mtDNA and the origin of the Icelanders: deciphering signals of recent population history

Previous attempts to investigate the origin of the Icelanders have provided estimates of ancestry ranging from a 98% British Isles contribution to an 86% Scandinavian contribution. We generated mitochondrial sequence data for 401 Icelandic individuals and compared these data with >2,500 other European sequences from published sources, to determine the probable origins of women who contributed to Iceland’s settlement. Although the mean number of base-pair differences is high in the Icelandic sequences and they are widely distributed in the overall European mtDNA phylogeny, we find a smaller number of distinct mitochondrial lineages, compared with most other European populations. The frequencies of a number of mtDNA lineages in the Icelanders deviate noticeably from those in neighboring populations, suggesting that founder effects and genetic drift may have had a considerable influence on the Icelandic gene pool. This is in accordance with available demographic evidence about Icelandic population history. A comparison with published mtDNA lineages from European populations indicates that, whereas most founding females probably originated from Scandinavia and the British Isles, lesser contributions from other populations may also have taken place. We present a highly resolved phylogenetic network for the Icelandic data, identifying a number of previously unreported mtDNA lineage clusters and providing a detailed depiction of the evolutionary relationships between European mtDNA clusters. Our findings indicate that European populations contain a large number of closely related mitochondrial lineages, many of which have not yet been sampled in the current comparative data set. Consequently, substantial increases in sample sizes that use mtDNA data will be needed to obtain valid estimates of the diverse ancestral mixtures that ultimately gave rise to contemporary populations.     

Distilling artificial recombinants from large sets of complete mtDNA genomes

Background

Large-scale genome sequencing poses enormous problems to the logistics of laboratory work and data handling. When numerous fragments of different genomes are PCR amplified and sequenced in a laboratory, there is a high immanent risk of sample confusion. For genetic markers, such as mitochondrial DNA (mtDNA), which are free of natural recombination, single instances of sample mix-up involving different branches of the mtDNA phylogeny would give rise to reticulate patterns and should therefore be detectable.

Methodology/Principal Findings

We have developed a strategy for comparing new complete mtDNA genomes, one by one, to a current skeleton of the worldwide mtDNA phylogeny. The mutations distinguishing the reference sequence from a putative recombinant sequence can then be allocated to two or more different branches of this phylogenetic skeleton. Thus, one would search for two (or three) near-matches in the total mtDNA database that together best explain the variation seen in the recombinants. The evolutionary pathway from the mtDNA tree connecting this pair together with the recombinant then generate a grid-like median network, from which one can read off the exchanged segments.

Conclusions

We have applied this procedure to a large collection of complete human mtDNA sequences, where several recombinants could be distilled by our method. All these recombinant sequences were subsequently corrected by de novo experiments – fully concordant with the predictions from our data-analytical approach.     

Median networks: speedy construction and greedy reduction, one simulation, and two case studies from human mtDNA

Molecular data sets characterized by few phylogenetically informative characters with a broad spectrum of mutation rates, such as intraspecific control-region sequence variation of human mitochondrial DNA (mtDNA), can be usefully visualized in the form of median networks. Here we provide a step-by-step guide to the construction of such networks by hand. We improve upon a previously implemented algorithm by outlining an efficient parametrized strategy amenable to large data sets, greedy reduction, which makes it possible to reconstruct some of the confounding recurrent mutations. This entails some postprocessing as well, which assists in capturing more parsimonious solutions. To simplify the creation of the resulting network by hand, we describe a speedy approach to network construction, based on a careful planning of the processing order. A coalescent simulation tailored to human mtDNA variation in Eurasia testifies to the usefulness of reduced median networks, while highlighting notorious problems faced by all phylogenetic methods in this context. Finally, we discuss two case studies involving the comparison of characters in the two hypervariable segments of the human mtDNA control region in the light of the worldwide control-region sequence database, as well as additional restriction fragment length polymorphism information. We conclude that only a minority of the mutations that hit the second segment occur at sites that would have a mutation rate comparable to those at most sites in the first segment. Discarding the known "noisy" sites of the second segment enhances the analysis.     

Median-joining networks for inferring intraspecific phylogenies.

Reconstructing phylogenies from intraspecific data (such as human mitochondrial DNA variation) is often a challenging task because of large sample sizes and small genetic distances between individuals. The resulting multitude of plausible trees is best expressed by a network which displays alternative potential evolutionary paths in the form of cycles. We present a method ("median joining" [MJ]) for constructing networks from recombination-free population data that combines features of Kruskal's algorithm for finding minimum spanning trees by favoring short connections, and Farris's maximum-parsimony (MP) heuristic algorithm, which sequentially adds new vertices called "median vectors", except that our MJ method does not resolve ties. The MJ method is hence closely related to the earlier approach of Foulds, Hendy, and Penny for estimating MP trees but can be adjusted to the level of homoplasy by setting a parameter epsilon. Unlike our earlier reduced median (RM) network method, MJ is applicable to multistate characters (e.g., amino acid sequences). An additional feature is the speed of the implemented algorithm: a sample of 800 worldwide mtDNA hypervariable segment I sequences requires less than 3 h on a Pentium 120 PC. The MJ method is demonstrated on a Tibetan mitochondrial DNA RFLP data set.     

Association studies on human mitochondrial DNA: methodological aspects and results in the most common age-related diseases

Mitochondrial DNA (mtDNA) follows direct maternal inheritance and, as such, can be used in phylogenetic studies to determine a human lineage tree. The presence of common polymorphisms allows a classification of mtDNA in haplogroups and sub-haplogroups, according to the branch they belong to. Thanks to the rapidly growing number of mtDNA sequences available, this classification is being corrected and redefined to be more accurate. In parallel with this process, several studies are trying to identify an association between common mtDNA polymorphisms and common complex traits, as hypothesized by the common disease-common variant theory. Here we review the associations already reported with the main age-related complex diseases and we identify the critical points (sample size, size of the recruiting area, careful matching between cases and controls regarding geographical origin and ethnicity, data quality checking) to be taken in account in planning such studies. On the whole, this research area is opening a new perspective as an important component of "mitochondrial medicine", capable of identifying new molecular targets for the diagnosis, prevention and treatment of common complex diseases.     

qPCR-based mitochondrial DNA quantification: influence of template DNA fragmentation on accuracy

Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.     

The landscape of mitochondrial DNA variation in human colorectal cancer on the background of phylogenetic knowledge

Recently, an increasing number of studies indicate that mutations in mitochondrial genome may contribute to cancer development or metastasis. Hence, it is important to determine whether the mitochondrial DNA might be a good, clinically applicable marker of cancer. This review describes hereditary as well as somatic mutations reported in mitochondrial DNA of colorectal cancer cells. We showed here that the entire mitochondrial genome mutational spectra are different in colorectal cancer and non-tumor cells. We also placed the described mutations on the phylogenetic context, which highlighted the recurrent problem of data quality. Therefore, the most important rules for adequately assessing the quality of mitochondrial DNA sequence analysis in cancer have been summarized. As follows from this review, neither the reliable spectrum of mtDNA somatic mutations nor the association between hereditary mutations and colorectal cancer risk have been resolved. This indicates that only high resolution studies on mtDNA variability, followed by a proper data interpretation employing phylogenetic knowledge may finally verify the utility of mtDNA sequence (if any) in clinical practice.     

The landscape of mitochondrial DNA variation in human colorectal cancer on the background of phylogenetic knowledge

Recently, an increasing number of studies indicate that mutations in mitochondrial genome may contribute to cancer development or metastasis. Hence, it is important to determine whether the mitochondrial DNA might be a good, clinically applicable marker of cancer. This review describes hereditary as well as somatic mutations reported in mitochondrial DNA of colorectal cancer cells. We showed here that the entire mitochondrial genome mutational spectra are different in colorectal cancer and non-tumor cells. We also placed the described mutations on the phylogenetic context, which highlighted the recurrent problem of data quality. Therefore, the most important rules for adequately assessing the quality of mitochondrial DNA sequence analysis in cancer have been summarized. As follows from this review, neither the reliable spectrum of mtDNA somatic mutations nor the association between hereditary mutations and colorectal cancer risk have been resolved. This indicates that only high resolution studies on mtDNA variability, followed by a proper data interpretation employing phylogenetic knowledge may finally verify the utility of mtDNA sequence (if any) in clinical practice.     

Mitochondrial DNA evidence for the spread of Pacific rats through Oceania

In the 10 years since we published our first full analysis of mitochondrial DNA (mtDNA) variation in Rattus exulans as a means for tracking human migration in Polynesia, we have extended the commensal approach through time and space with the use of ancient DNA (aDNA) and by analysing samples from across the Pacific. Not only can mtDNA phylogenies provide information regarding population origins and paths of migration, they have also provided information regarding degrees of contact and interaction between islands. An important extension of the R. exulans project is the creation and on-going development of a genetic database for the identification of Rattus species based on mtDNA sequences. The phylogenetic analysis of sequences from 18 species and 1 subspecies of Rattus thus far have raised some questions regarding species identification and species distributions in the Pacific.     

Low "penetrance" of phylogenetic knowledge in mitochondrial disease studies

An up-to-date view of the worldwide mitochondrial DNA (mtDNA) phylogeny together with an evaluation of the conservation of each site is a reliable tool for detecting errors in mtDNA studies and assessing the functional importance of alleged pathogenic mutations. However, most of the published studies on mitochondrial diseases make very little use of the phylogenetic knowledge that is currently available. This drawback has two inadvertent consequences: first, there is no sufficient a posteriori quality assessment of complete mtDNA sequencing efforts; and second, no feedback is provided for the general mtDNA database when apparently new mtDNA lineages are discovered. We demonstrate, by way of example, these issues by reanalysing three mtDNA sequencing attempts, two from Europe and another one from East Asia. To further validate our phylogenetic deductions, we completely sequenced two mtDNAs from healthy subjects that nearly match the mtDNAs of two patients, whose sequences gave problematic results.     

Mitochondrial phylogenies in the light of pseudogenes and Wolbachia: re-assessment of a bark beetle dataset

Phylogenetic studies based on mtDNA become increasingly questioned because of potential pitfalls due to mitochondrial pseudogenes and mitochondrial selective sweeps. While the inclusion of nuclear markers should preferentially be considered for future studies, there is no need to abandon mtDNA as long as tests for the known mtDNA artefacts are performed. In this study we presentadditionaldata and test previous phylogeographical studies of Pityogenes chalcographus. We did not detect nuclear copies (numts) of the previously used mitochondrial markers by performing a combined long range/nested PCR of the COI gene and by an in silico analysis of the COI sequence data. This confirms the robustness of our previous phylogenetic study of Pityogenes chalcographus. Results of an in-situ hybridization of Wolbachia in Pityogenes chalcographus confirm the presence of this endosysmbiont in this species. However, we did not detect a correlation between infection status, geographical region and mtDNA haplotypes. The hybridisation data also support a previous hypothesis that infections do not result from parasitoids or parasitic nematodes, insect surface or laboratory contaminations and are hence a true infection of Pityogenes chalcographus. We conclude that the deep structure found in mitochondrial populations of Pityogenes chalcographus indeed represents the evolutionary history of European populations.     

Genome skimming by shotgun sequencing helps resolve the phylogeny of a pantropical tree family

Whole genome sequencing is helping generate robust phylogenetic hypotheses for a range of taxonomic groups that were previously recalcitrant to classical molecular phylogenetic approaches. As a case study, we performed a shallow shotgun sequencing of eight species in the tropical tree family Chrysobalanaceae to retrieve large fragments of high‐copy number DNA regions and test the potential of these regions for phylogeny reconstruction. We were able to assemble the nuclear ribosomal cluster (nrDNA), the complete plastid genome (ptDNA) and a large fraction of the mitochondrial genome (mtDNA) with approximately 1000×, 450× and 120× sequencing depth respectively. The phylogenetic tree obtained with ptDNA resolved five of the seven internal nodes. In contrast, the tree obtained with mtDNA and nrDNA data were largely unresolved. This study demonstrates that genome skimming is a cost‐effective approach and shows potential in plant molecular systematics within Chrysobalanaceae and other under‐studied groups.     

High resolution analysis and phylogenetic network construction using complete mtDNA sequences in sardinian genetic isolates

For mitochondrial phylogenetic analysis, the best result comes from complete sequences. We therefore decided to sequence the entire mitochondrial DNA (mtDNA) (coding and D-loop regions) of 63 individuals selected in 3 small Ogliastra villages, an isolated area of eastern Sardinia: Talana, Urzulei, and Perdasdefogu. We studied at least one individual for each of the most frequent maternal genealogical lineages belonging to haplogroups H, V, J, K, T, U, and X. We found in our 63 samples, 172 and 69 sequence changes in the coding and in the D-loop region, respectively. Thirteen out of 172 sequence changes in the coding region are novel. It is our hypothesis that some of them are characteristic of the Ogliastra region and/or Sardinia. We reconstructed the phylogenetic network of the 63 complete mtDNA sequences for the 3 villages. We also drew a network including a large number of European sequences and calculated various indices of genetic diversity in Ogliastra. It appears that these small populations remained extremely isolated and genetically differentiated compared with other European populations. We also identified in our samples a never previously described subhaplogroup, U5b3, which seems peculiar to the Ogliastra region.     

Complete mitochondrial DNA of the hagfish, Eptatretus burgeri: the comparative analysis of mitochondrial DNA sequences strongly supports the cyclostome monophyly

The phylogenetic position of cyclostomes, i.e., the relationships between hagfishes, lampreys, and jawed vertebrates is an unresolved problem. Anatomical data support the paraphyly of cyclostomes, whereas nuclear genes data support monophyly of cyclostomes. Previous results obtained using mitochondrial DNA are ambiguous, presumably due to a lack of informative sequences. By adding the complete mtDNA of a hagfish, Eptatretus burgeri, we have generated a novel data set for sequences of hagfishes and of lampreys. The addition of this mtDNA sequence to the 12 taxa we have already used becomes sufficient to obtain unambiguous results. This data set, which includes sequences of mtDNA of animals closely related to the lamprey/hagfish node, was used in a phylogenetic analysis with two independent statistical approaches and unequivocally supported the monophyly of cyclostomes. Thus molecular data, i.e., our results and those obtained using nuclear genes, conclude that hagfishes and lampreys form a clade.     

Phylogeny and temporal diversification of darters (Percidae: Etheostomatinae)

Discussions aimed at resolution of the Tree of Life are most often focused on the interrelationships of major organismal lineages. In this study, we focus on the resolution of some of the most apical branches in the Tree of Life through exploration of the phylogenetic relationships of darters, a species-rich clade of North American freshwater fishes. With a near-complete taxon sampling of close to 250 species, we aim to investigate strategies for efficient multilocus data sampling and the estimation of divergence times using relaxed-clock methods when a clade lacks a fossil record. Our phylogenetic data set comprises a single mitochondrial DNA (mtDNA) gene and two nuclear genes sampled from 245 of the 248 darter species. This dense sampling allows us to determine if a modest amount of nuclear DNA sequence data can resolve relationships among closely related animal species. Darters lack a fossil record to provide age calibration priors in relaxed-clock analyses. Therefore, we use a near-complete species-sampled phylogeny of the perciform clade Centrarchidae, which has a rich fossil record, to assess two distinct strategies of external calibration in relaxed-clock divergence time estimates of darters: using ages inferred from the fossil record and molecular evolutionary rate estimates. Comparison of Bayesian phylogenies inferred from mtDNA and nuclear genes reveals that heterospecific mtDNA is present in approximately 12.5% of all darter species. We identify three patterns of mtDNA introgression in darters: proximal mtDNA transfer, which involves the transfer of mtDNA among extant and sympatric darter species, indeterminate introgression, which involves the transfer of mtDNA from a lineage that cannot be confidently identified because the introgressed haplotypes are not clearly referable to mtDNA haplotypes in any recognized species, and deep introgression, which is characterized by species diversification within a recipient clade subsequent to the transfer of heterospecific mtDNA. The results of our analyses indicate that DNA sequences sampled from single-copy nuclear genes can provide appreciable phylogenetic resolution for closely related animal species. A well-resolved near-complete species-sampled phylogeny of darters was estimated with Bayesian methods using a concatenated mtDNA and nuclear gene data set with all identified heterospecific mtDNA haplotypes treated as missing data. The relaxed-clock analyses resulted in very similar posterior age estimates across the three sampled genes and methods of calibration and therefore offer a viable strategy for estimating divergence times for clades that lack a fossil record. In addition, an informative rank-free clade-based classification of darters that preserves the rich history of nomenclature in the group and provides formal taxonomic communication of darter clades was constructed using the mtDNA and nuclear gene phylogeny. On the whole, the appeal of mtDNA for phylogeny inference among closely related animal species is diminished by the observations of extensive mtDNA introgression and by finding appreciable phylogenetic signal in a modest sampling of nuclear genes in our phylogenetic analyses of darters.     

Deep genetic subdivision within a continuously distributed and highly vagile marine mammal, the Steller's sea lion (Eumetopias jubatus)

The Steller's sea lion Eumetopias jubatus is an endangered marine mammal that has experienced dramatic population declines over much of its range during the past five decades. Studies using mitochondrial DNA (mtDNA) have shown that an apparently continuous population includes a strong division, yielding two discrete stocks, western and eastern. Based on a weaker split within the western stock, a third Asian stock has also been defined. While these findings indicate strong female philopatry, a recent study using nuclear microsatellite markers found little evidence of any genetic structure, implying extensive paternal gene flow. However, this result was at odds with mark–recapture data, and both sample sizes and genetic resolution were limited. To address these concerns, we increased analytical power by genotyping over 700 individuals from across the species' range at 13 highly polymorphic microsatellite loci. We found a clear phylogenetic break between populations of the eastern stock and those of the western and Asian stocks. However, our data provide little support for the classification of a separate Asian stock. Our findings show that mtDNA structuring is not due simply to female philopatry, but instead reflects a genuine discontinuity within the range, with implications for both the phylogeography and conservation of this important marine mammal.     

Forty Million Years of Independent Evolution: A Mitochondrial Gene and Its Corresponding Nuclear Pseudogene in Primates

Sequences from nuclear mitochondrial pseudogenes (numts) that originated by transfer of genetic information from mitochondria to the nucleus offer a unique opportunity to compare different regimes of molecular evolution. Analyzing a 1621-nt-long numt of the rRNA specifying mitochondrial DNA residing on human chromosome 3 and its corresponding mitochondrial gene in 18 anthropoid primates, we were able to retrace about 40 MY of primate rDNA evolutionary history. The results illustrate strengths and weaknesses of mtDNA data sets in reconstructing and dating the phylogenetic history of primates. We were able to show the following. In contrast to numt-DNA, (1) the nucleotide composition of mtDNA changed dramatically in the different primate lineages. This is assumed to lead to significant misinterpretations of the mitochondrial evolutionary history. (2) Due to the nucleotide compositional plasticity of primate mtDNA, the phylogenetic reconstruction combining mitochondrial and nuclear sequences is unlikely to yield reliable information for either tree topologies or branch lengths. This is because a major part of the underlying sequence evolution model — the nucleotide composition — is undergoing dramatic change in different mitochondrial lineages. We propose that this problem is also expressed in the occasional unexpected long branches leading to the “common ancestor” of orthologous numt sequences of different primate taxa. (3) The heterogeneous and lineage-specific evolution of mitochondrial sequences in primates renders molecular dating based on primate mtDNA problematic, whereas the numt sequences provide a much more reliable base for dating.[Reviewing Editor: Dr. Rafael Zardoya]     

Phylogeny of east Asian mitochondrial DNA lineages inferred from complete sequences

The now-emerging mitochondrial DNA (mtDNA) population genomics provides information for reconstructing a well-resolved mtDNA phylogeny and for discerning the phylogenetic status of the subcontinentally specific haplogroups. Although several major East Asian mtDNA haplogroups have been identified in studies elsewhere, some of the most basal haplogroups, as well as numerous minor subhaplogroups, were not yet determined or fully characterized. To fill the lacunae, we selected 48 mtDNAs from >2,000 samples across China for complete sequencing that cover virtually all (sub)haplogroups discernible to date in East Asia. This East Asian mtDNA phylogeny can henceforth serve as a solid basis for phylogeographic analyses of mtDNAs, as well as for studies of mitochondrial diseases in East and Southeast Asia.     

Monophyly of clade III nematodes is not supported by phylogenetic analysis of complete mitochondrial genome sequences

Background

The orders Ascaridida, Oxyurida, and Spirurida represent major components of zooparasitic nematode diversity, including many species of veterinary and medical importance. Phylum-wide nematode phylogenetic hypotheses have mainly been based on nuclear rDNA sequences, but more recently complete mitochondrial (mtDNA) gene sequences have provided another source of molecular information to evaluate relationships. Although there is much agreement between nuclear rDNA and mtDNA phylogenies, relationships among certain major clades are different. In this study we report that mtDNA sequences do not support the monophyly of Ascaridida, Oxyurida and Spirurida (clade III) in contrast to results for nuclear rDNA. Results from mtDNA genomes show promise as an additional independently evolving genome for developing phylogenetic hypotheses for nematodes, although substantially increased taxon sampling is needed for enhanced comparative value with nuclear rDNA. Ultimately, topological incongruence (and congruence) between nuclear rDNA and mtDNA phylogenetic hypotheses will need to be tested relative to additional independent loci that provide appropriate levels of resolution.

Results

For this comparative phylogenetic study, we determined the complete mitochondrial genome sequences of three nematode species, Cucullanus robustus (13,972 bp) representing Ascaridida, Wellcomia siamensis (14,128 bp) representing Oxyurida, and Heliconema longissimum (13,610 bp) representing Spirurida. These new sequences were used along with 33 published nematode mitochondrial genomes to investigate phylogenetic relationships among chromadorean orders. Phylogenetic analyses of both nucleotide and amino acid sequence datasets support the hypothesis that Ascaridida is nested within Rhabditida. The position of Oxyurida within Chromadorea varies among analyses; in most analyses this order is sister to the Ascaridida plus Rhabditida clade, with representative Spirurida forming a distinct clade, however, in one case Oxyurida is sister to Spirurida. Ascaridida, Oxyurida, and Spirurida (the sampled clade III taxa) do not form a monophyletic group based on complete mitochondrial DNA sequences. Tree topology tests revealed that constraining clade III taxa to be monophyletic, given the mtDNA datasets analyzed, was a significantly worse result.

Conclusion

The phylogenetic hypotheses from comparative analysis of the complete mitochondrial genome data (analysis of nucleotide and amino acid datasets, and nucleotide data excluding 3^rd positions) indicates that nematodes representing Ascaridida, Oxyurida and Spirurida do not share an exclusive most recent common ancestor, in contrast to published results based on nuclear ribosomal DNA. Overall, mtDNA genome data provides reliable support for nematode relationships that often corroborates findings based on nuclear rDNA. It is anticipated that additional taxonomic sampling will provide a wealth of information on mitochondrial genome evolution and sequence data for developing phylogenetic hypotheses for the phylum Nematoda.

     

Illumination of cryptic species boundaries in long-tailed shrew tenrecs (Mammalia: Tenrecidae; Microgale), with new insights into geographic variation and distributional constraints

The increasing use of mitochondrial DNA (mtDNA) to explore and test species limits among morphologically similar species is potentially compromised by phenomena poorly reflective of organismal history and speciation, including (but not limited to) stochastic lineage sorting and gene flow. In situations where molecular data are only available from a single gene or linkage partition (e.g. mtDNA), corroboration of suspected species boundaries should be sought from independent lines of evidence, such as morphology. Recent attempts to delimit species using mtDNA and morphology have either implicitly or explicitly ignored the possibility that distinct species can occur in direct sympatry throughout much of their range, presumably because such situations are believed to be rare. We examined phylogenetic relationships within the long‐tailed shrew tenrecs (Mammalia: Tenrecidae; Microgale spp.) from Madagascar. Current taxonomy recognizes two broadly sympatric species, though as many as six have been described. Given that alpha taxonomy within shrew tenrecs has been controversial, and that patterns of morphological variation can be especially difficult to assess for this group, some authors have suggested that additional cryptic species may exist. To examine this possibility, we conducted a phylogenetic study using the mitochondrial NADH dehydrogenase subunit 2 gene and a morphometric analysis of 29 craniodental, postcranial, and external measurements from a broad geographical sample of long‐tailed shrew tenrecs. The two data sets were nearly perfectly congruent in identifying four groups that can be classified as species, thereby doubling the currently recognized number of species. We present previously unrecognized distributional evidence consistent with our conclusions and provide an empirical example of how a revised understanding of species limits alters inferences of geographic variation and species coexistence, particularly with respect to fine‐scale habitat partitioning. The results of this study suggest that certain species pairs, previously assumed to be single species occupying broad elevational ranges, are actually reproductively isolated units that are partitioning their environment along elevational lines. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 1–22.