The effects of co-colonising ectomycorrhizal fungi on mycorrhizal colonisation and sporocarp formation in Laccaria japonica colonising seedlings of Pinus densiflora.

Forest trees are colonised by different species of ectomycorrhizal (ECM) fungi that interact competitively or mutualistically with one another. Most ECM fungi can produce sporocarps. To date, the effects of co-colonising fungal species on sporocarp formation in ECM fungi remain unknown. In this study, we examined host plant growth, mycorrhizal colonisation, and sporocarp formation when roots of Pinus densiflora are colonised by Laccaria japonica and three other ECM fungal species (Cenococcum geophilum, Pisolithus sp., and Suillus luteus). Sporocarp numbers were recorded throughout the experimental period. The biomass, photosynthetic rate, and mycorrhizal colonisation rate of the seedlings were also measured at 45 days, 62 days, and 1 year after seedlings were transplanted. Results indicated that C. geophilum and S. luteus may negatively impact mycorrhizal colonisation and sporocarp formation in L. japonica. Sporocarp formation in L. japonica was positively correlated with conspecific mycorrhizal colonisation but negatively correlated with the biomass of seedlings of P. densiflora. The co-occurring ECM fungi largely competed with L. japonica, resulting in various effects on mycorrhizal colonisation and sporocarp formation in L. japonica. A variety of mechanisms may be involved in the competitive interactions among the different ECM fungal species, including abilities to more rapidly colonise root tips, acquire soil nutrients, or produce antibiotics. These mechanisms need to be confirmed in further studies.

Development and characterization of polymorphic microsatellite markers (SSRs) for an endemic plant, Pseudolarix amabilis (Nelson) Rehd. (Pinaceae).

Pseudolarix (Pinaceae) is a vulnerable (sensu IUCN) monotypic genus restricted to southeastern China. To better understand levels of genetic diversity, population structure and gene flow among populations of P. amabilis, we developed five compound SSR markers and ten novel polymorphic expressed sequence tags (EST) derived microsatellites. The results showed that all 15 loci were polymorphic with the number of alleles per locus ranging from two to seven. The expected and observed heterozygosities varied from 0.169 to 0.752, and 0.000 to 1.000, respectively. The inbreeding coefficient ranged from -0.833 to 1.000. These markers will contribute to research on genetic diversity and population genetic structure of P. amabilis, which in turn will contribute to the species conservation.

Physiological and Transcriptome Responses of Pinus massoniana Seedlings Inoculated by Various Ecotypes of the Ectomycorrhizal Fungus Cenococcum geophilum during the Early Stage of Drought Stress.

The impact of drought stress on plant growth in arid regions is a critical concern, necessitating the exploration of strategies to enhance plant drought resistance, particularly during the early stages of drought stress. This study focuses on the ectomycorrhizal fungus Cenococcum geophilum, renowned for its extensive genetic diversity and broad host compatibility, making it a crucial ally for host plants facing external stresses. We utilized Pinus massoniana seedlings inoculated with different ecotypic strains of C. geophilum under drought stress. The results showed that the inoculation of most strains of C. geophilum enhanced the drought resistance of P. massoniana seedlings under the early stages of drought stress, by influencing the water content, photosynthesis, accumulation of osmotic adjustment substances, and antioxidant enzyme activities in both shoots and roots of seedlings. Transcriptome analysis showed that mycorrhizal seedlings mainly regulated energy metabolism and reduction-oxidation reaction to resist early drought stress. Notably, the level of drought resistance observed in mycorrhizal seedlings was irrespective of the level of drought tolerance of C. geophilum strains. This study contributes essential data for understanding the drought response mechanisms of mycorrhizal P. massoniana seedlings inoculated by distinct C. geophilum ecotypes and guidance on selecting candidate species of ectomycorrhizal fungi for mycorrhizal afforestation in drought areas.

Analysis of the distribution pattern of the ectomycorrhizal fungus Cenococcum geophilum under climate change using the optimized MaxEnt model.

Cenococcum geophilum (C. geophilum) is a widely distributed ectomycorrhizal fungus that plays a crucial role in forest ecosystems worldwide. However, the specific ecological factors influencing its global distribution and how climate change will affect its range are still relatively unknown. In this study, we used the MaxEnt model optimized with the kuenm package to simulate changes in the distribution pattern of C. geophilum from the Last Glacial Maximum to the future based on 164 global distribution records and 17 environmental variables and investigated the key environmental factors influencing its distribution. We employed the optimal parameter combination of RM = 4 and FC = QPH, resulting in a highly accurate predictive model. Our study clearly shows that the mean temperature of the coldest quarter and annual precipitation are the key environmental factors influencing the suitable habitats of C. geophilum. Currently, appropriate habitats of C. geophilum are mainly distributed in eastern Asia, west-central Europe, the western seaboard and eastern regions of North America, and southeastern Australia, covering a total area of approximately 36,578,300 km2 globally. During the Last Glacial Maximum and the mid-Holocene, C. geophilum had a much smaller distribution area, being mainly concentrated in the Qinling-Huaihe Line region of China and eastern Peninsular Malaysia. As global warming continues, the future suitable habitat for C. geophilum is projected to shift northward, leading to an expected expansion of the suitable area from 9.21% to 21.02%. This study provides a theoretical foundation for global conservation efforts and biogeographic understanding of C. geophilum, offering new insights into its distribution patterns and evolutionary trends.

Ocean Currents Drove Genetic Structure of Seven Dominant Mangrove Species Along the Coastlines of Southern China.

Mangrove forest ecosystems, which provide important ecological services for marine environments and human activities, are being destroyed worldwide at an alarming rate. The objective of our study was to use molecular data and analytical techniques to separate the effects of historical and contemporary processes on the distribution of mangroves and patterns of population genetic differentiation. Seven mangrove species (Acanthus ilicifolius, Aegiceras corniculatum, Avicennia marina, Bruguiera gymnorrhiza, Kandelia obovata, Lumnitzera racemosa, and Rhizophora stylosa), which are predominant along the coastlines of South China, were genotyped at nuclear (nSSR) and chloroplast (cpSSR) microsatellite markers. We estimated historical and contemporary gene flow, the genetic diversity and population structure of seven mangrove species in China. All of these seven species exhibited few haplotypes, low levels of genetic diversity (H E = 0.160-0.361, with the exception of K. obovata) and high levels of inbreeding (F IS = 0.104-0.637), which may be due to their marginal geographical distribution, human-driven and natural stressors on habitat loss and fragmentation. The distribution patterns of haplotypes and population genetic structures of seven mangrove species in China suggest historical connectivity between populations over a large geographic area. In contrast, significant genetic differentiation [F ST = 0.165-0.629 (nSSR); G ST = 0.173-0.923 (cpSSR)] indicates that populations of mangroves are isolated from one another with low levels of contemporary gene flow among populations. Our results suggest that populations of mangroves were historically more widely inter-connected and have recently been isolated, likely through a combination of ocean currents and human activities. In addition, genetic admixture in Beibu Gulf populations and populations surrounding Hainan Island and southern mainland China were attributed to asymmetric gene flow along prevailing oceanic currents in China in historical times. Even ocean currents promote genetic exchanges among mangrove populations, which are still unable to offset the effects of natural and anthropogenic fragmentation. The recent isolation and lack of gene flow among populations of mangroves may affect their long-term survival along the coastlines of South China. Our study enhances the understanding of oceanic currents contributing to population connectivity, and the effects of anthropogenic and natural habitat fragmentation on mangroves, thereby informing future conservation efforts and seascape genetics toward mangroves.

Rapid Genetic Divergence of an Invasive Species, Spartina alterniflora, in China.

Hundreds of plants and half a kilogram of seeds of Spartina alterniflora, which were collected from Morehead City in North Carolina, Sapelo Island in Georgia, and Tampa Bay in Florida, were introduced to China in 1979. However, according to documented records, S. alterniflora from different origins were introduced to different areas when the species was first introduced to the coastal areas of China in the 1980s. In order to understand the relationship between the invasive S. alterniflora populations of China and the native S. alterniflora populations of the United States, and whether the genetic structure and genetic diversity of the invasive populations of China were affected by different introductions in the 1980s, molecular markers were used to determine the levels of gene flow and its effect on population differentiation. A total of 715 samples of S. alterniflora were collected from nine invasive populations in China and nine native populations from the United States. The genetic diversity and genetic structure of invasive and native populations were compared using microsatellite markers. The heterozygosity of Chinese invasive populations of S. alterniflora (HO = 0.538, HE = 0.725) were similar with those of native populations (HO = 0.530, HE = 0.744), which may attribute to its multiple introductions with the multisource populations from different geographic areas of the United States. However, the lower allelic diversities of Chinese invasive populations were detected, which may be due to the founder effect, or the bottleneck, which supports the theory that the allelic diversity is more sensitive to population bottlenecks than heterozygosity. The results of the STRUCTURE analysis among all sampling sites showed that the value of ΔK was largest when K = 2, which indicated that the invasive S. alterniflora populations in China had completed differentiated from the native populations of the United States. This may be because of admixture and hybridization of three non-overlapping original populations, or the postintroduction rapid evolution in China, and reproductive isolation under long-term geographic isolation. There was significant differentiation among invasive populations, which was mainly affected by different human-mediated introductions in 1980s. Significant genetic structure (K = 7) and high genetic differentiation (Fst = 0.30193) were detected in Chinese invasive populations, which may due to the low natural gene flow among populations. The genetic structure of the invasive populations in China was still affected by the human-mediated introductions in the 1980s, and the different initial introductions might promote differentiation among the invasive populations. In fact, the human-mediated long-distance dispersal should take the most of responsibility for the rapid spread of S. alterniflora along the coast of China. Multisource introductions of S. alterniflora are perhaps helpful for local adaptation but itself cannot cause rapid spread along the whole coast of China. Meanwhile, we suggest that the prevention of gene exchange among populations of S. alterniflora is the first and most important step in the control of the species on the coast of China, because admixture and hybridization of isolated populations might generate new heterosis and increase the difficulty of managing S. alterniflora in China.

Development and characterization of 20 novel EST-SSR markers for Pteroceltis tatarinowii, a relict tree in China.

PREMISE: Pteroceltis tatarinowii (Ulmaceae), the only species of the genus Pteroceltis, is an endangered tree in China. Here, novel expressed sequence tag-simple sequence repeat (EST-SSR) markers were developed to illuminate its genetic diversity for conservation and assisted breeding. METHODS AND RESULTS: Based on Illumina transcriptome data from P. tatarinowii, a total of 70 EST-SSR markers were initially designed and tested. Forty-eight of 70 loci (68.6%) were successfully amplified, of which 20 were polymorphic. The number of alleles per locus ranged from two to six, and the levels of observed and expected heterozygosity ranged from 0.018 to 0.781 and from 0.023 to 0.702, respectively. Additionally, cross-amplification was successful for 17 loci in two related species, Ulmus gaussenii and U. chenmoui. CONCLUSIONS: These new EST-SSR markers are valuable transcriptomic resources for P. tatarinowii and will facilitate population genetics and molecular breeding of this species and its relatives in Ulmaceae.

Mating system, pollen and propagule dispersal, and spatial genetic structure in a high-density population of the mangrove tree Kandelia candel.

Mangrove tree species form ecologically and economically important forests along the tropical and subtropical coastlines of the world. Although low intrapopulation genetic diversity and high interpopulation genetic differentiation have been detected in most mangrove tree species, no direct investigation of pollen and propagule dispersal through paternity and/or parentage analysis and spatial genetic structure within populations has been conducted. We surveyed the mating system, pollen and propagule dispersal, and spatial genetic structure in a natural population of Kandelia candel, one of the typical viviparous mangrove tree species, using nuclear and chloroplast microsatellite markers. High diversity and outcrossing rates were observed. Paternity and parentage analysis and modelling estimations revealed the presence of an extremely short-distance component of pollen and propagule dispersal (pollen: 15.2+/-14.9 m (SD) by paternity analysis and 34.4 m by modelling; propagule: 9.4+/-13.8 m (SD) by parentage analysis, and 18.6 m by modelling). Genetic structure was significant at short distances, and a clumped distribution of chloroplast microsatellite genotypes was seen in K. candel adults. We conclude that the K. candel population was initiated by limited propagule founders from outside by long-distance dispersal followed by limited propagule dispersal from the founders, resulting in a half-sib family structure.

Development and Application of Genomic Resources in an Endangered Palaeoendemic Tree, Parrotia subaequalis (Hamamelidaceae) From Eastern China.

Parrotia subaequalis is an endangered palaeoendemic tree from disjunct montane sites in eastern China. Due to the lack of effective genomic resources, the genetic diversity and population structure of this endangered species are not clearly understood. In this study, we conducted paired-end shotgun sequencing (2 × 125 bp) of genomic DNA for two individuals of P. subaequalis on the Illumina HiSeq platform. Based on the resulting sequences, we have successfully assembled the complete chloroplast genome of P. subaequalis, as well as identified the polymorphic chloroplast microsatellites (cpSSRs), nuclear microsatellites (nSSRs) and mutational hotspots of chloroplast. Ten polymorphic cpSSR loci and 12 polymorphic nSSR loci were used to genotype 96 individuals of P. subaequalis from six populations to estimate genetic diversity and population structure. Our results revealed that P. subaequalis exhibited abundant genetic diversity (e.g., cpSSRs: Hcp = 0.862; nSSRs: HT = 0.559) and high genetic differentiation (e.g., cpSSRs: RST = 0.652; nSSRs: RST = 0.331), and characterized by a low pollen-to-seed migration ratio (r ≈ 1.78). These genetic patterns are attributable to its long evolutionary histories and low levels of contemporary inter-population gene flow by pollen and seed. In addition, lack of isolation-by-distance pattern and strong population genetic structuring in both marker systems, suggests that long-term isolation and/or habitat fragmentation as well as genetic drift may have also contributed to the geographic differentiation of P. subaequalis. Therefore, long-term habitat protection is the most important methods to prevent further loss of genetic variation and a decrease in effective population size. Furthermore, both cpSSRs and nSSRs revealed that P. subaequalis populations consisted of three genetic clusters, which should be considered as separated conservation units.

Understanding population structure and historical demography of Litsea auriculata (Lauraceae), an endangered species in east China.

Detecting how historical and contemporary factors contribute to genetic divergence and genetic structure is a central question in ecology and evolution. We examine this question by intergrating population genetics with ecological niche modelling of Litsea auriculata (Lauraceae), which is endangered and native to east China. Geographical and environmental factors including climatic fluctuations since the last glacial maximum (LGM) have also contribute to population demography and patterns of genetic structure. L. auriculata populations underwent expansion after divergence and dramatically decreased to the current small size with relative population bottlenecks due to climate changes. Populations separated by physical geographical barrier including geographic distance and Yangtze River, as a result contemporary gene flow among L. auriculata populations showed drastic declines in comparison with historical gene flow, resulting in a high level of population divergence. Thus, patterns of genetic structure of L. auriculata can result from both geographic and environmental factors including climate changes. This information is helpful in forming conservation strategies for L. auriculata in China.

Microsatellite markers for the critically endangered elm species Ulmus gaussenii (Ulmaceae).

The Anhui elm Ulmus gaussenii is listed as a critically endangered species by the International Union for Conservation of Nature and is endemic to China, where its only population is restricted to Langya Mountain in Chuzhou, Anhui Province. To better understand the population genetics of U. gaussenii, we developed 12 microsatellite markers using an improved technique. The 12 markers were polymorphic, with the number of alleles per locus ranging from two to nine. Observed and expected heterozygosities ranged from 0.021 to 0.750 and 0.225 to 0.744, respectively. The inbreeding coefficient ranged from -0.157 to 0.960. Significant linkage disequilibrium was detected for two pairs of loci, and significant deviations from Hardy-Weinberg equilibrium were found in nine loci. These microsatellite markers will contribute to the studies of population genetics in U. gaussenii, which in turn will contribute to species conservation and protection.

Effect of Yangtze River on population genetic structure of the relict plant Parrotia subaequalis in eastern China.

Parrotia subaequalis (Hamamelidaceae) is a Tertiary relic species endemic in eastern China. We used inter-simple sequence repeat (ISSR) markers to access genetic diversity and population genetic structure in natural five populations of P. subaequalis. The levels of genetic diversity were higher at species level (H = 0.2031) but lower at population level (H = 0.1096). The higher genetic diversity at species levels might be attributed to the accumulation of distinctive genotypes which adapted to the different habitats after Quaternary glaciations. Meanwhile, founder effects on the early stage, and subsequent bottleneck of population regeneration due to its biological characteristics, environmental features, and human activities, seemed to explain the low population levels of genetic diversity. The hierarchical AMOVA revealed high levels (42.60%) of among-population genetic differentiation, which was in congruence with the high levels of Nei's genetic differentiation index (G ST = 0.4629) and limited gene flow (N m = 0.5801) among the studied populations. Mantel test showed a significant isolation-by-distance, indicating that geographic isolation has a significant effect on genetic structure in this species. Unweighted pair-group method with arithmetic average clustering, PCoA, and Bayesian analyses uniformly recovered groups that matched the geographical distribution of this species. In particular, our results suggest that Yangtze River has served as a natural barrier to gene flow between populations occurred on both riversides. Concerning the management of P. subaequalis, the high genetic differentiation among populations indicates that preserving all five natural populations in situ and collecting enough individuals from these populations for ex situ conservation are necessary.

An improved technique for isolating codominant compound microsatellite markers.

An approach for developing codominant polymorphic markers (compound microsatellite (SSR) markers), with substantial time and cost savings, is introduced in this paper. In this technique, fragments flanked by a compound SSR sequence at one end were amplified from the constructed DNA library using compound SSR primer (AC)6(AG)5 or (TC)6(AC)5 and an adaptor primer for the suppression-PCR. A locus-specific primer was designed from the sequence flanking the compound SSR. The primer pairs of the locus-specific and compound SSR primers were used as a compound SSR marker. Because only one locus-specific primer was needed for design of each marker and only a common compound SSR primer was needed as the fluorescence-labeled primer for analyzing all the compound SSR markers, this approach substantially reduced the cost of developing codominant markers and analyzing their polymorphism. We have demonstrated this technique for Dendropanax trifidus and easily developed 11 codominant markers with high polymorphism for D. trifidus. Use of the technique for successful isolation of codominant compound SSR markers for several other plant species is currently in progress.