David H. Lunt

The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies

Insect mitochondrial cytochrome oxidase I (COI) genes are used as a model to examine the within‐gene heterogeneity of evolutionary rate and Its implications for evolutionary analyses. The complete sequence (1537 bp) of the meadow grasshopper (Chorthippus parallelus) COI gene has been determined, and compared with eight other Insect COI genes at both the DNA and amino acid sequence levels. This reveals that different regions evolve at different rates, and the patterns of sequence variability seems associated with functional constraints on the protein. The COOH‐terminal was found to be significantly more variable than Internal loops (I), external loops (E), transmembrane helices (M) or the NH2 terminal. The central region of COI (M5‐M8) has lower levels of sequence variability, which Is related to several Important functional domains In this region. Highly conserved primers which amplify regions of different variabilities have been designed to cover the entire insect COI gene. These primers have been shown to amplify COI in a wide range of species, representing all the major insect groups; some even In an arachnid. Implications of the observed evolutionary pattern for phylogenetic analysis are discussed, with particular regard to the choice of regions of suitable variability for specific phylogenetic projects.

Refugia within Refugia: Patterns of Phylogeographic Concordance in the Iberian Peninsula

The Iberian Peninsula was one of the most important Pleistocene glacial refugia in Europe. A number of recent studies have documented the phylogeography of Iberian taxa and their relationship to more widely distributed species that expanded from this southern European refugium. We use a comparative approach to review the literature that challenges the paradigm of Iberia as a single refuge during Pleistocene glacial maxima and instead supports the occurrence of several Iberian refugia for a range of flora and fauna. Some patterns of phylogeo-graphic concordance were found between the refugial areas identified by different case studies and these broadly overlapped with previously recognized areas of high endemism in the Iberian Peninsula. Such patterns help to illustrate the internal complexity of the Iberian Peninsula as a glacial refugium, and show that for many species, populations with a high degree of genetic structure have existed throughout the Pleistocene. Importantly, the occurrence of these ‘refugia-within-refugia’ may confound the interpretation of phylogeographic patterns of European species, and can misleadingly support the occurrence of northern refugia. We discuss these and other consequences, especially when a limited number of samples from the southern Euro-pean refugia are used.

SPECIATION IN ANCIENT CRYPTIC SPECIES COMPLEXES: EVIDENCE FROM THE MOLECULAR PHYLOGENY OF BRACHIONUS PLICATILIS (ROTIFERA)

Abstract Continental lake‐dwelling zooplanktonic organisms have long been considered cosmopolitan species with little geographic variation in spite of the isolation of their habitats. Evidence of morphological cohesiveness and high dispersal capabilities support this interpretation. However, this view has been challenged recently as many such species have been shown either to comprise cryptic species complexes or to exhibit marked population genetic differentiation and strong phylogeographic structuring at a regional scale. Here we investigate the molecular phylogeny of the cosmopolitan passively dispersing rotifer Brachionus plicatilis (Rotifera: Monogononta) species complex using nu‐cleotide sequence variation from both nuclear (ribosomal internal transcribed spacer 1, ITS1) and mitochondrial (cytochrome c oxidase subunit I, COI) genes. Analysis of rotifer resting eggs from 27 salt lakes in the Iberian Peninsula plus lakes from four continents revealed nine genetically divergent lineages. The high level of sequence divergence, absence of hybridization, and extensive sympatry observed support the specific status of these lineages. Sequence divergence estimates indicate that the B. plicatilis complex began diversifying many millions of years ago, yet has showed relatively high levels of morphological stasis. We discuss these results in relation to the ecology and genetics of aquatic invertebrates possessing dispersive resting propagules and address the apparent contradiction between zooplanktonic population structure and their morphological stasis.

Mitochondrial DNA variable number tandem repeats (VNTRs) : Utility and problems in molecular ecology

Analysis of mitochondrial (mt)DNA size polymorphism in the form of variable number tandem repeats (mtVNTRs) has become an increasingly popular methodology for addressing questions in molecular ecology. When detected by PCR, mtVNTR analysis can provide a sensitive, rapid, and cost‐effective measure of genetic variability that may be exploited in studies of population differentiation and biogeography. Despite the emergence of this approach, there has been little critical evaluation of its success or utility as a practical tool. In this review, we identify problematic methodological, theoretical and interpretive factors that can influence the utility of mtVNTR analysis. The reliability of the procedure is considered in terms of both detection of alleles and scoring of intra‐individual allele frequencies. While many of the potential technical problems of the technique do not raise serious practical concerns, this rapid and sensitive methodology is seriously compromised by the difficulty of reliably assessing allele frequencies, of assaying only germline tissue, and in our ignorance of the mechanisms generating mtVNTR diversity. Thus, although there is a considerable potential for mtVNTR pilot studies to assess genetic diversity, the utility of the technique to resolve broader questions in molecular ecology should be treated cautiously until such a time as the system is better understood.

An extant cichlid fish radiation emerged in an extinct Pleistocene lake

The haplochromine cichlid fish of the East African Great Lakes represent some of the fastest and most species-rich adaptive radiations known, but rivers in most of Africa accommodate only a few morphologically similar species of haplochromine cichlid fish. This has been explained by the wealth of ecological opportunity in large lakes compared with rivers. It is therefore surprising that the rivers of southern Africa harbour many, ecologically diverse haplochromines. Here we present genetic, morphological and biogeographical evidence suggesting that these riverine cichlids are products of a recent adaptive radiation in a large lake that dried up in the Holocene. Haplochromine species richness peaks steeply in an area for which geological data reveal the historical existence of Lake palaeo-Makgadikgadi. The centre of this extinct lake is now a saltpan north of the Kalahari Desert, but it once hosted a rapidly evolving fish species radiation, comparable in morphological diversity to that in the extant African Great Lakes. Importantly, this lake seeded all major river systems of southern Africa with ecologically diverse cichlids. This discovery reveals how local evolutionary processes operating during a short window of ecological opportunity can have a major and lasting effect on biodiversity on a continental scale.

Phylogeography and regional endemism of a passively dispersing zooplankter: mitochondrial DNA variation in rotifer resting egg banks

We investigated the phylogeography of the salt water rotifer Brachionus plicatilis, a cyclical parthenogen with passive dispersal mechanisms, using resting eggs recovered from saline lake sediments. Individual resting eggs were obtained from a large selection of lakes which were representative of five endorheic basins and the chain of coastal ponds in the Iberian Peninsula. The novel use of resting eggs allows the integration of seasonal and annual variations as well as the impact of stochastic effects such as drift and local extinction. A 653 bp fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene was sequenced from 98 eggs. Our results revealed a deep phylogeographical structure in this species, with a division into two main lineages with distinct geographical distributions, which probably diverged at the beginning of the Pleistocene period. Most of the mitochondrial DNA haplotypes were restricted to single lakes. Nested clade analysis supported Early Pleistocene fragmentation of populations, low gene flow and some long–distance colonization. These conclusions contrast strongly with previous ideas on rotifer biogeography and this pattern is consistent with a recolonization of the Iberian Peninsula from two glacial refugia. The results provide new insights into the processes responsible for the genetic diversification of passive dispersers, a life–history trait typical of zooplanktonic biotas.

Mating trials validate the use of DNA barcoding to reveal cryptic speciation of a marine bryozoan taxon.

Despite increasing threats to the marine environment, only a fraction of the biodiversity of the oceans has been described, owing in part to the widespread occurrence of cryptic species. DNA-based barcoding through screening of an orthologous reference gene has been proposed as a powerful tool to uncover biological diversity in the face of dwindling taxonomic expertise and the limitations of traditional species identification. Although DNA barcoding should be particularly useful in the sea, given the prevalence of marine cryptic species, the link between taxa identified through DNA barcodes and reproductively isolated taxa (biological species) has rarely been explicitly tested. Here, we use an integrated framework comparing breeding compatibility, morphology and mitochondrial (cytochrome c oxidase 1) and nuclear (elongation factor-1-alpha) DNA sequence variation among globally distributed samples of the cosmopolitan marine bryozoan Celleporella hyalina (L.). Our results reveal that C. hyalina comprises numerous deep, mostly allopatric, genetic lineages that are reproductively isolated, yet share very similar morphology, indicating rampant cryptic speciation. The close correspondence between genetic lineages and reproductively isolated taxa in the context of minimal morphological change suggests that DNA barcoding will play a leading role in uncovering the hidden biodiversity of the oceans and that the sole use of morphologically based taxonomy would grossly underestimate the number of marine species.

mtDNA phylogeography and postglacial patterns of subdivision in the meadow grasshopper Chorthippus parallelus

A 300 bp portion of the mitochondrial cytochrome oxidase subunit I (COI) gene was used to investigate the intraspecific genetic structure of the European meadow grasshopper Chorthippus parallelus. Levels of genetic subdivision between geographical regions in Europe were assessed using the KST statistic and revealed patterns indicative of the postglacial history of this organism. Northern European populations are shown to share a very recent common ancestor with, and hence originate from, a Balkan expansion. Genetic distances between these areas and both southern Italy and central Spain are shown to be approximately equal, even though only the Spanish form is recognized as a distinct subspecies. Distance estimates indicate that the common ancestor of the two subspecies lived more than one glacial cycle ago, and the refugial populations have probably been diverging in isolation for five or six glacial cycles (≈550 000 years). Comparisons are made to the analysis of a similar data set using an anonymous nuclear DNA marker (Cooper et al., 1995). Although the structure revealed is generally very similar, differences in the location of transition zones between different genetic forms of C. parallelus may reflect the different historical assortment of these molecules.

The interplay between colonization history and gene flow in passively dispersing zooplankton: microsatellite analysis of rotifer resting egg banks

Zooplanktonic organisms that disperse passively as diapausing eggs often exhibit surprisingly strong population subdivision given their high colonization ability. Here we attempt to disentangle the impacts of colonization history and gene flow on these organisms by studying the population genetic structure of the rotifer Brachionus plicatilis. The resting egg banks of B. plicatilis in fourteen salt lake populations in the Iberian Peninsula were examined using seven microsatellite loci. A remarkably high degree of geographical structuring was found (Fst=0.43), with a significant pattern of isolation by distance. Microsatellite loci were in genetic equilibrium, ruling out inbreeding as an important force in population structuring. Comparisons are drawn with previously published phylogeographical data. Surprisingly, introgression of nuclear genes was detected in neighbouring populations with divergent mtDNA haplotypes. These results stress the long lasting impact of colonization history and the modulating effect of gene flow at local scales in these organisms.

Probing marine Gammarus (Amphipoda) taxonomy with DNA barcodes

Abstract The genus Gammarus (Amphipoda) is one of the most speciose genera of Crustacea, yet much uncertainty remains concerning taxonomy and systematic relationships, particularly for brackish and marine forms. We used DNA barcode sequences from the mitochondrial cytochrome c oxidase I (COI) gene to probe the taxonomy of prominent members of marine and brackish water Gammarus of the North Atlantic, Baltic, Mediterranean and Black Seas. We investigated 16 putative Gammarus spp. at an average number of 9 specimens per species. This constitutes the most taxonomically and geographically comprehensive molecular study of marine Gammarus to date. Average between‐species sequence divergence (26.8%) was much higher than intraspecific distances (0.8%), enabling clear molecular species identification and highlighting several possible misidentifications from previously published studies. Specimens of Gammarus aequicauda and G. insensibilis from the Black Sea were at least 14% distant from their putative conspecifics elsewhere. Placing these findings in a geographic context provides strong indication of cryptic speciation. Further, we detected phylogeographic splits in G. oceanicus and G. duebeni. Our analyses also suggest phylogenetic positioning of G. marinus with members of the genus Echinogammarus, thus confirming its classification as Echinogammarus marinus. We have demonstrated that comprehensive analyses of taxonomically complex groups using DNA barcodes can result in a diversity of complementary data on taxonomy, phylogeography and phylogenetics. The combination of these results, with further morphological and ecological data, will enable significant progress in our understanding of this ecologically important group of crustaceans.