Northern richness and southern poverty: contrasting genetic footprints of glacial refugia in the relictual tree Sciadopitys verticillata (Coniferales: Sciadopityaceae)

Sciadopitys verticillata is amongst the most relictual of all plants, being the last living member of an ancient conifer lineage, the Sciadopityaceae, and is distributed in small and disjunct populations in high rainfall regions of Japan. Although mega‐fossils indicate the persistence of the species within Japan through the Pleistocene glacial–interglacial cycles, how the species withstood the colder and drier climates of the glacials is not well known. The present study utilized phylogeography and palaeodistribution modelling to test whether the species survived within pollen‐based coastal temperate forest glacial refugia or within previously unidentified refugia close to its current range. Sixteen chloroplast haplotypes were found that displayed significant geographical structuring. Unexpectedly, northern populations in central Honshu most distant from coastal refugia had the highest chloroplast diversity and were differentiated from the south, a legacy of glacial populations possibly in inland river valleys close to its current northern range. By contrast, populations near putative coastal refugia in southern Japan, harboured the lower chloroplast diversity and were dominated by a single haplotype. Fragment size polymorphism at a highly variable and homoplasious mononucleotide repeat region in the trnT‐trnL intergenic spacer reinforced the contrasting patterns of diversity observed between northern and southern populations. The divergent histories of northern and southern populations revealed in the present study will inform the management of this globally significant conifer. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 108 , 263–277.     

Phylogeography of the Japanese giant flying squirrel, Petaurista leucogenys (Rodentia: Sciuridae): implication of glacial refugia in an arboreal small mammal in the Japanese Islands

To test the association between temperate forest dynamics and glacial refugia for arboreal small mammals, we studied the phylogeography of the Japanese giant flying squirrel ( Petaurista leucogenys ) using complete mitochondrial cytochrome b gene sequences (1140 bp). This squirrel is endemic to three of Japan's main islands: Honshu, Shikoku, and Kyushu. We examined 58 specimens of P. leucogenys collected from 40 localities in Japan. Additionally, two individuals with unknown sampling localities were included in phylogenetic analyses. There were 54 haplotypes of P. leucogenys. We found five major phylogroups (Northern, Central, South-eastern, South-western, and Southern). These phylogroups may have originated from glacial refugia during the Late Pleistocene. After the last glaciation, the Northern phylogroup, widely distributed in eastern Japan, could have extensively expanded northward from its refugia. By contrast, in western Japan, population expansion was restricted to western Japan. All members of four phylogroups existed in western Japan during glaciations. The complicated phylogeographical pattern of P. leucogenys populations originating from western Japan may have resulted from the long history.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 47–60.     

Phylogeography and genetic structure of disjunct Salix arbutifolia populations in Japan

Disjunct geographic distributions of boreal plant species at the southern edges of their ranges are expected to result in low genetic diversity and high genetic differentiation in the disjunct populations. This prediction was tested in a riparian willow, Salix arbutifolia, distributed in the northeastern Eurasian continent and the Sakhalin, Hokkaido, and Honshu Islands, using chloroplast DNA haplotypes and nuclear microsatellite genotypes. Hokkaido and Honshu populations shared a chloroplast haplotype identical to a closely related species, S. cardiophylla. This haplotype was divergent from haplotypes in the Eurasian continent (Primorsky) and the Sakhalin Island. In the nuclear microsatellites, most Hokkaido populations were genetically closer to Primorsky populations than to Sakhalin populations in spite of the geographical vicinity between Sakhalin and Hokkaido. The unexpected genetic divergence between Sakhalin and Hokkaido implies a complicated history of migration and colonization. The most peripheral populations in Honshu had the lowest genetic diversity and were most differentiated from the others. Thus, low genetic diversity and high genetic differentiation at the range periphery suggest substantial effects of genetic drift on genetic structure in the disjunct populations of Salix arbutifolia at the southern edge of its range.     

Glacial history of high alpine Ranunculus glacialis (Ranunculaceae) in the European Alps in a comparative phylogeographical context

There is considerable controversy concerning the fate of Alpine plants during Pleistocene glaciations. While some studies have found evidence for nunatak survival, others have explained the present genetic patterns by survival only in peripheral refugia. We investigated 75 populations of high alpine Ranunculus glacialis from its entire Alpine distribution. Phylogeographical analyses of AFLP data revealed four groups of populations. Two of them, located in the western Alps, were genetically isolated from each other and from the eastern groups, whereas the two eastern Alpine groups were genetically more similar to each other. This suggests longer isolation and/or lower levels of gene flow in the two western groups. As all groups are close to, or overlap with, presumed glacial refugia, invoking glacial survival on nunataks is unnecessary to explain the present genetic pattern. Similar to the phylogeographical patterns of R. glacialis , the previously investigated alpine Phyteuma globulariifolium and Androsace alpina , which are also confined to siliceous bedrock, showed strong geographical affinities to peripheral refugial areas and there were large-scale congruencies in the location of these refugia for all three species.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 183–195.     

Distinct geographical structure across species units evidenced by chloroplast DNA haplotypes and nuclear ribosomal ITS genotypes of Corylopsis (Hamamelidaceae) in the Japanese islands

The geographical distribution of chloroplast DNA (cpDNA) haplotypes and nuclear ribosomal internal transcribed spacer (nrITS) genotypes of Japanese Corylopsis (Hamamelidaceae), which consists of four species, was investigated. Two hundred and five individuals belonging to four species from 30 populations, covering the entire geographical range, were studied. Based on approximately 1108 bp of the three non-coding regions of cpDNA, nine haplotypes were detected, and each was distinguished from adjacent haplotypes by one substitution. Based on approximately 507-bp nrITS sequences, 47 genotypes were detected, for which three clades were identified in the phylogenetic analysis. There was inconsistency between the cpDNA haplotypes, nrITS genotypes, and classification of Corylopsis taxa, possibly because of incomplete lineage sorting or introgressive hybridization. The distribution of the haplotypes was highly structured geographically, and N _ST (0.893) was significantly greater than G _ST (0.819), implying that the current distribution of Corylopsis species was structured phylogeographically during Quaternary climatic oscillations. The haplotype composition and results of analysis of molecular variance showed that the populations in Hokuriku were highly divergent, suggesting that they are long-term persistent populations arising from refugia during the Quaternary climatic oscillations. Refugial populations in Chugoku and Shikoku may have lost genetic diversity because of a bottleneck resulting from a small population size, followed by post-glacial range expansion. Pre-existing refugia may have been so small that the subsequent range expansion replaced the pre-existing genetic structure.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 501–518.     

Contrasting patterns of nuclear microsatellite genetic structure of Fraxinus mandshurica var. japonica between northern and southern populations in Japan

Aim The aim of this study is to detect extant patterns of population genetic structure of Fraxinus mandshurica var. japonica in Japan, and to provide insights into the post‐glacial history of this species during the Holocene. Location Hokkaido and Honshu islands, Japan (including the Oshima and Shimokita peninsulas). Methods We examined nine polymorphic nuclear microsatellite loci to assess genetic variation within and among 15 populations across almost the entire range of the species in Japan. Extant patterns of geographical structure were analysed using Bayesian clustering, Monmonier’s algorithm, analysis of molecular variance, Mantel tests and principal coordinates analysis. Recent bottlenecks within populations and regional genetic variation were also assessed. Results Northern populations (Hokkaido Island and the Shimokita Peninsula) formed a single homogeneous deme, maintaining the highest level of allelic diversity on the Oshima Peninsula. By contrast, southern populations (Honshu Island) demonstrated strong substructure on both coasts. Specifically, populations on the Pacific side of Honshu exhibited significant bottlenecks and erosion of allelic diversity but preserved distinct subclusters diverging from widespread subclusters on the Japan Sea side of this island. Main conclusions Genetic evidence and life history traits suggest that F. mandshurica occupied cryptic northern refugia on the Oshima Peninsula during the Last Glacial Maximum, which is reflected in the species’ extant northern distribution. Strong geographical structure in southern populations, in agreement with fossil pollen records, suggests geographical isolation by mountain ranges running north–south along Honshu. Given that this tree species is cold‐adapted and found in riparian habitats, populations on the Pacific side of Honshu probably contracted into higher‐elevation swamps during warm post‐glacial periods, leading to a reduction of effective population sizes and rare allelic richness.     

Glacial history of Saxifraga paniculata (Saxifragaceae): molecular biogeography of a disjunct arctic-alpine species from Europe and North America

The molecular biogeography of the disjunctly distributed and morphologically highly variable species Saxifraga paniculata Mill. was analysed using amplified fragment length polymorphism (AFLP) and chloroplast microsatellites. The study comprised 77 samples from mountain regions in Europe and North America throughout the complete range of distribution. AFLP data revealed clear genetic differentiation between samples from the Arctic, the Caucasus, and the eastern European mountains. Samples from the Alps were divided into two groups. One group clustered with the samples from central Europe and the Pyrenees, whereas another group with individuals from southern Norway. AFLP diversity was lowest in the Arctic and highest in the Alps. Chloroplast microsatellite analysis revealed eight haplotypes but no unequivocal phylogeographical pattern. However, haplotype diversity was highest in the Alps and central Europe whereas, in the Arctic, only few widespread haplotypes could be found. The results indicate in situ survival of S. paniculata in the Caucasus, the eastern European mountains, and the Alps. The Arctic has presumably been colonized postglacially from North American refugia south of the ice shield. Southern Norway and the Pyrenees have most likely been colonized from two phylogeographically different groups in the Alps. The origin of the central European samples remains ambiguous. In situ survival seems to be as possible as several postglacial recolonization events from the Alps. The obtained molecular data clearly support the subdivision of S. paniculata into three subspecies: ssp. cartilaginea from the Caucasus, ssp. laestadii from northern Norway, Iceland, and North America, and ssp. paniculata from the other geographical regions.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 385–398.     

Risk of predation as a promoting factor of species divergence in threespine sticklebacks (Gasterosteus aculeatus L.)

Icelandic freshwater systems are geologically young and contain only six species of freshwater fish. As these species colonized Icelandic fresh waters they were presented with a diversity of unique, uncontested habitats and food resources, promoting the evolution of new behaviour strategies crucial to the formation of new morphs and speciation. To determine the likelihood that predation threat could affect the antipredator behaviour and possibly the sympatric divergence of prey populations, we analysed antipredator behaviour of seven groups of Icelandic threespine sticklebacks ( Gasterosteus aculeatus ): two marine groups, one group from a lake without piscine predators, and two polymorphic lake populations, each with two groups occupying unique habitats. Shoaling cohesion, school formation and duration, and vigilance in predator inspection/avoidance behaviour varied greatly among groups. The differences appeared to be related to the risk of predation as well as to opportunities and constraints set by the different habitats. Antipredator behaviour was especially pronounced and differed extensively in two polymorphic forms from the lake Thingvallavatn, where predation risk is very high. By keeping the two morphs separate in their respective habitats, high predation risk may be a contributing factor in promoting the habitat-specific divergence of G. aculeatus seen in the lake. This suggests that in situations where refuge habitats are spatially separated, the risk of predation may contribute to the evolution of separate sympatric forms of small fish such as G. aculeatus .  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 189–203.     

Population genetics of the black ant Formica lemani (Hymenoptera: Formicidae)

Colony kin structure and spatial population structure were studied in multiple populations of the ant Formica lemani , using allozymes and DNA microsatellites. Average genetic relatedness between nestmate workers varied little between populations ( r  = 0.51–0.76), indicating that the average colony kin structure was relatively simple. Worker genotypes could not be explained with a single breeding pair in all nests, however, and the distribution of relatedness estimates across nests was bimodal, suggesting that single- and multi-queen colonies co-occur. We studied spatial population structure in a successional boreal forest system, which is a mixture of different aged habitats. Newly clear-cut open habitat patches are quickly colonized by F. lemani , where it is able to persist for a limited number of generations. Newly-founded populations showed signs of a founder effect and spatial substructuring, whereas older populations were more homogenous. This suggests that new populations are founded by a limited number of colonizers arriving from more than one source. Genetic differentiation among local populations was minor, indicating strong migration between them. There were, however, indications of both isolation by distance and populations becoming more isolated as habitat patches grew older.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 247–258.     

Chloroplast DNA variation in the shrub Justicia areysiana (Acanthaceae) endemic to the monsoon affected coastal mountains of the southern Arabian Peninsula

The monsoon affected mountains of the southern Arabian Peninsula harbour in climatically favoured refugia vegetation elements of palaeo-African origin. To understand better the temporal and spatial differentiation of these refugia, chloroplast variation in Justicia areysiana Deflers (Acanthaceae), a shrub species endemic to the Yemeni and Omani mountains close to the Arabian Sea, was studied using PCR-RFLP and chloroplast microsatellite diversity. Eleven haplotypes were characterized and show a distinct geographical distribution pattern with a deep split between populations from south Yemeni fog oases and those from Hawf Mountains/Dhofar region in east Yemen and south Oman. Very limited haplotype diversity within populations (h_S = 0.15) and a high level of population differentiation (G_ST = 0.81) demonstrate the strong genetic isolation of populations from each other. Past oscillations between humid and arid periods connected with glacial and interglacial episodes in the Pleistocene and Holocene are considered responsible for the observed patterns of genetic variation.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 148 , 437–444.     

Geographic allozyme variation in the Japanese clouded salamander, Hynobius nebulosus (Amphibia: Urodela)

Using starch gel electrophoresis, geographical enzyme variation was surveyed in 46 populations of the Japanese salamander Hynobius nebulosus , which occurs widely in western Japan. This species exhibits substantial local genetic differentiation and is more diverse in inland regions than on small islands. In several different analyses, two groups of populations, one from Kyushu Island to the westernmost part of Chugoku district, Honshu (western group), and another from Shikoku through Kinki to Chubu district, Honshu (eastern group), were consistently recognized. Most of the remaining populations were placed in two less clearly-defined groups: the montane group from the Chugoku Mountains and the Chugoku group from the coastal regions of Chugoku district. The divergence patterns of H. nebulosus are thought to be related to the geological history of its range. The populations near the boundary of the western group were found to share several alleles that were predominant on both sides of the boundary, indicating past secondary contact. Differentiation in the montane group did not follow the isolation by distance model, probably because several populations included were genetically mixed with lowland populations of the Chugoku group.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 311–330.     

Spartina anglica C. E. Hubbard: a natural model system for analysing early evolutionary changes that affect allopolyploid genomes

Spartina anglica arose during the end of the 19th century in England by hybridization between the indigenous Spartina maritima and the introduced East American Spartina alterniflora and following genome duplication of the hybrid ( S.  ×  townsendii ). This system allows investigations of the early evolutionary changes that accompany stabilization of a new allopolyploid species in natural populations. Various molecular data indicate that S. anglica has resulted from a unique parental genotype. This young species contains two distinctly divergent homoeologous genomes that have not undergone extensive change since their reunion. No burst of retroelements has been encountered in the F₁ hybrid or in the allopolyploid, suggesting a 'structural genomic stasis' rather than 'rapid genomic changes'. However, modifications of the methylation patterns in the genomes of S.  ×  townsendii and S. anglica indicate that in this system, epigenetic changes have followed both hybridization and polyploidization.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 475–484.     

Habitat-associated morphological divergence in two Neotropical fish species

We examined intraspecific morphological diversification between river channel and lagoon habitats for two Neotropical fish ( Bryconops caudomaculatus , Characidae; Biotodoma wavrini , Cichlidae). We hypothesized that differences between habitats (e.g. flow regime, foraging opportunities) might create selective pressures resulting in morphological divergence between conspecific populations. We collected fish from four channel-lagoon habitat pairs in the Río Cinaruco, Venezuela, and compared body morphology using geometric morphometrics. There were two aspects of divergence in both species: (1) placement of maximum body depth and (2) orientation of the mouth. For both species, maximum body depth was positioned more anteriorly (i.e. fusiform) in the river channel than in lagoons. Both species exhibited a relatively terminal mouth in lagoons compared to the channel. The mouth of B. caudomaculatus was relatively upturned, whereas the mouth of B. wavrini was relatively subterminal, in channel habitats. Observed morphological patterns are consistent with functional morphological principles suggesting adaptive divergence. We also show that spatial distance between habitats, presumably reflecting rates of population mixing, appears to have constrained diversification. For both species, morphological divergence increased with distance between habitats. Thus morphological divergence between channel and lagoon habitats apparently reflects a balance between diversification driven by natural selection, and homogenization driven by population mixing.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 689–698.     

Cryptic speciation in liverworts – a case study in the Aneura pinguis complex

Bryophytes are amongst the most ancestral terrestrial plants and often have large distribution ranges across continents. Recent biochemical and molecular studies have suggested that many worldwide morphological species of bryophyte may represent genetically divergent and reproductively isolated cryptic species. We tested the cryptic species hypothesis in the thalloid liverwort Aneura pinguis complex. We applied analyses of chloroplast DNA (cpDNA) sequence variation and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods to discriminate between specimens of A. pinguis derived from various habitats in five distant geographical regions in Poland. Of the 19 specimens sequence characterized for the cpDNA tRNA^Leu region, seven haplotypes were identified divided into three nonmonophyletic clusters. The application of developed PCR-RFLP markers confirmed the existence of three tRNA^Leu types of A. pinguis (A–C) within the specimens derived from 21 populations. Sympatric populations of different tRNA^Leu types were found in lowland and mountain regions. No clear correlation between stand type and the presence of two tRNA^Leu types (A, B) was observed, as both were growing on soil, humus, and rocks. The tRNA^Leu type C was found only on humus and its distribution was restricted to low-lying northern populations. The above results indicate that the A. pinguis complex is highly differentiated at the molecular level and may represent three cryptic species.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 273–282.     

Implications of contrasting patterns of genetic variability in two vespertilionid bats from the Indonesian archipelago

Wallacean island populations of two Vespertilioninae bats, Myotis muricola and Scotophilus kuhlii , which have similar geographical ranges, showed marked contrast in the amount and pattern of genetic variation. Within islands, genetic variation was on average much higher in M. muricola but declined from west to east, whereas all populations of S. kuhlii had uniformly low levels of genetic variation by mammalian standards. S. kuhlii showed little genetic differentiation between islands and estimates of gene flow were substantial whereas island populations of M. muricola differed markedly and there was a strong isolation-by-distance effect associated with the extent of the sea crossing between islands. Furthermore, the lower mean heterozygosity and small genetic distances between eastern island populations of M. muricola is evidence that there has been a bottleneck associated with the colonization of this area. The attenuation of genetic diversity to the east is also seen in some other mammalian species and may indicate limits to dispersal and have implications for species management. The patterns of variability in S. kuhlii may be a consequence of its strong dispersal capacity and close association with human activity, which, together with other factors, suggest a panmictic population.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 421–431.     

Mitochondrial introgression and replacement between yellowhammers (Emberiza citrinella) and pine buntings (Emberiza leucocephalos) (Aves: Passeriformes)

In studies of phylogeography and taxonomy, strong emphasis is usually placed on the study of mitochondrial (mt)DNA. In the present study, we present a remarkable case in which highly phenotypically divergent species have almost no divergence in mtDNA. Yellowhammers ( Emberiza citrinella Linnaeus) and pine buntings ( Emberiza leucocephalos S. G. Gmelin) differ noticeably in appearance and song but hybridize in some areas of contact. They share a variety of closely-related mtDNA haplotypes, with little divergence in frequencies, indicating a mitochondrial divergence time sometime during or after the last major glacial period. By contrast, nuclear DNA (amplified fragment length polymorphism markers and CHD1Z gene sequences) differs more strongly between the species, and these differences can be used to identify intermediate genetic signatures of hybrids. The combined amount of mitochondrial diversity within yellowhammers and pine buntings is very low compared to other Emberiza species pairs, whereas the level of variation at the nuclear gene CHD1Z is comparable to that within other species pairs. Although it is difficult to completely reject the possibility that the two species split extremely recently and experienced rapid nuclear and phenotypic differentiation, we argue that the evidence better supports another possibility: the two species are older and mtDNA has recently introgressed between them, most likely as a result of a selective sweep. Mismatches between mitochondrial and nuclear phylogeographic patterns may occur more commonly than previously considered, and could have important implications for the fields of phylogeography and taxonomy.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 422–438.     

Comparative analysis of biological and ecological differentiation of Anemone palmata L. (Ranunculaceae) in the western Mediterranean (France and Spain): an assessment of rarity and population persistence

The western Mediterranean geophyte Anemone palmata L. is one of the most endangered angiosperm taxa in France. The biological and ecological characteristics of French populations are contrasted with those of populations from near the centre of the species distribution in east Spain. A Correspondence Analysis discriminates the French and Spanish populations according to substrate and the composition of the plant communities where they grow. The karyological study reveals that east Spanish plants are autotetraploid, whilst the French are all diploid. In addition, morphological differences were registered, Spanish plants generally being more vigorous. French populations consist mostly of senile plants, without or with very low rates of sexual reproduction, whereas the populations from Spain include all age groups with an important percentage of juvenile plants. The main factors which could affect the French populations are habitat destruction and modification of plant communities due to land-abandonment. Conservation measures should be supported by immediate protection of the French localities and habitats to prevent the total decline of this rare species.  © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 140 , 95–114.     

Phylogenetic relationships based on morphology among the Diospyros (Ebenaceae) species endemic to the Mascarene Islands

The Diospyros (Ebenaceae) species which colonized the Mascarene Islands, namely Mauritius, Reunion and Rodrigues, have been decimated over the years by human settlements. Of the 14 endemic species that have been described and collected for herbaria, Diospyros angulata is now believed to be extinct in Mauritius. The phylogenetic relationships of the 14 Diospyros species were determined using maximum parsimony analysis of 35 morphological characters. This analysis separated the Mascarene Diospyros into two major clades, with D. revaughanii , D. egrettarum and D. leucomelas grouped in the same strongly supported most basal clade while the rest of the species formed the other major clade. High bootstrap values were obtained for the sister species D. angulata and D. boutonania , and the clade clustering the upland species D. neraudii , D. nodosa and D. pterocalyx . There was also relatively strong support for the clade comprising D. hemiteles and D. melanida , which are located in mid altitude regions. These results indicate that Diospyros species most probably colonized the coastal areas of Mauritius and then moved to mid altitude habitats before finally reaching the upland regions. There are also strong indications that D. borbonica and D. diversifolia , endemic to Reunion and Rodrigues, respectively, resulted from migrations from Mauritius.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 307–313.     

Impact of ocean barriers, topography, and glaciation on the phylogeography of the catfish Trichomycterus areolatus (Teleostei: Trichomycteridae) in Chile

We examined the role of several earth history events on the phylogeographic distribution of the catfish Trichomycterus areolatus in Chile using the cytochrome b gene. We explored three biogeographic hypotheses: that sea level changes have resulted in the isolation of populations by drainages; that glaciation has impacted genetic diversity; and that ichthyological subprovince boundaries correspond to phylogeographic breaks in our focal species. We found seven well-supported clades within T. areolatus with high levels of genetic divergence. The strongest signal in our data was for an important role of sea level changes structuring populations. Five of the seven clades mapped cleanly to the geographic landscape and breaks corresponded closely to areas of narrowest continental shelf. In addition, few haplotypes were shared between rivers within clades, suggesting that only limited local movement of individuals has occurred. There was no relationship between the levels of genetic diversity and the proportion of individual drainages covered by glaciers during the last glacial maximum. Two phylogeographic breaks within T. areolatus did match the two previously identified faunal boundaries, but we found three additional breaks, which suggests that faunal breaks have only limited utility in explaining phylogeographic patterns. These results imply that the narrow continental shelf coupled with sea level changes had a strong influence on the obligate freshwater fishes in Chile.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 876–892.