| Emmenopterys henryi Oliv. (Rubiaceae), which is the only extant species of its genus, is native to subtropical China, where it occurs in disjunct montane valleys of mainly warm-temperate deciduous (WTD) forests, at elevations ranging from c.400-1600 (2000) m above sea level. Landscape characteristics, climatic conditions, and soil types vary between regions within the distribution range of E. henryi. Well-preserved infructescences of now-extinct Emmenopterys species are known from the Eocene of North America and Germany, but there are no reliable fossils of E. henryi. There is dated molecular evidence to suggest that the origin of this species dates back to the early Miocene. However, there is unknown how historical and contemporary geographic and environmental factors contribute to genetic divergence at different evolutionary scales. Here, we examine this key question by investigating how environmental and geographic factors across different epochs have driven genetic divergence at deeper (phylogeographic) and shallower (landscape genetic) evolutionary scales in E. henryi. We integrate genetic markers that capture signatures from historical (chloroplast DNA) and contemporary (amplified fragment length polymorphisms; AFLPs) divergence, ENM, and spatial genetic modelling approaches to disentangle the relative roles of geography, climate, and ecology in shaping the population genetic structure of E. henryi across subtropical China. The major results are shown as below:(1) Population genetic diversity and structure based on cpDNA and ITSBased on the cpDNA set, the Bayesian haplotype tree inferred from BEAST supported two main clades (Northern and Southern lineages). Notably, the distribution pattern of the phylogeographic break between Northern and Southern lineages overlapped with the Yangtze River. At the species level, the cpDNA and ITS data revealed high estimates of haplotype and nucleotide diversity (cpDNA:hT= 0.928, πT= 0.271×10-2; ITS:hT= 0.675; πT= 0.195×10-2) as well as pronounced population genetic structure (cpDNA:FST= 0.779; ITS:FST= 0.558). By contrast, estimates of within population haplotype (hs) and nucleotide (πs) diversity was low (hs = 0.291, πs= 0.054×10-2; ITS:hs= 0.347,;πs= 0.083×10-2). The population genetic diversity of the Southern lineage (cpDNA:hT= 0.889, πT= 0.215×10-2; ITS:hT= 0.687,πT= 0.220×10-2) was higher than that of the Northern lineage (cpDNA:hT= 0.818, πT= 0.196×102; ITS:hT= 0.561, πT= 0.085×10-2). Meanwhile, the population genetic divergence of the Southern lineage (cpDNA: FST= 0.823; ITS:FST= 0.558) was obviously larger than that of the Northern lineage (cpDNA: FST= 0.551; ITS:FST= 0.184). SAMOVA analysis of cpDNA data showed that Northern and Southern lineages could be further divided into Northeast group vs. Northwest group and Southeast group vs. Central-Southwest group, respectively. Significant phylogeographic structure for cpDNA was observed at the range-wide scale (GST/NST= 0.704/0.802, P< 0.01) and in the Southern lineage (GST/NST= 0.757/0.868, P< 0.05). There was significant effect of Isolation-by-Distance throughout the range (r= 0.237, P= 0.001), and the same applied to Southern (r= 0.293, P= 0.001) and Northern (r= 0.506, P= 0.001) lineages.(2) Molecular dating and historical demography based on cpDNA data setBased on cpDNA haplotype data set, by using Bayesian phylogenetic analysis with a relaxed molecular clock in BEAST software, the divergence time between the Northern and Southern lineages was estimated as c.5.06 Ma (95% HPD:1.68-8.91 Ma). The onset of lineage diversification was estimated as c.3.42 Ma in the Northern lineage (95% HPD:0.99-6.40 Ma) and c.3.64 Ma in the Southern lineage (95% HPD:1.18-6.42 Ma). Refugial isolation associated with climate oscillation during the Miocene/Pliocene likely promoted allopatric lineage divergence of E. henryi. The Southeast, Northeast and Northwest groups identified in the SAMOVA analysis were found to be in accordance with the expected distributions under a spatial expansion model. All these spatial expansion events were dated back to the penultimate (Riss) glacial (c.0.12-0.35 Ma):Southeast:c.0.23 Ma,95% CI:0.000-0.811 Ma; Northwest:c.0.19 Ma,95% CI:0.111-0.314 Ma; Northeast:c.0.26 Ma,95% CI:0.133-0.376 Ma).(3) Ecological niche modelling and niche identity analysisBased on a total of 114 occurance records, the potential distribution of E. henryi across its range under present (1950-2000), past [the Last Glacial Maximum (LGM, c.21 kya), the Last Interglacial (LIG, c.130-114 kya)] and future (2080) climatic conditions were reconstructed in Maxent v.3.3.3 using 6 bioclimatic data layers. Results indicated that the predicted current distributional were similar to its actual distribution, except for southeastern Qinghai-Tibetan Plateau and Taiwan. Palaeodistribution modeling suggested the ranges of the species was narrower during the LIG compared with its current distribution, particularly in north-central China (e.g. northern Sichuan Basin and Daba/Qinling Mts.), with subsequent expansion during the LGM to cover a slightly greater area than that predicted under current climatic conditions. However, climate change will result in a reduction of the potential range in the future (2080). Most evident is a loss of suitable habitat in areas south of the Yangtze Delta, where only small and disjunct mountain areas are predicted suitable for E. henryi. Moreover, randomization tests of niche identity (based on nineteen bioclimatic variables or six non-correlated variables) indicated that these Southern and Northern lineages are not ecologically equivalent, regardless of the measure of similarity used (Schoener’s D or Hellinger’s I). And SEEVA furtherly showed that the significant phylogenetic splits between Southern and Northern lineages in terms of the bioclimatic variables of precipitation.(4) The population genetic structure and diversity based on AFLPsThe nine primer combinations employed with samples from 37 populations (394 individuals) of E. henryi generated a total of 457 fragments, of which 431 (94.31%) were polymorphic. At the species level, the AFLPs data revealed high estimates of genetic diversity (HE= 0.217,1-0.394) and genetic divergence among population (FST= 0.344). The population genetic diversity of the cpDNA Southern lineage (HE= 0.212,I= 0.323) was almost equal to that of the cpDNA Northern lineage (HE= 0.203,I= 0.338), but the population genetic divergence of the Southern lineage (FST = 0.416) was larger than that of the Northern lineage (FST= 0.175). All populations from the cpDNA Northern lineage formed one cluster, but populations from the cpDNA Southern lineage were divided into nine clusters in BAPS. The results of PCoA (principal coordinate analysis) and NJ (neighbour-joining) analysis were generally in accordance with that by BAPS, except that populations from the cpDNA Southern lineage formed two regional groups (central-southeastern regional group & southwestern regional group).(5) Ecological and geographic factors responsible for spatial genetic divergenceGenome scanning (GS), multiple linear regression (MLR) and univariate regressions (UR) were employed to identify the potentially adaptive loci and the bioclimatic variables associated to the outlier loci. Using Mcheza and BayeScan v.2.0,67 and 16 (out of 457) loci were identified as outlier loci among the nine BAPS clusters, respectively, while only 6 outlier loci were confirmed by both of them. Eventually,4 potential loci (L128, L144, L294, and L305) were confirmed under selection by the MLR analysis. Furthermore, the UR analysis showed that, BIO2 (mean diurnal temperature range), BIO4 (temperature seasonality), BIO5 (maximum temperature of the warmest month), BIO 12 (annual precipitation) and BIO15 (precipitation seasonality) were highly associated with the potential loci under selection.Based on the neutral data set of AFLPs, Multiple Matrix Regression with Randomization (MMRR) and Structural Equation Modelling (SEM) were used to quantify the effects of geographic versus environmental isolation and divergent selection on population divergence of E.henryi. Results showed that IBD (MMRR:0.307; SEM:0.360 ± 0.037) accounted for more variation in genetic differentiation than IBE (Isolation-by-Environment) (MMRR:0.247; SEM: 0.181 ± 0.151). By quantifying the relative contributions of each environmental variables, it revealed that BIO4 (temperature seasonality) and BIO7 (temperature annual range) were the primary contributors, followed by BIO1 (annual mean temperature), BIO5 (max temperature of warmest month), BIO9 (mean temperature of driest quarter), and BIO15 (precipitation seasonality). By contrast, slope, soil, and BIO12 (annual precipitation) had very limited contributions. Thus, geographic isolation, environmental resistance and natural selection have all contributed to presently observed patterns of genetic divergence in E. henryi.In conclusion, E. henryi harboured high levels of intraspecific genetic diversity and significant phylogeographic structure. Refugial isolation associated with climate oscillation during the Miocene/Pliocene probably promoted allopatric lineage divergence of E. henryi. Geography played a predominant role at all levels-phylogeographic clades are broadly geographically structured and IBD primarily explained population genetic structure. However, environmental factors are clearly also important-climatic fluctuations since the LIG have likely contributed to phylogeographic structure, and the population genetic structure (in our AFLP dataset) was partly explained by IBE, which may have resulted from natural selection in environments with divergent climates. Thus, historical and contemporary geography and historical and contemporary environments have all shaped patterns of genetic structure in E. henryi. Finally, our findings not only provided the theoretical foundation about the genetic monitoring and genetic rescue of E. henryi by confirming the evolutionarily significant units (ESUs) and populations protected with priority, but also offered new perspectives to understand how the Tertiary climate change had impacted on the evolution processes of the East Asian temperate plant. |
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