(with Dr. David Lunt and Prof. Gary Carvalho)

(Research grant funded by the Natural Environment Research Council (NERC))

Project Summary

Phylogeographic analysis is crucial to understanding processes such as population subdivision, speciation, ecological adaptation and historical climate change, and has consequently become increasingly common in recent years. It is apparent from the literature however that there is a very biased sampling of the component species of existing biotas, with most species studied in detail able to disperse widely through relatively continuous habitat for a significant part of their recent history (1). It would be very valuable therefore to examine organisms for whom dispersal mechanisms, degree of habitat fragmentation, and life-history characteristics do not fit these well described models.

Zooplanktonic organisms are important components of continental aquatic ecosystems, and often present dormant stages in their life cycles, which allow only for passive dispersal. In addition, they exhibit a diversity of life cycles, including sexuals, parthenogens and cyclical parthenogens. Finally, the patchy nature of their habitats is likely to influence both their colonisation patterns and local adaptive divergence (2). However, despite the high number of major taxa involved, there have been relatively few studies addressing the zoogeography of genetic lineages of lacustrine zooplankters (3). In particular, the detailed studies of Daphnia have revealed much about the incidence of breeding system variation and hybridisation on patterns of genetic variability (4). However, these very factors, widespread in this genus, have often obscured the interpretation of its intraspecific phylogeographic patterns (5).

Here we propose to use DNA sequence variation to investigate the phylogeography of a planktonic rotifer through its resting egg banks. Rotifers are a major group of zooplankters and will provide an excellent model system because, although they share the low dispersal abilities and the habitat patchiness of Daphnia, do not suffer from the compounding effects of hybridisation and obligate asexuality which seems to complicate the intraspecific analysis of variability. These organisms exhibit cyclical parthenogenesis, and disperse passively via resting eggs. Brachionus plicatilis in particular, is a rotifer species complex inhabiting saline lakes and ponds world-wide (6), habitats of a highly patchy nature. At least three sibling species have also been recognised to inhabit the Iberian Peninsula (7).

The use of resting eggs banks to infer the phylogeographic patterns of an organism is a novel approach. Resting egg banks are archives of genetic variability, integrating the seasonal and annual variation of a species (8). As sampling success of individuals in the water column is often highly unpredictable, due to the fact that species, as well as their habitats, are often ephemeral or seasonal, sampling from sediments provides a more robust sampling strategy. In addition, the presence of a resting egg bank in B. plicatilis will remove or reduce the stochastic effects of drift and local extinction allowing the data collected to more clearly represent long-term genetic subdivision.

The Iberian Peninsula has a rich diversity in saline lakes. It presents five distinct isolated endorheic basins of Early Miocene origin containing saline lakes, and a chain of coastal saline lagoons, of more recent origin (Holocene) (9) (Fig.1). These lakes vary widely in their degree of seasonal permanency and chemical characteristics (10), with a significant ecological division between the inland and coastal lakes (11). Such geographical and ecological heterogeneity in saline lakes across a relatively small spatial scale allows us to test the degree to which passive dispersers are affected by the degree of habitat patchiness, ecological transitions and historical processes.

This rotifer system allows the formulation and testing of clear phylogeographic hypotheses: (i) in terms of genetic structure, low gene flow should favour the genetic divergence of populations in each endorheic basin or lake group (deep phylogeographic structure, type I phylogeographic structure (12), (ii) coastal lakes are much more continuous and will not be characterised by extensive subdivision over small to medium distances (type III, IV, or V (12)).

These hypotheses will be tested by careful geographic sampling and use of resting egg DNA sequence variation for the analysis intraspecific phylogeography for the rotifer Brachionus plicatilis in Iberia. We will (1) construct a regional phylogeography including both coastal and inland populations, (2) test the influence of geographic isolation and habitat patchiness on historical subdivision, and (3) relate observed patterns of subdivision to regional ecological characteristics.

Superficial sediment samples, containing resting eggs from recent years, have already been collected from five Iberian inland lakes (from two basins) and four coastal lagoons. The proposed additional sampling will allow us to complete the collections in a more structured manner. In addition, an extensive collection of Iberian clones from this species is already available for analysis and several colleagues have kindly offered to send samples from outside Spain and related species. We have previously optimised methods for efficient retrieval of resting eggs from sediments, and extraction of high quality DNA suitable for PCR analysis. From a range of suitable primers available to us, those for the rRNA ITS region 1 have proved to be appropriate for this study. We will sequence the entire ITS1 region (600 bp, 1 sequencing run) from a minimum of 10 individual resting eggs per location (population) in 12 locales representing the inland endorheic basins and the coastal saline ponds. To place the observed genetic divergence in context we will also sequence ITS1 from 3 other European locations and 2 other species in this genus. The Molecular Ecology laboratory at Hull University is very well equipped with 2 automated sequencers and 9 PCR machines so the total of ca. 130 sequences necessary is a figure which we know from considerable previous experience to be realistic in the time and budget proposed.

This proposal will represent the first survey of phylogeographic patterns using resting egg stages in a zooplanktonic organism. We are able to address specific hypotheses which will enable us to determine the interaction of gene flow, distance and habitat patchiness for passive dispersers. Moreover, the resultant phylogeographic data will build upon the investigation of mating behaviour investigated previously, and provide novel insights into the speciation processes of these cyclical parthenogens.