No Tertiary relicts? A biogeographical study on the Macaronesian laurel forest species in Daucus (Apiaceae), Geranium (Geraniaceae), Gesnouinia (Urticaceae), Phyllis (Rubiaceae), Semele (Asparagaceae) and Visnea (Pentaphylacaceae)

The Macaronesian laurel forest is characterized by humidity-adapted, evergreen trees with glossy, entire and elongated leaves. Based on fossil data, this vegetation type has been regarded as a relict of Tertiary, European/Mediterranean forests since at least the middle of the 19th century. In contrast to that, more recent studies indicate that the Macaronesian laurel forest species may be much younger than previously thought, with the majority of the analyzed species dating to the Plio-/Pleistocene. Furthermore, they recovered a rather heterogeneous geographical origin, suggesting that the Mediterranean region, other Macaronesian vegetation zones as well as tropical areas have served as source areas for the corresponding species. Although previous analyses included quite characteristic taxa, e.g. all of the Macaronesian Lauraceae, only a small number (around 26%) of laurel forest genera has been studied to this day, most of them are woody. In this dissertation, the biogeography of six typical and widespread Macaronesian laurel forest genera (Daucus, Geranium, Gesnouinia, Phyllis, Semele and Visnea) is studied, covering different life-forms and ecologies. Conducting molecular phylogenetic and dating analyses as well as ancestral area estimations, a) the timeframes for the colonization of Macaronesia and the laurel forest, b) the geographical origin of the colonizers and c) the timeframes for inter-archipelago and inter-island dispersal were studied. Furthermore, the usefulness of stem ages and crown ages for inferring the colonization times is tested. Additional analyses were conducted for Gesnouinia and Visnea. In Gesnouinia, the wood anatomy was studied as the genus was considered as potentially insular woody in previous studies, which would contradict a relict status. For Visnea, fossils of the extinct V. germanica from the Miocene to Pliocene of Germany and Italy were analyzed regarding their affinity to laurel forest V. mocanera using MicroCT scans. The results obtained here provide further support for the heterogeneous origin of the Macaronesian laurel forest and indicate that stem ages should be preferred over crown ages for inferring the relict status. A relict origin of Visnea (Oligocene age) and the laurel forest taxa of Geranium (Miocene age) is very likely, whereas the situation is ambiguous in Semele and Daucus. The latter two are of Miocene age, but their phylogenetic position is poorly resolved. Laurel forest Gesnouinia and Phyllis originated within Macaronesia and are clearly no relicts from the Tertiary by their source area. Dispersal from or into the dry infra-Canarian vegetation is indicated for both genera, with the time frames differing. In Phyllis, dispersal falls into the Early Pliocene, whereas in Gesnouinia, an overlap with range-shifts associated with the Pleistocene glaciation cycles is recovered. The non-relictual trait of insular woodiness could not be unambiguously inferred for Gesnouinia. While woodiness in Gesnouinia probably is derived, it may have evolved prior to island colonization. Interarchipelago colonization between Madeira and the Canary Islands is inferred to be young in most taxa, overlapping with Pleistocene sea-level fluctuations and the timeframes recovered for species from other Macaronesian vegetation zones. The same is found for inter-island colonization within the archipelagos. For the Macaronesian laurel forest as a whole, the newly generated data as well as literature data indicate that there is likely no obvious relationship between time of colonization and life-form or time of colonization and the extant ecological niche occupied within the forest. Instead, data points towards a link between time of colonization and the main source area of the colonizers. In the humid climate of the Late Miocene, habitat conservative dispersal from the Mediterranean/Europe to newly emerged islands and habitat space created by catastrophic events seems to have predominated. In the still humid Early Pliocene, the influx from the Mediterranean/Europe decreased and the majority of colonizers originated within Macaronesia. During the Late Pliocene climatic deterioration (cooler, drier and increasing seasonal), dispersal from the Mediterranean, probably non-habitat conservatively, was prevalent. In the course of the Pleistocene (Early and Middle), climatic changes and range shifts associated with the glaciation cycles possibly promoted the arrival of a large amount of Macaronesian taxa. Pleistocene establishment is also indicated for a number of Mediterranean/European taxa, but restricted to the Early Pleistocene. Colonization events from Asia, the New World and (Eastern) Africa seem to be rare and likely occurred prior to the Pleistocene. They may have been facilitated by the lack of e.g. climatic, tectonic or marine barriers during certain periods of time

Biogeography of Mediterranean hotspot biodiversity: re-evaluating the 'Tertiary relict' hypothesis of Macaronesian laurel forests

The Macaronesian laurel forests (MLF) are dominated by trees with a laurophyll habit comparable to evergreen humid forests which were scattered across Europe and the Mediterranean in the Paleogene and Neogene. Therefore, MLF are traditionally regarded as an old, 'Tertiary relict' vegetation type. Here we address the question if key taxa of the MLF are relictual. We evaluated the relict hypothesis consulting fossil data and analyses based on molecular phylogenies of 18 representative species. For molecular dating we used the program BEAST, for ancestral trait reconstructions BayesTraits and Lagrange to infer ancestral areas. Our molecular dating showed that the origins of four species date back to the Upper Miocene while 14 originated in the Plio-Pleistocene. This coincides with the decline of fossil laurophyllous elements in Europe since the middle Miocene. Ancestral trait and area reconstructions indicate that MLF evolved partly from pre-adapted taxa from the Mediterranean, Macaronesia and the tropics. According to the fossil record laurophyllous taxa existed in Macaronesia since the Plio- and Pleistocene. MLF are composed of species with a heterogeneous origin. The taxa dated to the Pleistocene are likely not 'Tertiary relicts'. Some species may be interpreted as relictual. In this case, the establishment of most species in the Plio-Pleistocene suggests that there was a massive species turnover before this time. Alternatively, MLF were largely newly assembled through global recruitment rather than surviving as relicts of a once more widespread vegetation. This process may have possibly been triggered by the intensification of the trade winds at the end of the Pliocene as indicated by proxy data

Functional Domain Analysis of the Remorin Protein LjSYMREM1 in <em>Lotus japonicus</em>

<div><p>In legumes rhizobial infection during root nodule symbiosis (RNS) is controlled by a conserved set of receptor proteins and downstream components. MtSYMREM1, a protein of the Remorin family in <em>Medicago truncatula</em>, was shown to interact with at least three receptor-like kinases (RLKs) that are essential for RNS. Remorins are comprised of a conserved C-terminal domain and a variable N-terminal region that defines the six different Remorin groups. While both N- and C-terminal regions of Remorins belonging to the same phylogenetic group are similar to each other throughout the plant kingdom, the N-terminal domains of legume-specific group 2 Remorins show exceptional high degrees of sequence divergence suggesting evolutionary specialization of this protein within this clade. We therefore identified and characterized the MtSYMREM1 ortholog from <em>Lotus japonicus</em> (LjSYMREM1), a model legume that forms determinate root nodules. Here, we resolved its spatio-temporal regulation and showed that over-expression of <em>LjSYMREM1</em> increases nodulation on transgenic roots. Using a structure-function approach we show that protein interactions including Remorin oligomerization are mainly mediated and stabilized by the Remorin C-terminal region with its coiled-coil domain while the RLK kinase domains transiently interact <em>in vivo</em> and phosphorylate a residue in the N-terminal region of the LjSYMREM1 protein <em>in vitro</em>. These data provide novel insights into the mechanism of this putative molecular scaffold protein and underline its importance during rhizobial infection.</p> </div

The C-terminal domain of the LjSYMREM1 protein mainly contributed to protein interactions.

<p>The yeast split-ubiquitin assay was used to test interactions between the LjSYMREM1 variants and the RLKs NFR1, NFR5 and SYMRK. The coding regions were fused to the C-terminal half (Cub) and the N-terminal half (NubG) of ubiquitin and interaction was tested on an individual basis. Yeast growth on medium lacking leucine and tryptophan (−LW) indicates presence of both constructs. Interaction was tested on medium additionally lacking histidine (−LWH) that was supplemented with 15 mM 3-amino-1,2,4-triazole (3-AT) to suppress residual levels of endogenous histidine biosynthesis. The yeast resident ER protein Alg5 was used as negative control (Alg5:NubG and Alg5:Cub). Yeast growth was sustained on –LWH medium indicating strong interaction of the RLKs and Remorins variants with LjSYMREM1_C (A). Weak interaction of LjSYMREM1_N with the RLKs and Remorins variants indicates minor or transient contribution of the N-terminal region to protein interactions (B). Pigmentation of yeast indicates severe adenine deficiency as a consequence of lacking interaction. A series of three dilutions (non-diluted, 10⁻¹ and 10⁻²) are shown in each panel from left to right).</p

Overexpression of <i>LjSYMREM1</i> leads to increased nodule numbers.

<p>LjSYMREM1 was overexpressed as a mOrange fusion protein in transgenic <i>L. japonicus</i> roots (A). Nodule number and morphology was assessed 28 dpi with <i>M. loti</i> (MAFF303099-DsRed). Nodules were grouped into mature/pink and immature/white nodules and counted. Nodule morphology was not altered as indicated by the representative inlets above. Scale bars indicate 500 µm. Error bars represent standard errors and significance levels that were determined by student's t-test are indicated by an asterisk (p<0.01). Western Blot analysis to determine expression levels of <i>LjSYMREM1</i> in transgenic roots (B). Proteins from transgenic roots of chimeric plants expressing a <i>pUbi:LjSYMREM1:mOrange</i> construct (left) and stable transgenic plants expressing a <i>pLjSYMREM1:LjSYMREM1:YFP</i> construct (right) were transferred to a PVDF membrane and probed with the respective antibodies. Amounts of proteins loaded transferred the membrane is indicated by Amido black staining.</p

Identification and analysis of orthologous SYMREM1 genes and proteins.

<p>The <i>LjSYMREM1</i> sequence is similar to the previously published one of <i>MtSYMREM1</i> Both genes show the same exon-intron structure even though the <i>MtSYMREM1</i> gene is comprised of longer introns (A). Phylogenetic analysis based on 147 amino acid Remorin sequences using 101 unambiguously aligned residues in the conserved C-terminal region identifies the group 2 (B). Amino acid sequences of 11 group 2 Remorins from legumes and poplar were aligned and analyzed in 172 positions (C). MtSYMREM1 and LjSYMREM1 clearly cluster indicating that these proteins are orthologous to each other. Names marked with an asterisk were introduced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030817#pone.0030817-Raffaele1" target="_blank">[16]</a>.</p

Expression of LjSYMREM1 variants in <i>L. japonicus</i> roots and <i>N. benthamiana</i> leaves.

<p>Clones derived from cDNA of LjSYMREM1 were C-terminally tagged with the mOrange fluorophore and expressed under control of the <i>Lotus</i> polyubiquitin promoter in transgenic <i>L. japonicus</i> roots (A–C) and as a CaMV-35S promoter-driven construct in leave epidermal cells of <i>N. benthamiana</i> (D,E,G). The full-length (FL) protein and the C-terminal region of LjSYMREM1 (LjSYMREM1_C) are associated to the PM while the N-terminal region (LjSYMREM1_N) is cytosolic indicated by visible cytoplasmatic strands. In addition NFR1:Cerulean (F) and free Cerulean (H) were expressed in <i>N. benthamiana</i> leaves resulting in PM and cytosolic localization, respectively. Bars indicate 200 µm (A–C) and 50 µm (D–I).</p

NFR1 and SYMRK kinase domains are able to phosphorylate LjSYMREM1 <i>in vitro</i>.

<p>Recombinant proteins purified from <i>E. coli</i> were tested for phosphorylation <i>in vitro</i>. LjSYMREM1 was N-terminally fused to the maltose binding protein (MBP). While NFR1 and NFR5 kinase domains (CD; cytosolic domains of the RLKs were used) were used as untagged proteins, SYMRK-CD contained a His-tag at its C-terminal end. Phosphorylation was visualized by detection of integrated radioactively labeled γ-³²P-ATP. Both CDs were able to phosphorylate LjSYMREM1 even though NFR1 to a lower extent than SYMRK (A). Autophosphorylation of NFR1 and SYMRK kinase domains as well as trans-phosphorylation of NFR5-CD (inactive) by NFR1 were observed. Presence of NFR5 did not change the level of LjSYMREM1 phosphorylation. Protein staining of the SDS-PAGE shows presence of used proteins. Due to high kinase activity of SYMRK-CD protein amounts used for the assay were decreased to 0.25 µg and thus not visible on the gel (A). Representative MS/MS spectra of phosphorylated peptide ESQNAESSNSpTLTITR (NFR1-LjSYMREM1) (B) and ESQNAESSNpSTLTITR (SYMRK-LjSYMREM1) (C) were obtained when mapping the phosphorylation sites S48 and T49 on the LjSYMREM1 protein, respectively. While SYMRK was able to phosphorylate the C-terminal part of the protein, the LjSYMREM1 N-terminal region alone could not be phosphorylated <i>in vitro</i> (D).</p

Analysis of <i>LjSYMREM1</i> promoter activity during rhizobial infection.

<p>Uninoculated transgenic roots transformed with the promoter:GUS construct (A). Application of 10⁻⁸M purified Nod Factors for 24 hours induced promoter activity in the root infection zone (1–5 cm above the root tip) (B). Root 48 hours after inoculation with DsRed expressing <i>M. loti</i> MAFF303099 (no infections) (C). Root segment with nodule primordia at 4dpi (D). Red fluorescence deriving from the bacteria shows their presence at the root surface. Young nodule at 6dpi with bacteria infecting the cortex (E). Mature nodules at 21dpi that are entirely infected by rhizobia (F). Bars indicate 500 µm.</p