Living the high life: high altitude adaptation in earthworms

Altitude provides a mix of challenging physiological conditions: lower temperatures, shorter seasonal duration, and reduced oxygen availability (hypoxia). The climatic conditions also vary along a latitudinal gradient, such that temperate and tropical mountain ranges provide a variety of hostile environments to which associated biota must locally adapt. This project will address the deficiency of research on invertebrate physiological adaptations to life at high altitude utilising earthworms as models exploiting their sedentary behaviour and intimate physical/functional contact with soil. We will investigate adaptations to altitude focusing on the mechanistic aspects of how organisms cope when oxygen and seasonal duration is diminished.

Despite the lay perception that oxygen availability is the key driver for adaptation at high altitudes, temperature, aridity and seasonal variation provide greater pressures in all but those of the highest and warmest alpine environments. Earthworms are found across altitudinal transects acting synergistically to support vegetation within these most hostile of environments. Reduced temperature, and soil depth together with loss of moisture provide significant challenges for these ecosystem engineers. Unlike larger vertebrates that migrate to avoid seasonal extremes the sedentary behaviour of these invertebrates means that at altitudinal extremes, they must accelerate their lifecycle to adapt to a restricted viable breeding season. An earthworm seasonal migration distance is only measured in the tens of meters (Marinissen and Bosch 1992). The Latitudinal Biodiversity Gradient (LBG) is a widely recognised phenomenon in ecology where a loss of diversity and key adaptive changes allows only a limited number of species to survive at high latitudes (Dowle et al. 2013). The rates these physical parameters change with latitude occur over thousands of kilometres and produce vast and complex ecological linkages. In contrast, changes in altitude mirror many of the same environmental stressors but these can alter over mere tens of kilometres and therefore provide a potentially highly tractable system. As such, investigating alpine environments negate some of the confounding complications and allow a clearer understanding of genomic adaptation.

Every 1000 meters above sea level temperature drops by 6.5⁰C providing a temperature gradient scaled by the latitudinal location and local climate. In temperate latitudes higher altitudes exhibit reduced seasonal duration with winter conditions providing ground frost compatible only with the survival of earthworm cocoons. This latter restriction can, as studies on earthworms in seasonally inundated flood plains in Holland indicate (Zorn et al. 2005), create an adaptive driver selecting for earthworms exhibiting accelerated lifecycles that can exploit the reduced alpine breeding season. In mountains within the tropics where sea level temperatures are higher, earthworms can colonize much higher altitudes where temperature is favourable, but they encounter restricted oxygen availability (oxygen available at 3500m is only 66% that of sea level). Earthworms do not possess ‘dedicated’ respiratory organs; instead relying on gaseous diffusion through their skin, a passive event facilitated by a relatively efficient closed circulatory system. The presence of erythrocruorins, (large macromolecules of globin subunits), in free solution in earthworm blood co-operatively bind oxygen delivering it efficiently to respiring tissues.

We will test the overarching hypothesis that at temperate latitudes seasonal duration is the major selective driver whilst within the tropics adaptation to oxygen availability is observed. Using a combination of phylogeography and functional genomics to identify the genetic structure of earthworm populations along altitudinal transects and endeavour to determine the functional basis of acclimatisation to the prevailing conditions.

Methodology

Earthworm biota will be collected along altitudinal transects within temperate and tropical mountain ranges. Temperate transects will incorporated two sites in the Western Alps have been selected based on their altitude change over very short horizontal distances, Les Deux Alpes’s grass line ranging from 800m to around 2200m and the mountain pass of Col de l’Iseran, near Savoie in France which has a grass-line at ~3000m. Both are located between 45 and 46 degrees latitude and are just over 50 miles apart and in between sites at Bardonecchia, previous investigation have identified L. terrestris at 1500m, (Bernier and Ponge 1998). In contrast two independent tropical alpine transects will be conducted on Mount Kilimanjaro, -3o latitude, which has a grass line up to 4000m and has virtually no seasonal temperature variation. Earthworms have not been speciated here, however have been observed at 3840m (Moore 2008). Samples will be collected in the Western Alps during summer months and winter months for Mount Kilimanjaro at regular intervals along designated transects.

Samples will be provisionally washed, speciated, and preserved using RNAlater on-site and transported to Cardiff for analysis. Individuals will be taxonomically identified prior to performing stratified molecular analysis. We will determine earthworm community composition using a singular mitochondrial barcode (COI), characterise the population structure of an individual species found through-out the altitude transects using multiple mitochondrial loci together with nuclear markers (SNPs or microsatellites) and then investigate functional loci selection using global SNP analysis (RAD-tagging) for individuals of the target species at key altitudinal sites.  Furthermore, we will explore the signals of functional acclimatisation using global transcript analysis using RNAseq as well as targeted transcript analysis of genes associated with lifecycle control and hypoxia using qPCR comparing individuals of target species sampled from the same key altitudinal sites.

Deliverables

  1. Earthworm community structure
    1. Identify the earthworm species found in the two Western Alps sites over the altitude transects between ~800m to 2500m.
    2. Identify the earthworm species found on Mount Kilimanjaro over two altitude transects between ~1500 to 4000m.
  2. Ascertain and establish any relationship between altitude and population structure of a specific species of earthworm at temperate and tropical locations, identifying any specific altitudinal traits.
  • Determine whether there are signals for genetic selection within earthworms across temperate and tropical altitudinal gradients and test whether traits selected for are the same in temperate and tropical zones.
  1. Derive the functional signals of acclimatisation within earthworms across temperate and tropical altitudinal gradients.
  2. Critically, we aim to detail if key functional changes in gene expression are needed to live at high altitudes and whether these are acquired through genetic adaptation, phenotypic plasticity driven by acclimatization or epigenetic change.

In addition we will test whether there is an Altitudinal Biodiversity Gradient (ABG) for soil biota, focusing on earthworm speciation that is equivalent to ABG found in plants (Grythnes and Vetaas, 2002). This sees incremental changes in diversity, that increase up to 1500m but starts reducing after 2500m.