Genome and Transcriptome Analysis of the Torreya grandis WRKY Gene Family during Seed Development.

Torreya grandis, an economically significant evergreen tree species exclusive to subtropical China, is highly valued for its seeds. However, the seed development process of T. grandis remains relatively unexplored. Given the pivotal role WRKY transcription factors (TFs) play in coordinating diverse cellular and biological activities, as well as crucial signaling pathways essential for plant growth and development, and the lack of comprehensive investigation into their specific functions in T. grandis, our study investigated its genome and successfully isolated 78 WRKY genes and categorized them into three distinct clades. A conserved motif analysis unveiled the presence of the characteristic WRKY domain in each identified TgWRKY protein. The examination of gene structures revealed variable numbers of introns (ranging from zero to eight) and exons (ranging from one to nine) among TgWRKY genes. A chromosomal distribution analysis demonstrated the presence of TgWRKY across eight chromosomes in T. grandis. Tissue-specific expression profiling unveiled distinctive patterns of these 78 TgWRKY genes across various tissues. Remarkably, a co-expression analysis integrating RNA-seq data and morphological assessments pinpointed the pronounced expression of TgWRKY25 during the developmental stages of T. grandis seeds. Moreover, a KEGG enrichment analysis, focusing on genes correlated with TgWRKY25 expression, suggested its potential involvement in processes such as protein processing in the endoplasmic reticulum, starch, and sucrose metabolism, thereby modulating seed development in T. grandis. These findings not only underscore the pivotal role of WRKY genes in T. grandis seed development but also pave the way for innovative breeding strategies.

TgLUT1 regulated by TgWRKY10 enhances the tolerance of Torreya grandis to drought stress.

Drought stress is a major abiotic stress which severely reduces the plant growth and limits agricultural productivity. Previous studies have demonstrated that lutein directly synthesized by the carotenoid epsilon-ring hydroxylase gene (LUT1) played crucial roles in regulating drought response. Notwithstanding the myriad studies on LUT1's response to drought stress in certain plant species such as Arabidopsis, the precise function mechanisms within tree species remain ambiguously understood. Our study reveals that under drought stress, TgLUT1, a novel LUT gene instrumental in ß-lutein biosynthesis, was markedly up-regulated in Torreya grandis. Subcellular localization assay indicated that TgLUT1 protein was localized to chloroplasts. Phenotypic analysis showed that overexpression of TgLUT1 enhanced the tolerance of tomato to drought stress. Overexpressing of TgLUT1 increased the values of maximal photochemical efficiency of photosystem II (Fv/Fm), net photosynthetic rate (Pn) and non-photochemical quenching (NPQ), and reduced the accumulation of hydrogen peroxide (H2O2), malondialdehyde (MDA) content and electrolyte leakage percentage in response to drought stress. Furthermore, overexpression of TgLUT1 decreased the stomatal conductance to reduce the water loss rate exposed to drought stress. In addition, yeast one-hybrid assay, dual luciferase assay system and qRT-PCR results showed that TgWRKY10 down-regulated by drought stress inhibited the expression of TgLUT1 by directly binding to the TgLUT1 promoter. Collectively, our results show that TgWRKY10, down-regulated by drought stress, negatively regulates the expression of TgLUT1 to modulate the drought stress response. This study contributes to a more comprehensive understanding of LUT1's function in the stress responses of economically significant forest plants.

Peripatric speciation within Torreya fargesii (Taxaceae) in the Hengduan Mountains inferred from multi-loci phylogeography.

BACKGROUND: The Hengduan Mountains (HDM) are one of the major global biodiversity hotspots in the world. Several evolutionary scenarios, especially in-situ diversification, have been proposed to account for the high species richness of temperate plants. However, peripatric speciation, an important mode of allopatric speciation, has seldom been reported in this region. RESULTS: Here, two chloroplast DNA regions and 14 nuclear loci were sequenced for 112 individuals from 10 populations of Torreya fargesii var. fargesii and 63 individuals from 6 populations of T. fargesii var. yunnanensis. Population genetic analyses revealed that the two varieties are well differentiated genetically (FST, 0.5765) and have uneven genetic diversity (π, 0.00221 vs. 0.00073 on an average of nuclear loci). The gene genealogical relationship showed that T. fargesii var. yunnanensis is inferred as derived from T. fargesii var. fargesii, which was further supported by the coalescent simulations (DIYABC, fastsimcoal2 and IMa2). By the coalescent simulations, the divergence time (~ 2.50-3.65 Ma) and the weak gene flow between the two varieties were detected. The gene flow was asymmetrical and only occurred in later stages of divergence, which is caused by second contact due to the population expansion (~ 0.61 Ma) in T. fargesii var. fargesii. In addition, niche modeling indicated that the two varieties are differentiated geographically and ecologically and have unbalanced distribution range. CONCLUSIONS: Overall, T. fargesii var. fargesii is always parapatric with respect to T. fargesii var. yunnanensis, and the latter derived from the former in peripatry of the HDM following a colonization from central China during the late Pliocene. Our findings demonstrate that peripatric speciation following dispersal events may be an important evolutionary scenario for the formation of biodiversity hotspot of the HDM.

Multi-omics analysis reveals the molecular responses of Torreya grandis shoots to nanoplastic pollutant.

Micro/nanoplastic has become an emerging pollutant of global concern. At present, ecotoxic researches on micro/nanoplastics mostly focus on marine aquatic organisms and freshwater algae. Research on the ecological impacts of plastics on higher terrestrial plants, especially on forest plants, is relatively limited. Torreya grandis cv. Merrillii, a species of conifer in the family Taxaceae, is a unique and economically valuable tree species in China. The physiological and biochemical responses of T. grandis seedlings to polystyrene nanoplastics (PSNPs) with a diameter of 100 nm were systematically studied inthe present study. The results showed that nanoplastics enhanced the accumulation of the thiobarbituric acid reactive substance and the activities of catalase and peroxidase. The concentrations of iron, sulfur, and zinc were reduced after nanoplastic exposure. PSNP treatment had an important effect on a series of chemical and genetic indicators of T. grandis, includingantioxidants, small RNA, gene transcription, protein expressions, and metabolite accumulation. Multi-omic analysis revealed that PSNPs modulate terpenoid- and flavonoid-biosynthesis pathways by regulating small RNA transcription and protein expression. Our study provided novelty insights into the responses of forest plants to nanoplastic treatment.

Characterization and Analysis of the Full-Length Transcriptomes of Multiple Organs in Pseudotaxus chienii (W.C.Cheng) W.C.Cheng.

Pseudotaxus chienii, a rare tertiary relict species with economic and ecological value, is a representative of the monotypic genus Pseudotaxus that is endemic to China. P. chienii can adapt well to habitat isolation and ecological heterogeneity under a variety of climate and soil conditions, and is able to survive in harsh environments. However, little is known about the molecular and genetic resources of this long-lived conifer. Herein, we sequenced the transcriptomes of four organs of P. chienii using the PacBio Isoform Sequencing and Illumina RNA Sequencing platforms. Based on the PacBio Iso-Seq data, we obtained 44,896, 58,082, 50,485, and 67,638 full-length unigenes from the root, stem, leaf, and strobilus, respectively, with a mean length of 2692 bp, and a mean N50 length of 3010.75 bp. We then comprehensively annotated these unigenes. The number of organ-specific expressed unigenes ranged from 4393 in leaf to 9124 in strobilus, suggesting their special roles in physiological processes, organ development, and adaptability in the different four organs. A total of 16,562 differentially expressed genes (DEGs) were identified among the four organs and clustered into six subclusters. The gene families related to biotic/abiotic factors, including the TPS, CYP450, and HSP families, were characterized. The expression levels of most DEGs in the phenylpropanoid biosynthesis pathway and plant-pathogen interactions were higher in the root than in the three other organs, suggesting that root constitutes the main organ of defensive compound synthesis and accumulation and has a stronger ability to respond to stress. The sequences were analyzed to predict transcription factors, long non-coding RNAs, and alternative splicing events. The expression levels of most DEGs of C2H2, C3H, bHLH, and bZIP families in the root and stem were higher than those in the leaf and strobilus, indicating that these TFs may play a crucial role in the survival of the root and stem. These results comprise the first comprehensive gene expression profiles obtained for different organs of P. chienii. Our findings will facilitate further studies on the functional genomics, adaptive evolution, and phylogeny of P. chienii, and lay the foundation for the development of conservation strategies for this endangered conifer.

Full-Length Transcriptome Analysis of the Genes Involved in Tocopherol Biosynthesis in Torreya grandis.

The seeds of Torreya grandis (Cephalotaxaceae) are rich in tocopherols, which are essential components of the human diet as a result of their function in scavenging reactive oxygen and free radicals. Different T. grandis cultivars (10 cultivars selected in this study were researched, and their information is shown in Table S1 of the Supporting Information) vary enormously in their tocopherol contents (0.28-11.98 mg/100 g). However, little is known about the molecular basis and regulatory mechanisms of tocopherol biosynthesis in T. grandis kernels. Here, we applied single-molecule real-time (SMRT) sequencing to T. grandis (X08 cultivar) for the first time and obtained a total of 97 211 full-length transcripts. We proposed the biosynthetic pathway of tocopherol and identified eight full-length transcripts encoding enzymes potentially involved in tocopherol biosynthesis in T. grandis. The results of the correlation analysis between the tocopherol content and gene expression level in the 10 selected cultivars and different kernel developmental stages of the X08 cultivar suggested that homogentisate phytyltransferase coding gene ( TgVTE2b) and γ-tocopherol methyltransferase coding gene ( TgVTE4) may be key players in tocopherol accumulation in the kernels of T. grandis. Subcellular localization assays showed that both TgVTE2b and TgVTE4 were localized to the chloroplast. We also identified candidate regulatory genes similar to WRI1 and DGAT1 in Arabidopsis that may be involved in the regulation of tocopherol biosynthesis. Our findings provide valuable genetic information for T. grandis using full-length transcriptomic analysis, elucidating the candidate genes and key regulatory genes involved in tocopherol biosynthesis. This information will be critical for further molecular-assisted screening and breeding of T. grandis genotypes with high tocopherol contents.

Diversity and evolution of leaf anatomical characters in Taxaceae s.l.-fluorescence microscopy reveals new delimitating characters.

Taxaceae s.l. comprise six genera (including Cephalotaxus) and about 35 species; The present study aims to give new insights into the evolution of this family, especially into the phylogenetic position of Cephalotaxus. Moreover, only little is known about comparative leaf anatomy of this family and this study aims to expose and interpret the diversity and evolution of leaf anatomical characters and to assess their applicability to identify taxa at the generic and species level. A detailed phylogeny was reconstructed, using both maximum likelihood and Bayesian inference, with a combined dataset of four molecular markers from the plastid and nuclear genomes. Leaf sections from 132 specimens, representing 32 species and four varieties (fresh and herbarium material) were inspected, using fluorescence microscopy. Ancestral characters were reconstructed using Mesquite. The phylogenetic analyses provided full support for Cephalotaxus as sister group to Taxaceae s.str. Within the latter, two monophyletic tribes Taxeae (comprising Austrotaxus, Pseudotaxus, and Taxus) and Torreyeae (comprising Amentotaxus and Torreya) were fully supported. Fluorescence microscopy was shown to be very useful for identifying leaf tissues and their constitution. We were able to show that particularly sclerified tissues have highest potential for the discrimination of both freshly collected samples and rehydrated herbarium specimens at the generic and species level. A correlation between the presence of different sclereid types could be shown and sclereids were hypothesized to pose a primitive trait in the evolution of Taxaceae s.l. New identification keys were generated on the basis of leaf anatomical characters. The microscopic method presented here is applicable for further studies within gymnosperms and probably in angiosperms, as well.

The complete chloroplast genome of Torreya fargesii (Taxaceae).

The complete chloroplast genome sequence of Torreya fargesii (Taxaceae), a relic plant endemic to China, is presented in this study. The genome is 137 075 bp in length, with 35.47% average GC content. One copy of the large inverted repeats is lost from this genome. The T. fargesii chloroplast genome encodes 118 unique genes, in which trnI-CAU, trnQ-UUG, trnN-GUU are duplicated. Protein-coding, tRNA and rRNA genes represent 54.7%, 1.9% and 3.4% of the genome, respectively. There are 17 intron-containing genes, of which 6 are tRNA genes. A maximum likelihood phylogenetic analysis revealed a strong sister relationship between Torreya and Amentotaxus.

Cross-Species, Amplifiable EST-SSR Markers for Amentotaxus Species Obtained by Next-Generation Sequencing.

Amentotaxus, a genus of Taxaceae, is an ancient lineage with six relic and endangered species. Four Amentotaxus species, namely A. argotaenia, A. formosana, A. yunnanensis, and A. poilanei, are considered a species complex because of their morphological similarities. Small populations of these species are allopatrically distributed in Asian forests. However, only a few codominant markers have been developed and applied to study population genetic structure of these endangered species. In this study, we developed and characterized polymorphic expressed sequence tag-simple sequence repeats (EST-SSRs) from the transcriptome of A. formosana. We identified 4955 putative EST-SSRs from 68,281 unigenes as potential molecular markers. Twenty-six EST-SSRs were selected for estimating polymorphism and transferability among Amentotaxus species, of which 23 EST-SSRs were polymorphic within Amentotaxus species. Among these, the number of alleles ranged from 1-4, the polymorphism information content ranged from 0.000-0.692, and the observed and expected heterozygosity were 0.000-1.000 and 0.080-0.740, respectively. Population genetic structure analyses confirmed that A. argotaenia and A. formosana were separate species and A. yunnanensis and A. poilanei were the same species. These novel EST-SSRs can facilitate further population genetic structure research of Amentotaxus species.

The complete chloroplast genome sequence of Amentotaxus argotaenia (Taxaceae).

The complete chloroplast genome sequence of Amentotaxus argotaenia was determined in this study. The genome is 136 657 bp in length and lacks one inverted repeat region. The overall GC content of the genome is 35.85% (protein-coding genes, 36.90%; tRNA genes, 53.31%; rRNA genes, 52.99%; introns, 36.10%; intergenic spacers, 31.03%). The A. argotaenia chloroplast genome contains 118 unique genes, including 83 protein-coding genes, 31 tRNA genes, and four rRNA genes. Ten protein-coding genes and six tRNA genes have one intron, while ycf3 contains two. The coding regions occupy 60.27% of the genome length and the gene density is estimated to be 0.88 genes/kb. A maximum likelihood phylogenetic analysis suggested that Amentotaxus is sister to Taxus within the Taxaceae family.

Ancient nuclear plastid DNA in the yew family (taxaceae).

Plastid-to-nucleus DNA transfer provides a rich genetic resource to the complexity of plant nuclear genome architecture. To date, the evolutionary route of nuclear plastid DNA (nupt) remain unknown in conifers. We have sequenced the complete plastomes of two yews, Amentotaxus formosana and Taxus mairei (Taxaceae of coniferales). Our comparative genomic analyses recovered an evolutionary scenario for plastomic reorganization from ancestral to extant plastomes in the three sampled Taxaceae genera, Amentotaxus, Cephalotaxus, and Taxus. Specific primers were designed to amplify nonsyntenic regions between ancestral and extant plastomes, and 12.6 kb of nupts were identified based on phylogenetic analyses. These nupts have significantly accumulated GC-to-AT mutations, reflecting a nuclear mutational environment shaped by spontaneous deamination of 5-methylcytosin. The ancestral initial codon of rps8 is retained in the T. nupts, but its corresponding extant codon is mutated and requires C-to-U RNA-editing. These findings suggest that nupts can help recover scenarios of the nucleotide mutation process. We show that the Taxaceae nupts we retrieved may have been retained because the Cretaceous and they carry information of both ancestral genomic organization and nucleotide composition, which offer clues for understanding the plastome evolution in conifers.

Genetic linkage map construction and QTL identification of juvenile growth traits in Torreya grandis.

Torreya grandis Fort. ex Lindl, a conifer species widely distributed in Southeastern China, is of high economic value by producing edible, nutrient seeds. However, knowledge about the genome structure and organization of this species is poorly understood, thereby limiting the effective use of its gene resources. Here, we report on a first genetic linkage map for Torreya grandis using 96 progeny randomly chosen from a half-sib family of a commercially cultivated variety of this species, Torreya grandis Fort. ex Lindl cv. Merrillii. The map contains 262 molecular markers, i.e., 75 random amplified polymorphic DNAs (RAPD), 119 inter-simple sequence repeats (ISSR) and 62 amplified fragments length polymorphisms (AFLP), and spans a total of 7,139.9 cM, separated by 10 linkage groups. The linkage map was used to map quantitative trait loci (QTLs) associated with juvenile growth traits by functional mapping. We identified four basal diameter-related QTLs on linkage groups 1, 5 and 9; four height-related QTLs on linkage groups 1, 2, 5 and 8. It was observed that the genetic effects of QTLs on growth traits vary with age, suggesting the dynamic behavior of growth QTLs. Part of the QTLs was found to display a pleiotropic effect on basal diameter growth and height growth.

Development and characterization of microsatellite markers for the endangered Chinese yew Taxus chinensis var. mairei (Taxaceae).

We isolated and characterized 11 microsatellite loci for the Chinese yew, Taxus chinensis var. mairei, an endangered tree species in China, by constructing a (CA)(12)-enriched library. The number of alleles per locus ranged from 5 to 10. The observed heterozygosities ranged from 0.2500 to 0.8333 and the expected heterozygosities ranged from 0.5196 to 0.8680. No significant linkage disequilibrium was detected at these loci. However, four loci significantly deviated from Hardy-Weinberg equilibrium. The null alleles were found to be present at locus Tach9 and locus Tach11 by the Micro-checker test (P < 0.001). These polymorphic loci could be employed in research of gene flow and spatial genetic patterns of T. chinensis var. mairei.

Isolation and characterization of 15 microsatellite loci in four endangered Amentotaxus species (Taxaceae).

PREMISE OF THE STUDY: Fifteen microsatellite loci were developed in an endangered species, Amentotaxus formosana, and were tested in an additional three species, A. argotaenia, A. yunnanensis, and A. poilanei, to evaluate the population structure for conservation efforts and reconstruct the phylogeographic patterns of this ancient lineage. METHODS AND RESULTS: Polymorphic primer sets were developed from A. formosana; the number of alleles ranged from two to 10, with an observed heterozygosity ranging from 0 to 0.60. All of the loci were found to be interspecifically amplifiable. CONCLUSIONS: These polymorphic and transferable loci will be potentially useful for future studies that will focus on identifying distinct genetic units within species and establishing the phylogeographic patterns and the process of speciation among closely related species.

Development and characterization of microsatellites in Torreya jackii (Taxaceae), an endangered species in China.

PREMISE OF THE STUDY: Polymorphic microsatellite loci were developed in Torreya jackii, an endangered species in China, to provide markers for further studies on the genetic diversity of this species. METHODS AND RESULTS: Eight polymorphic loci and one monomorphic locus were developed and characterized in four T. jackii populations (Xianju, Songyang, Pujiang, and Tonglu) from Zhejiang Province, China. The number of alleles per locus ranged from one to eight across 80 T. jackii individuals. At the eight polymorphic loci, the observed heterozygosity ranged from 0.150 to 1.000 and the expected heterozygosity ranged from 0.185 to 0.796. CONCLUSIONS: The microsatellite loci developed and characterized in this study will facilitate future analyses of the genetic diversity of T. jackii. Such information will aid in designing strategies to conserve this currently endangered species.

[Genetic diversity of natural and planted Glyptostrobus pensilis populations: a comparative study].

Glyptostrobus pensilis is a rare and endangered relict species in China. To make a comparative study on the genetic diversity and genetic structure of natural and planted G. pensilis populations would have significance in the conservation and proliferation of the species. Samples from the main distribution regions of G. pensilis were analyzed by ISSR (inter simple sequence repeats) molecular marker. A total of 95 discernible DNA fragments were detected with 10 ISSR primers, of which, polymorphic loci occupied 39.0%, suggesting that the genetic variation in the test G. pensilis populations was at a very low level, compared with other endangered gymnosperm. The genetic differential index (G(st) = 0.3982) and the gene flow (N(m) = 0.3778) indicated that there existed genetic differentiation among populations but the differentiation dominated within populations. There was a significant positive correlation between genetic distance and geographical distance. The mean values of polymorphic loci (P), Nei's gene index (H(e)), and Shannon information index (I) of natural populations (P = 39.9%, H(e) = 0.1499, I = 0.2202) were much higher than those of planted G. pensilis populations (P = 30.7%, H(e) = 0.1265, I = 0.1759), and the coefficient of gene differentiation (G(st)) and genetic distance (D) of natural populations (G(st) = 0.4513, D = 0.0301) were also much higher than those of the planted populations (G(st) = 0.3025, D = 0.0192).

Evolution of the chloroplast trnL-trnF region in the gymnosperm lineages Taxaceae and Cephalotaxaceae.

The trnL-trnF region is located in the large single-copy region of the chloroplast genome. It consists of the trnL gene, a group I intron, and the trnL-F intergenic spacer. We analyzed the evolution of the region in three gymnosperm families, Taxaceae, Cephalotaxaceae, and Podocarpaceae, with especially dense sampling in Taxaceae and Cephalotaxaceae, for which we sequenced 43 accessions, representing all species. The trnL intron has a conserved secondary structure and contains elements that are homologous across land plants, and the spacer is highly variable in length and composition. The spatial distribution of nucleotide diversity along the trnL-F region suggests that different portions of this region have different evolutionary patterns. Tandem repeats that form stem-loop structures were detected in both the trnL intron and the trnL-F spacer, and the spacer sequences contain promoter elements for the trnF gene. The presence of promoters and stem-loop structures in the trnL-F spacer and high sequence variation in this region suggest that trnL and trnF are independently transcribed. Stem-loop regions P6, P8, and P9 of the trnL intron and the trnL-F spacer (except the promoter elements) might undergo neutral evolution with respect to their escape from functional constraints.

[Genetic diversity of endangered plant Torreya jackii: a study with RAPD markers].

With random amplified polymorphic DNA (RAPD) techniques, this paper studied the genetic diversity and genetic differentiation of Torreya jackii, an endangered plant endemic to China. In the 180 individuals of 9 T. jackii natural populations, 180 repetitive loci were detected by using 12 random primers, among which, 119 were polymorphic. At species level, the percentage of polymorphic loci (P) was 66.11%, and the genetic diversity estimated by Shannon information index (I) and Nei's index (h) was 0.3087 and 0.2015, respectively, suggesting that the genetic diversity at species level was relatively high. However, at population level, the genetic diversity was relatively low (P = 23.76% , I = 0.1221, and h = 0.0813). The analysis of molecular variance (AMOVA) showed that 42.57% of genetic variance was found within populations, and 57.43% of it was resided among populations. The coefficient of gene differentiation (G(st)) was 0.5965, indicating the high genetic differentiation among the populations of T. jackii, and the gene flow among the populations was quite low, being 0.3382. The bottleneck effect, population isolation, and low gene flow among populations would have contributed to such a population genetic structure in T. jackii. The averaged genetic distance among 9 T. jackii populations was 0.1630. By using the unweighted pair group method with arithmetic mean (UPGMA), the 9 populations could be divided into two groups, i. e., Zhejiang group and Fujian group. It was proposed that in the ex situ conservation of T. jackii, the gcrmplasm transferring among populations should be avoided.

Low genetic diversity and significant population structuring in the relict Amentotaxus argotaenia complex (Taxaceae) based on ISSR fingerprinting.

Amentotaxus, a genus of the Taxaceae, represents an ancient lineage that has long existed in Eurasia. All Amentotaxus species experienced frequent population expansion and contraction over periodical glaciations in Tertiary and Quaternary. Among them, Amentotaxus argotaenia complex consists of three morphologically alike species, A. argotaenia, Amentotaxus yunnanensis, and Amentotaxus formosana. This complex is distributed in the subtropical region of mainland China and Taiwan where many Pleistocene refugia have been documented. In this study, genetic diversity and population structuring within and between species were investigated based on the inter-simple sequence repeats (ISSR) fingerprinting. Mean genetic diversity within populations was estimated in three ways: (1) the percentage of polymorphic loci out of all loci (P) (2) Nei's unbiased expected heterozygosity (He), and (3) Shannon's index of phenotypic diversity. For a total of 310 individuals of 15 populations sampled from the three species, low levels of ISSR genetic variation within populations were detected, with P=4.66-16.58%, He=0.0176-0.0645 and Hpop=0.0263-0.0939, agreeing with their seriously threatened status. AMOVA analyses revealed that the differences between species only accounted for 27.38% of the total variation, whereas differences among populations and within populations were 57.70 and 14.92%, respectively, indicating substantial isolation between the patch-like populations. A neighbor-joining tree identified a close affinity between A. yunnanensis and A. formosana. Genetic drift due to small population size, plus limited current gene flow, resulted in significant genetic structuring. Low levels of intra-populational genetic variation and considerable inter-populational divergence were also attributable to demographic bottlenecks during and/or after the Pleistocene glaciations.