Evaluating Ecosystem Services Provided by Non-Native Species: An Experimental Test in California Grasslands

<div><p>The concept of ecosystem services – the benefits that nature provides to human's society – has gained increasing attention over the past decade. Increasing global abiotic and biotic change, including species invasions, is threatening the secure delivery of these ecosystem services. Efficient evaluation methods of ecosystem services are urgently needed to improve our ability to determine management strategies and restoration goals in face of these new emerging ecosystems. Considering a range of multiple ecosystem functions may be a useful way to determine such strategies. We tested this framework experimentally in California grasslands, where large shifts in species composition have occurred since the late 1700's. We compared a suite of ecosystem functions within one historic native and two non-native species assemblages under different grazing intensities to address how different species assemblages vary in provisioning, regulatory and supporting ecosystem services. Forage production was reduced in one non-native assemblage (medusahead). Cultural ecosystem services, such as native species diversity, were inherently lower in both non-native assemblages, whereas most other services were maintained across grazing intensities. All systems provided similar ecosystem services under the highest grazing intensity treatment, which simulated unsustainable grazing intensity. We suggest that applying a more comprehensive ecosystem framework that considers multiple ecosystem services to evaluate new emerging ecosystems is a valuable tool to determine management goals and how to intervene in a changing ecosystem.</p></div

The three grassland species assemblages investigated in this study.

<p>The (a) three original planted species assemblages are clearly distinguishable based on their species composition as shown by (b) results of CCA of species abundances from 2008–2010 with year and study site as covariate. (c) Residual dry matter measured in 2010 to describe grazing gradient within the three ecosystem types. Letter above bars indicate significant differences among grazing levels as determined by planned orthogonal contrasts at P<0.01.</p

Ecosystem service measurements taken in three different grassland ecosystem types.

<p>All measurements were taken in 2010.</p>†<p> excluded non-palatable species: <i>Aegilops triunciales</i> L., <i>Brachypodium distachyon</i> (L.) Beauv., <i>Bromus diandrus</i> Roth, <i>Carduus pycnocephalus</i> L., <i>Centaurea solistitialis</i> L., <i>Taeniatherum caput-medusae</i> L.</p>‡<p> weighting factor to take into account that non-palatable species provide some low quality forage during their early development stages which we estimated to be approx. 30% of their total biomass.</p><p>* as number of initially planted species varied among the grassland types, we did not use species richness as a diversity index.</p><p>Ecosystem service measurements taken in three different grassland ecosystem types.</p

PRMT6 and subunits of PRC1/PRC2 co-occupy the HOXA gene cluster.

<p>(<b>A</b>) Schematic representation of the <i>HOXA</i> gene cluster consisting of 11 genes (<i>HOXA1-13</i>) and located on chromosome 7. According to their expression pattern, the 3’-located genes <i>HOXA1-5</i> belong to the rostral genes and the 5’-located <i>HOXA9-13</i> to the caudal genes. (<b>B</b>) NT2/D1 cells were left untreated (0 d) or treated for 2 days (2 d) and 6 days (6 d) with 0.1 μM ATRA. Subsequently, the cells were subjected to ChIP analysis using PRMT6-, CBX8- as well as EZH2-specific antibodies and antibodies recognising the H3R2me2a and H3K27me3 marks. Isotype-specific IgG served as control antibody. Immunoprecipitated DNA was analysed in triplicates by qPCR with primers for the <i>HOXA1</i>, <i>A2</i>, <i>A5</i>, <i>A9</i> and <i>A10</i> genes. Mean values were calculated as percentage input (% Input) and error bars represent mean +/- S.D. of the triplicates.</p

Rostral <i>HOXA</i> genes are common target genes of PRMT6- and PRC-mediated repression.

<p>(<b>A</b>) NT2/D1 cells were transfected with control siRNAs (siControl) or siRNAs directed against PRMT6 (siPRMT6 = equal mixture of siPRMT6_1 and siPRMT6_2), CBX8 (siCBX8) or EZH2 (siEZH2). Subsequently, cells were harvested and 30 μg total protein of each sample were analysed by Western blot with the indicated antibodies. The asterisk indicates the specific signals for the CBX8 protein. (<b>B</b>) NT2/D1 cells were transfected as in (A). Forty-eight hours post transfection cells were left untreated (-) or treated for 2 days (+) with 0.1 μM ATRA. Total RNA was then prepared and analysed in triplicates by RT-qPCR for transcript levels of <i>HOXA1</i>, <i>A2</i> and <i>A5</i> normalised to <i>UBIQUITIN</i> transcription. Error bars represent mean +/- S.D. of the triplicates. Transcript levels of untreated and siControl-transfected NT2/D1cells were set to 1.</p

Seedling and species recruitment of sown plant species

Seedling and species recruitment data for exotic ("EXO") and native ("NAT") sown species were recorded in the field on 0.5 x 0.5 square meter subplots in seed addition and control subplots (separate column "Control") on 18 experimental sites in two regions (8 Central California ("CA"), 10 Central Germany ("GE")). Data were recorded in 2012 (census 1 and 2) and 2013 (census 3). Subplots were either located in rodent exclosure (Rod), gastropod exclosure (Gas) or control plots (Con). The number of added seeds per species ranged from 50 to 175 (column "Number"), depending on the seed size. The number of sown species ranged from 17 (California) to 20 (Germany). Acronyms for the species can be derived by taking the first 3 letters of the genus and species names (see Table A 4)

PRMT6 represses the ATRA-mediated transcriptional activation of rostral <i>HOXA</i> genes, but has no influence on the transcription of caudal <i>HOXA</i> genes.

<p>(<b>A</b>) NT2/D1 cells were left untreated (-) or treated for 2 days (+) with 0.1 μM ATRA. Subsequently, total RNA was prepared and analysed in triplicates by RT-qPCR for transcript levels of <i>HOXA1</i>, <i>A2</i>, <i>A5</i>, <i>A9</i> and <i>A10</i> normalised to <i>UBIQUITIN</i> transcription. Error bars represent mean +/- S.D. of the triplicates. Transcript levels of untreated cells were set to 1. (<b>B</b>) NT2/D1 cells were transfected with control siRNAs (siControl) or siRNA directed against PRMT6 (siPRMT6 = siPRMT6_1). Forty-eight hours post transfection cells were left untreated (-) or treated for 2 days (+) with 0.1 μM ATRA. Subsequently, total RNA was prepared and analysed in triplicates by RT-qPCR for transcript levels of <i>PRMT6</i>, <i>HOXA1</i>, <i>A2</i>, <i>A5</i>, <i>A9</i> and <i>A10</i> normalised to <i>UBIQUITIN</i> transcription. Error bars represent mean +/- S.D. of the triplicates. Transcript levels of untreated and siControl-transfected NT2/D1 cells were set to 1.</p

The Arginine Methyltransferase PRMT6 Cooperates with Polycomb Proteins in Regulating <i>HOXA</i> Gene Expression

<div><p>Protein arginine methyltransferase 6 (PRMT6) catalyses asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a), which has been shown to impede the deposition of histone H3 lysine 4 trimethylation (H3K4me3) by blocking the binding and activity of the MLL1 complex. Importantly, the genomic occurrence of H3R2me2a has been found to coincide with histone H3 lysine 27 trimethylation (H3K27me3), a repressive histone mark generated by the Polycomb repressive complex 2 (PRC2). Therefore, we investigate here a putative crosstalk between PRMT6- and PRC-mediated repression in a cellular model of neuronal differentiation. We show that PRMT6 and subunits of PRC2 as well as PRC1 are bound to the same regulatory regions of rostral <i>HOXA</i> genes and that they control the differentiation-associated activation of these genes. Furthermore, we find that PRMT6 interacts with subunits of PRC1 and PRC2 and that depletion of PRMT6 results in diminished PRC1/PRC2 and H3K27me3 occupancy and in increased H3K4me3 levels at these target genes. Taken together, our data uncover a novel, additional mechanism of how PRMT6 contributes to gene repression by cooperating with Polycomb proteins.</p></div

PRMT6 influences PRC-mediated gene silencing at the <i>HoxA</i> gene locus.

<p>(<b>A</b>) NT2/D1 cells were transfected with control siRNAs (siControl) or siRNAs directed against PRMT6 (siPRMT6 = equal mixture of siPRMT6_1 and siPRMT6_2). Forty-eight hours post transfection cells were left untreated (-) or treated for 2 days (+) with 0.1 μM ATRA. Subsequently, cells were subjected to ChIP analysis using PRMT6-, CBX8- as well as EZH2-specific antibodies. IgG served as control antibody for these three antibodies and is displayed together with the PRMT6 ChIP in the upper graph. Immunopreciptiated DNA was analysed in triplicates by qPCR with primers for the <i>HOXA2</i> and <i>A5</i> genes. Mean values are calculated as percentage input (% input) and error bars represent mean +/- S.D. of the triplicates. (<b>B, C</b>) NT2/D1 cells were transfected and treated as described in (A). Cells were then subjected to ChIP analysis using antibodies recognising histone H3 and the histone marks H3K27me3 (B) and H3K4me3 (C). Immunoprecipitated DNA was analysed in triplicates by qPCR with primers for the <i>HOXA2</i> and <i>A5</i> genes. Mean values are calculated as enrichment relative to histone H3 and error bars represent mean +/- S.D. of the triplicates.</p

PRMT6 interacts with subunits of PRC1 and PRC2.

<p>(<b>A</b>) HEK293 cells were transfected with untagged HPH1 or Myc-tagged HPH2 constructs (OE = overexpressed) and harvested 48 hours after transfection. Protein extracts were subjected to immunoprecipitation using antibodies against PRMT6 (α-PRMT6) or isotype-specific IgG as control. Inputs (2%) and precipitates were subjected to Western blot analysis using antibodies against HPH1 (α-HPH1), Myc-tag (α-Myc for HPH2 detection) and PRMT6 (α-PRMT6). The asterisk indicates an unspecific signal in the α-PRMT6 staining. (<b>B</b>) HEK293 cells were transfected with GFP-tagged CBX2, CBX4 and CBX8 constructs (OE = overexpressed) and harvested 48 hours after transfection. Protein extracts were subjected to immunoprecipitation using antibodies against PRMT6 (α-PRMT6) or as control isotype-specific IgG. Input (2%) and precipitates were subjected to Western blot analysis using antibodies against GFP (α-GFP for CBX detection) and PRMT6 (α-PRMT6). (<b>C</b>) For the detection of endogenous interactions, HEK293 protein extracts were subjected to immunoprecipitation using antibodies against PRMT6 (α-PRMT6) or isotype-specific IgG as control. Input (2%) and precipitates were subjected to Western blot analysis using antibodies against HPH1 (α-HPH1), HPH2 (α-HPH2), BMI1 (α-BMI1) and PRMT6 (α-PRMT6). (<b>D</b>) For size fractionation by gel filtration chromatography, whole-cell protein extracts were prepared from HEK293 cells. Protein extracts were applied to a Superose 6 column and 6 ml fractions were collected. Five % of each fraction (no. 6–17) were analysed by Western blot using the indicated antibodies to detect PRMT6, HPH2, CBX8, BMI1, RING1A and RING1B, endogenously. The column was calibrated using standard protein markers. Accordingly, the molecular weight included in the fractions and the void volume (V₀) are indicated. (<b>E</b>) For the detection of endogenous and exogenous interaction between PRMT6 and EZH2, HEK293 cells were either not transfected or transfected with Flag-tagged EZH2 construct (OE = overexpressed). Subsequently, protein extracts were subjected to immunoprecipitation using antibodies against PRMT6 (α-PRMT6) or isotype-specific IgG as control. Input (2%) and precipitates were analysed by Western blot using the indicated antibodies. (<b>F</b>) Whole-cell protein extracts were generated from HEK293 cells transfected with Flag-tagged EZH2 construct. Size fractionation by gel filtration chromatography was performed as described in (D) and fractions 6–16 were subjected to Western blot analysis using the indicated antibodies.</p