DFG project TransTibet - PhD position to be filled…

“TransTibet - Phylogenetic reconstruction of trans-Tibet dispersal events in wingless ground beetles” is a new exciting project where we aim to deepen our understanding of the paleoenvironmental evolution of the Tibetan Plateau (TP) through the study of wingless ground beetles’ phylogeny. Over the past decade, we have established ground beetles as a significant paleoenvironmental proxy, particularly for high mountain regions in low latitudes. By combining ecological and phylogenetic data from species endemic to the Himalayan-Tibetan orogeny, we have developed scenarios for the spatio-temporal evolution of paleoenvironments, contributing to our understanding of regional surface uplift in the area. TransTibet addresses the controversial debate surrounding the timing, quantity, and sequence of surface uplift of the TP, a key factor in understanding atmospheric circulation, paleoenvironments, and the evolution of megadiverse biota. Despite evidence suggesting a high elevated proto-Plateau as early as the Paleogene, based on stable isotope paleoaltimetry, and fossil findings, there are still significant uncertainties and disagreements regarding these elevations and the timing of alpine biota’s presence on the TP.

Figure 1: Oligocene-Miocene paleoenvironmental evolution of the HTO.

Our study proposes using wingless ground beetles as a source of paleoecological and paleotopographic information, leveraging the identification of trans-Tibet disjunctions in ground beetle distributions to test and develop new spatio-temporal scenarios for the paleoenvironmental evolution of the TP. By examining the phylogenetic structure of these beetles and dating cladogenetic events, the researchers aim to uncover the topographic and environmental history of the TP.

Figure 2: Development of trans-Tibet disjunct distributional patterns in a hypothetical species group.

Our approach, combining field research, taxonomy, molecular genetics, and biogeographic modeling, aims to resolve some of the controversies surrounding the paleoenvironmental history of the TP and contribute significantly to our understanding of its complex geological and biological evolution.

The overall objective of this project is to provide new insights into the Cenozoic paleoenvironmental evolution of the HTO in context of its uplift history by means of proxy-based data. More specifically, the project aims to date the period when temperate environments appeared in the HTO for the first time, in which part of the HTO this happened, and when the temperate forests disappeared in the central HTO (paleo-Tibet) due to the emergence of subalpine-alpine environments. We will use dated phylogenies of species groups of wingless ground beetles which today are characterized by trans-Tibetan distributional patterns, as paleoenvironmental proxy. The particular objectives result from the answers to the following questions:

Research Objectives:

1. How old are the crown-groups of the oldest endemic extra-tropical elements in the ground beetle fauna of the HTO?
   According to island biogeography theory, it has to be assumed that flight-active ground beetles adapted to temperate environments dispersed to the HTO immediately after its uplift to significant heights and evolved winglessness. Therefore, we date the earliest branching in extratropical groups of different beetle lineages endemic to the HTO to estimate the age for the emergence of temperate environments in the mountain system.

2. Are there significant differences in the crown-group ages of extra-tropical beetles endemic to certain parts of the HTO? Are crown-group ages of species groups characterized by trans-Tibet distributions younger than those with geographically restricted distributions?
   Winglessness markedly reduces the dispersal ability of beetles in high mountains and results in the evolution of geographically separated lineages in the course of regional uplift. Therefore, we compare dating results in wingless species groups endemic to different parts of the HTO with those with trans-Tibet distributions and derive spatially resolved models for the development of temperate paleoenvironments in the different HTO parts.

3. What are the ages of the respective geographically separated lineages in temperate adapted beetle groups that are characterized by disjunct trans-Tibetan distributional patterns?
   We assume that the observed phenomenon of trans-Tibet disjunctions was finalized by the uplift of its central part (TP) into subalpine-alpine heights. Therefore, we date the cladogenetic events in these groups to estimate the age of the uplift-induced break-up of the continuous distributional areas (indirect age estimation of large-scale development of subalpine-alpine environments).

4. What are the ages of strictly alpine groups endemic to the HTO which are characterized by trans-Tibet distributional patterns?
   The crown group ages of these species groups will lead us to date the emergence of alpine environments on the TP and therewith, the onset of the final uplift phase in the central HTO (direct age estimation of the emergence of alpine environments).

Working Hypotheses:

1. Crown-groups ages of extra-tropical elements in the HTO ground beetle fauna date back to the Paleogene-Neogene boundary, resulting from uplift-induced Late Oligocene emergence of temperate forests in paleo-Tibet. Confirmation of this hypothesis would reject uplift models which assume the presence of significant elevations in Tibet already during the Eocene.

2. In beetle groups adapted to the temperate climate: Maximum ages of the crown-groups of taxa endemic to certain parts of the HTO are younger than those characterized by trans-Tibet distributions. Confirmation of this hypothesis would support that the earliest significant HTO uplift occurred in paleo-Tibet’s interior, followed by subsequent surface uplift on Plateau margins. Therefore, paleotopographic scenarios which assume significant pre-Miocene elevations in NE- and E-Tibet will be rejected.

3. Crown ages of the respective geographically separated clades of beetles adapted to the temperate climate and characterized by trans-Tibetan disjunctions date to the end of the Miocene. Confirmation of this hypothesis would corroborate that the development of an extensive uplift-induced alpine area in central TP did not start before the Late Miocene, thus rejecting HTO uplift models which assume the presence of an Early Neogene alpine Plateau.

4. Maximum ages of crown groups of beetles which are strictly adapted to the alpine belt and characterized by trans-Tibet distributional patterns date to the Late Miocene. Confirmation of this hypothesis would corroborate the view that the final uplift phase of the HTO with the development of an extensive alpine TP did not start before the Late Miocene.

The Phd project

Reference genome

To facilitate subsequent phylogenomic analyses, the new Phd student will create a chromosome-level reference genome at the start of the project. For this purpose, he/she will extract high molecular weight DNA for PacBio HiFi sequencing at the functional genomic centre (Zürich). Scaffolding will be performed with Hi-C sequencing by PhaseGenomics (Seattle) to achieve chromosome level resolution. Because our proposed taxon sampling covers a wide spectrum of the ground beetle diversity from “basal-grade” to so called higher carabids, the Phd candidate will use a species of the Patrobini genus Deltomerodes for reference genome which represents a “middle grade” Carabidae (Schmidt et al. 2021). We expect a genome size of ~200 Mb (Weng et al. 2021). The annotation of the genome will be performed by IGATech (Udine). For this purpose, RNA-Seq data will be generated at very high coverage (1000M reads) for a multi-tissue RNA pool followed by an ab initio gene model prediction and a de novo functional annotation (InterProScan GO/motif + Blast of SwissProt entries to attach a semantic description). The Phd student will publish the new reference genome in collaboration with among other the PIs.

Phylogenomic and biogeographic analyses

Given that a significant portion of the samples are museum specimen (see table 1), the Phd project will be based on two approaches, a targeted sequencing approach reflecting samples with potential issues in DNA quality, as well as whole genome sequencing approach using low coverage sequencing at 5x coverage (lcWGS, Lou et al. 2021) for fresh and well-preserved museum beetle materials. In combination with the reference genome, this will allow the Phd student to design additional baits.

Because a genotype call at such low coverage has some uncertainty, genotype likelihoods will be used for downstream analysis (Lou et al. 2021). In detail the Phd student will proceed as follows: DNA will be extracted using the Blood and Tissue Kit from Qiagen (Hilden). A diluted Nextera (Illumina, Sandiego CA, USA) protocol (Baym et al. 2015) will be used to produce a du-al-indexed whole-genome sequencing library of each sample to enable multiplexing of 96 individuals. Different i5 and i7 primers will be used to tag multiplex-libraries for further multiplexing, and will be sequenced at 5x coverage using 150bp paired-end reads on the Novaseq-Sequencing platform (Illumina). The raw reads will be trimmed to remove polyG tails, quality controlled using FastQC (Andrews et al. 2010) and mapped to the de novo assembled reference genome (see previous chapter) using BWAmem. After applying quality filters (e.g. base quality, mapping quality, minimum depth or minimum number of individuals with coverage) genotype likelihoods will be calculated using the SAMtools genotype likelihood model implemented in ANGSD.

The targeted sequencing will be based on two sources of baits. First, the Phd student will use ~1200 ultraconserved elements (UCEs) based on a set of 13,674 published baits (Faircloth 2017). As the name indicates, UCEs are highly conserved regions of organismal genomes shared among evolutionary distant taxa and have proven useful in beetles for reconstructing the evolutionary history (Zhang et al. 2018) and on museum specimen (Derkarabetian et al. 2019). Second, we will design additional baits based on the new reference genome and the resequencing of our fresh beetle collection, allowing for more specific baits. Then, total genomic DNA will be hybridized following the NimbleGen SeqCap EZ Library User’s guide (Roche NimbleGen, see Wang et al. 2022), and sequenced on an HiSeq X Ten platform producing 150 bp paired end reads (Illumina). Raw reads will be filtered using the software Trimmomatic and then assembled to capture sequences using HybPiper. Both data sets will subsequently be merged for downstream analyses.

All genes will be aligned on the basis of translated amino acid sequences and will be concatenated. The protein and nucleotide data sets will be analyzed with Maximum Likelihood as well as Bayesian inference methods using RAxML (Stamatakis 2014) and ExaBayes (Aberer et al., 2014) respectively, given their capabilities to handle large datasets efficiently. For each gene, the best ML tree and 200 bootstrapping trees will be inferred with RAxML. The species tree analysis will then be conducted using ASTRAL III (Zhang et al. 2018) taking the unrooted best ML gene trees and corresponding bootstrapping trees as input, under the multilocus bootstrapping option. We will use the approximately unbiased test as implemented in CONSEL to evaluate the different phylogenetic hypotheses, and will calculate site-wise log likelihoods of all alternative topologies with RAxML, using these as input to estimate the P-values for each alternative hypothesis.

Using the genomic data and fossil information we will perform BEAST analyses (Bouckaert et al. 2019) for the molecular dating of cladogenetic events. An important strength of this proposal is our strategy to improve the age constraining of the phylogeny by substantially increasing the calibrating fossil data base as explained above. Because carabids are unknown from HTO fossil deposits, we will identify and deeply investigate fossils from close related outgroup taxa of the HTO endemics. Fossil species of the tribes Bembidiini, Pterostichini, Sphodrini, Trechini, Zabrini and many Harpalinae lineages are already available for study, additional promising amber fossils will be loaned from museums and private collectors. To enable the utilization of the markedly enlarged fossil data base in our phylogenetic analyses, we will use a large outgroup set of ~100 taxa (table 1) across the ground beetle diversity. We are thus optimistic, that we will be able to provide a very thorough age constraints resulting in small confidence intervals. To infer and compare different scenarios of the biogeographic history of the ground beetle species groups, the Phd student will follow two approaches. First, he/she will perform range evolution analysis using the R package BioGeoBAERS (Matzke 2013). The complex geological history of the HTO will be incorporated into the analyses by different time-stratification, information on connectivity between HTO parts according to the three currently widely discussed uplift scenarios, plus the just recently proposed scenario by us (Schmidt et al. 2022), the current topography as null model, as well as the choice of dispersal multipliers. Based on these different assumptions, several models that accommodate vicariance (and for reasons of comparison also dispersal) will be implemented in BioGeoBEARS by means of among-area probability matrices (Wang et al. 2022). Second, we will utilize the RevBayes (Höhna et al. 2016) framework to jointly model the range and biome evolution based on the cladogenetic state change framework (ClaSSE, Goldberg and Igić 2012). Again, we will set up the four different models (table 2) plus the current topography as a null model. Given niche conservatism of the beetles, we assume constant biome occupancy throughout all mountain ranges.

Co-PIs

The Phd student will be based at the University of Marburg and will be supervsied by Lars Opgenoorth. He/she will be co-supervised by Joachim Schmidt and Kangshan Mao with regular online meetings:

• Dr. Joachim Schmidt, University of Rostock Joachim is an entomologist specializing in the evolution of ground beetles. He has described and revised substantial parts of the high Asian and Afro-alpine ground beetle fauna, as well as the globally available ground beetle fossils, spearheading the use of micro CT technology to describe amber fossils.

• Prof. Dr. Kangshan Mao, Sichuan University, SCU College of Life Sciences, www.researchgate.net/profile/Kangshan-Mao Kangshan has been a leader on the evolution and phylogenomics of conifers in High-Asia.

To apply

in German

Uni-Marburg Job Posting 1

in English

Uni-Marburg Job Posting 2