplast genome, F. engeleriana (subgenus Engleriana) shows that the genomes are relatively conserved with no inversions or rearrangements observed while the proportion of nucleotide sites differing between the two species was equal to 0.0018. The six most variable regions were, in increasing order of variability, psbK-psbI, trnG-psbfM, rpl32, trnV, ndhI-ndh and ndhD-psaC. These highly variable chloroplast regions in addition to 160 chloroplast microsatellites identified (of which 46 were variable between the two species) will provide useful genetic resources for studies of the inter- and intra-specific genetic structure and diversity of this important northern hemisphere tree genus.

Development of chloroplast microsatellite markers for Glyptostrobus pensilis (Cupressaceae).

PREMISE: Glyptostrobus pensilis (Cupressaceae) is a critically endangered conifer native to China, Laos, and Vietnam, with only a few populations remaining in the wild. METHODS AND RESULTS: Using a complete chloroplast genome sequence, we designed 70 cpSSR loci and tested them for amplification success and polymorphism in 16 samples. Ten loci were found to be polymorphic and their genetic diversity was characterized using a total of 83 individuals from three populations in China. A total of 43 haplotypes were present, the effective number of haplotypes varied from 4.55 to 13.36, and the haplotypic richness ranged from 8.04 to 16.00. Gene diversity ranged from 0.81 to 0.97 (average 0.89). The number of alleles per locus and population ranged from one to eight, and the effective number of alleles ranged from 1.00 to 3.90. All polymorphic loci were successfully amplified in the related species Cryptomeria japonica var. sinensis, Taxodium distichum, T. ascendens, and Cunninghamia lanceolata. CONCLUSIONS: These newly developed chloroplast microsatellites will be useful for population genetic and phylogeographic analyses of G. pensilis and related species.

Development of 15 nuclear EST microsatellite markers for the paleoendemic conifer Pherosphaera hookeriana (Podocarpaceae).

PREMISE OF THE STUDY: Nuclear microsatellite markers were developed for population genetic analysis of the threatened paleoendemic conifer Pherosphaera hookeriana (Podocarpaceae). METHODS AND RESULTS: Fifteen variable loci were identified showing one to 13 alleles per population, with seven loci displaying at least four alleles in all populations, and the average number of alleles per locus ranging from 4.80 to 5.93 per population. Levels of observed heterozygosity per locus varied from 0.00 to 0.91, while average heterozygosity across all loci varied from 0.54 to 0.63 between populations. All loci also amplified in the endangered congener P. fitzgeraldii, but only five of the loci had more than one allele. CONCLUSIONS: These 15 loci are the first microsatellite markers developed in the genus Pherosphaera. These loci will be useful for investigating the species' extant genetic diversity and structure, the impact of past environmental change, and the significance of asexual reproduction.

The Complete Chloroplast Genome of Chinese Bayberry (Morella rubra, Myricaceae): Implications for Understanding the Evolution of Fagales.

Morella rubra (Myricaceae), also known as Chinese bayberry, is an economically important, subtropical, evergreen fruit tree. The phylogenetic placement of Myricaceae within Fagales and the origin of Chinese bayberry's domestication are still unresolved. In this study, we report the chloroplast (cp) genome of M. rubra and take advantage of several previously reported chloroplast genomes from related taxa to examine patterns of evolution in Fagales. The cp genomes of three M. rubra individuals were 159,478, 159,568, and 159.586 bp in length, respectively, comprising a pair of inverted repeat (IR) regions (26,014-26,069 bp) separated by a large single-copy (LSC) region (88,683-88,809 bp) and a small single-copy (SSC) region (18,676-18,767 bp). Each cp genome encodes the same 111 unique genes, consisting of 77 different protein-coding genes, 30 transfer RNA genes and four ribosomal RNA genes, with 18 duplicated in the IRs. Comparative analysis of chloroplast genomes from four representative Fagales families revealed the loss of infA and the pseudogenization of ycf15 in all analyzed species, and rpl22 has been pseudogenized in M. rubra and Castanea mollissima, but not in Juglans regia or Ostrya rehderiana. The genome size variations are detected mainly due to the length of intergenic spacers rather than gene loss, gene pseudogenization, IR expansion or contraction. The phylogenetic relationships yielded by the complete genome sequences strongly support the placement of Myricaceae as sister to Juglandaceae. Furthermore, seven cpDNA markers (trnH-psbA, psbA-trnK, rps2-rpoC2, ycf4-cemA, petD-rpoA, ndhE-ndhG, and ndhA intron) with relatively high levels of variation and variable cpSSR loci were identified within M. rubra, which will be useful in future research characterizing the population genetics of M. rubra and investigating the origin of domesticated Chinese bayberry.

Gondwanan conifer clones imperilled by bushfire.

Global increases in fire frequency driven by anthropogenic greenhouse emissions and land use change could threaten unique and ancient species by creeping into long-term fire refugia. The perhumid and mountainous western half of Tasmania is a globally important refugium for palaeo-endemic, fire intolerant lineages, especially conifers. Reproductive strategy will be crucial to the resilience of these organisms under warmer, dryer and more fire prone climates. This study analysed clonal versus sexual reproduction in old growth plots dominated by the palaeo-endemic conifer Athrotaxis cupressoides (Cupressaceae), a species that lacks any traits to tolerate frequent landscape fire. Across most of the seven plots the amount of sexually derived individuals was lower than clonally derived with, on average, 60% of all stems belonging to the same multi-locus lineage (MLL) (i.e. were clonal). Some MLLs were large spanning over 10 s of metres and consisted of up to 62 stems. The high mortality after fire and the rarity of sexual regeneration means that the range of this fire-intolerant species is likely to contract under enhanced fire regimes and has a limited capacity to disperse via seed to available fire refugia in the landscape.