Separating Population Structure from Population History: A Cladistic Analysis of the Geographical Distribution of Mitochondrial DNA Haplotypes in the Tiger Salamander, Ambystoma Tigrinum

Nonrandom associations of alleles or haplotypes with geographical location can arise from restricted gene flow, historical events (fragmentation, range expansion, colonization), or any mixture of these factors. In this paper, we show how a nested cladistic analysis of geographical distances can be used to test the null hypothesis of no geographical association of haplotypes, test the hypothesis that significant associations are due to restricted gene flow, and identify patterns of significant association that are due to historical events. In this last case, criteria are given to discriminate among contiguous range expansion, long-distance colonization, and population fragmentation. The ability to make these discriminations depends critically upon an adequate geographical sampling design. These points are illustrated with a worked example: mitochondrial DNA haplotypes in the salamander Ambystoma tigrinum. For this example, prior information exists about restricted gene flow and likely historical events, and the nested cladistic analyses were completely concordant with this prior information. This concordance establishes the plausibility of this nested cladistic approach, but much future work will be necessary to demonstrate robustness and to explore the power and accuracy of this procedure.     

The phylogeography and connectivity of the latitudinally widespread scleractinian coral Plesiastrea versipora in the Western Pacific

Whereas terrestrial animal populations might show genetic connectivity within a continent, marine species, such as hermatypic corals, may have connectivity stretching to all corners of the planet. We quantified the genetic variability within and among populations of the widespread scleractinian coral, Plesiastrea versipora along the eastern Australian seaboard (4145 km) and the Ryukyu Archipelago (Japan, 681 km) using sequences of internal transcribed spacers (ITS1-2) from ribosomal DNA. Geographic patterns in genetic variability were deduced from a nested clade analysis (NCA) performed on a parsimony network haplotype. This analysis allowed the establishment of geographical associations in the distribution of haplotypes within the network cladogram, therefore allowing us to deduce phylogeographical patterns based under models of restricted gene flow, fragmentation and range expansion. No significant structure was found among Ryukyu Archipelago populations. The lack of an association between the positions of haplotypes in the cladogram with geographical location of these populations may be accounted for by a high level of gene flow of P. versipora within this region, probably due to the strong Kuroshio Current. In contrast, strong geographical associations were apparent among populations of P. versipora along the south-east coast of Australia. This pattern of restricted genetic connectivity among populations of P. versipora on the eastern seaboard of Australia seems to be associated with the present surface ocean current (the East Australian Current) on this side of the south-western Pacific Ocean.     

Phylogeography of Mabuya maculilabris (Reptilia) from São Tomé Island (Gulf of Guinea) inferred from mtDNA sequences

The pattern of genetic variation of the lizard Mabuya maculilabris from S?o Tomé Island (Gulf of Guinea) was investigated using a combination of three mitochondrial DNA gene fragments. Forty-eight haplotypes were recovered among 66 individuals covering the whole island. The genealogy inferred from the most parsimonious network of haplotypes allows us to detect two main and long branches departing from the putative group of oldest haplotypes. The tips of these branches exhibit star-like phylogenies, which may indicate of recently expanded populations, most probably from a small number of founders. A nested clade analysis suggests a complex pattern of past events that gave rise to the extant geographical pattern found in the haplotype distribution: past and allopatric fragmentation, range expansion, restricted gene flow and long-distance dispersal. These results are consistent with the complex geological history of the island where important volcanic activity with extensive lava flows has occurred during several periods. Mismatch-distribution analysis and AMOVA also support these conclusions. Substantial genetic structuring among these lizards was detected as well as high levels of differentiation between the southern edge populations (particularly those from the Rolas Islet) and the remaining ones. However, variation is low relative to the geological age of the island. Our results indicate that patterns of variation observed in reptiles in other oceanic islands are not indicative of those observed in the islands of the Gulf of Guinea.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Population structure in the endangered cyprinid fish Pararasbora moltrechti in Taiwan, based on mitochondrial and microsatellite DNAs

The genetic structure of four populations of Pararasbora moltrechti, an endemic species of the Cyprinidae in Taiwan, was investigated based on the genetic variation of mtDNA Cyt b gene and five microsatellite loci. High haplotype diversity (h = 0.92) but low nucleotide diversity (0.004) in mtDNA was detected in this endangered species. In total, 33 haplotypes and four clusters were identified in its mtDNA. Nevertheless, low correspondence was found between geographical division and mtDNA clusters. In contrast, Bayesian cluster analysis of the microsatellite data identified four genetic groups and revealed highly structured populations. Significantly negative Tajima's D statistics and mismatch distribution analyses suggest that P. moltrechti populations may have experienced a demographic expansion. In light of the results of a nested clade analysis of mtDNA haplotypes, we conclude that recent population fluctuations and restricted gene flow played major roles in shaping the spatial genetic structure of P. moltrechti populations.     

Nested clade analyses of phylogeographic data: testing hypotheses about gene flow and population history

Since the 1920s, population geneticists have had measures that describe how genetic variation is distributed spatially within a species' geographical range. Modern genetic survey techniques frequently yield information on the evolutionary relationships among the alleles or haplotypes as well as information on allele frequencies and their spatial distributions. This evolutionary information is often expressed in the form of an estimated haplotype or allele tree. Traditional statistics of population structure, such as F statistics, do not make use of evolutionary genealogical information, so it is necessary to develop new statistical estimators and tests that explicitly incorporate information from the haplotype tree. One such technique is to use the haplotype tree to define a nested series of branches (clades), thereby allowing an evolutionary nested analysis of the spatial distribution of genetic variation. Such a nested analysis can be performed regarding the geographical sampling locations either as categorical or continuous variables (i.e. some measure of spatial distance). It is shown that such nested phylogeographical analyses have more power to detect geographical associations than traditional, nonhistorical analyses and, as a consequence, allow a broader range of gene-flow parameters to be estimated in a precise fashion. More importantly, such nested analyses can discriminate between phylogeographical associations due to recurrent but restricted gene flow vs. historical events operating at the population level (e.g. past fragmentation, colonization, or range expansion events). Restricted gene flow and historical events can be intertwined, and the cladistic analyses can reconstruct their temporal juxtapositions, thereby yielding great insight into both the evolutionary history and population structure of the species. Examples are given that illustrate these properties, concentrating on the detection of range expansion events.     

Phylogeography of southern Asian Dolly Varden char <Emphasis Type="Italic">Salvelinus malma krascheninnikovi</Emphasis>: Genealogical analysis of mitochondrial DNA

Phylogeography of southern Asian Dolly Varden char was studied using the data on mtDNA variation (regions ND1/ND2, ND5/ND6, and Cytb/D loop) obtained using PCR-RFLP analysis. Analysis of contemporary population genetic structure showed that Salvelinus malma krascheninnikovi throughout the whole species range was characterized by high population differentiation in combination with rather small differences between the populations from remote regions. The genealogy of mtDNA haplotypes was reconstructed and nested clade analysis of geographical distances was performed. Geographical distribution of mtDNA haplotypes of S. m. krascheninnikovi was explained by population genetic processes (restricted gene flow), as well as by historical demographic events (range expansion and fragmentation). It was demonstrated that the main demographic events were associated with cyclic processes of the geological formation of the Sea of Japan and adjacent territories. Furthermore, genealogical tree of S. m. krascheninnikovi contained the traces of secondary contact between isolated phylogeographical lineages.     

Phylogeography of the Vosges mountains populations of Gonioctena pallida (Coleoptera: Chrysomelidae): a nested clade analysis of mitochondrial DNA haplotypes

The pattern of genetic variation in the leaf beetle Gonioctena pallida was investigated inside the Vosges mountains using a highly variable 363 bp DNA fragment of the mitochondrial control region. Sequencing of 242 individuals, sampled in a geographical area of 100 x 40 km, identified 61 haplotypes whose genealogy was inferred. The resulting haplotype network exhibits four star-like phylogenies, two of which may be indicative of a population having recently expanded in size from a small number of founders. Nested clade analysis suggested multiple past expansion events, but also isolation by distance and possibly past fragmentation events, as the causes of the detected geographical associations of haplotypes. These results indicate the existence of effective barriers to gene flow inside the investigated area. Because the oldest demographic events inferred in the nested clade analysis were identified as expansion events, we hypothesize that a small population of founders have expanded not only in size, but also in geographical range from the south towards the north and east of the Vosges.     

Mitochondrial DNA signatures of restricted gene flow within divergent lineages of an atyid shrimp (Paratya australiensis)

We measured spatial genetic structure within three previously described mitochondrial lineages of the atyid shrimp, Paratya australiensis, occurring in upland streams of two major catchments within the Sydney Water Supply Catchment, New South Wales, Australia. In all three lineages, there was significant spatial structuring of genetic variation between catchments. In two lineages, recurrent but restricted maternal gene flow has apparently predominated in shaping within-catchment genetic structure, although this framework may be overlaid with episodic contiguous/long-distance expansion events. In the third lineage, there was no evidence of spatial genetic structuring within one of the catchments, because one haplotype was both common and widespread throughout the sampled area. High-frequency haplotypes were also shared among subcatchments in the other two lineages, and we discuss both historical and contemporary processes that may have left these genetic signatures. Our results are generally concordant with previous reports of significant population structuring in P. australiensis, occurring in upland river reaches elsewhere in eastern Australia. We propose that restricted dispersal and gene flow among upland populations of P. australiensis is linked to dramatic architectural structuring within and among mountain streams.     

Population structure and gene flow among wild populations of the black muntjac (Muntiacus crinifrons) based on mitochondrial DNA control region sequences

The black muntjac (Muntiacus crinifrons) is a rare species endemic to China and its current distribution is confined to partial mountain ranges in eastern China. To assess the population structure and gene flow among the extant populations, we sequenced a portion of the mitochondrial control region (424 bp) for 47 samples collected from its current three large populations (Huangshan, Tiammushan and Suichang). A total of 18 unique haplotypes were defined based on 22 polymorphic sites. Of these haplotypes, 15 ones were population-specific and only one haplotype was shared among the three populations. Significant genetic differentiation was detected between Suichang and Huangshan populations (?(ST)=0.1677, P<0.001) or between Suichang and Tianmushan populations (?(ST)=0.2002, P<0.001), indicating that the Suichang population may be spatially structured from other two populations along matriline. The Mantel test revealed that this significant differentiation was not driven by geographic distance (P=0.14), implying that genetic divergence of Suichang population might result from recent human disturbances. Phylogenetic analyses suggested the mitochondrial control region haplotypes were split into two well divergent clades (Clade I and Clade II). Interestingly, the two distinct haplotype clades were found to coexist in Suichang area. The nested clade analysis revealed a significant phylogeographic structure among the black muntjac populations (total cladogram: chi2=18.68; P<0.001), which was inferred to result from past fragmentation followed by range expansion. The population expansion was supported by the analysis of mismatch distribution and the tests of neutrality. Therefore, we suggest that the coexistence of distinct haplotypes in Suichang population was induced by historical population expansion after fragmentation and that the current genetic differentiation should be attributed to the reduction of female-mediated gene flow due to recent habitat fragmentation and subsequent loss.     

Phylogeography and monophyly of the swordtail fish species Xiphophorus birchmanni (Cyprinodontiformes, Poeciliidae)

We used sequences of the mitochondria control region to assess the distribution of genetic variation within and among populations of the poeciliid fish species Xiphophorus birchmanni . We collected 122 X. birchmanni samples from 11 sites in three drainage systems comprising the distribution of the species. We found low levels of polymorphism among aligned sequences and low levels of genetic variation within populations but high levels of genetic differentiation among populations. Haplotypes are exclusive to three river drainages (Los Hules, Calabozo and San Pedro). Mantel tests revealed correlations between geographical (both straight-line and river distances) and genetic distance, consistent with an isolation by distance scenario, while nested clade analysis suggested allopatric fragmentation between haplotypes from two of the major drainages, and isolation by distance with restricted gene flow within those drainages. Finally, monophyly of X. birchmanni is strongly supported while the previous hypothesis of the evolutionary origin of this species from X. malinche is not.     

Phylogeography of Lagochilus ilicifolius (Lamiaceae) in relation to Quaternary climatic oscillation and aridification in northern China

Using two chloroplast DNA intergenic spacers, a phylogeographical study of Lagochilus ilicifolius with 168 individuals from 14 populations was performed to assess geographical patterns and genetic variation in relation to past climate change. Population structure and history were inferred by nested clade analysis, neutrality tests, divergence time estimates, and mismatch distribution analysis. Twelve haplotypes were identified. Genetic differentiation among populations within groups was also elevated (F_SC = 0.772), suggesting restricted gene flow among populations within groups. Divergence time was at approximately 0.71 Ma, consistent with aridification and desert expansion during the middle Pleistocene. The Helan Mountains contained a large proportion of the haplotypes, which implies that the region may be the center of diversification for the species, whereas the Loess Plateau as a dispersal corridor for postglacial re-colonization northward. Climatic oscillation, aridification, and desert expansion within these regions have molded current distribution and biodiversity of L. ilicifolius.     

Dispersal, habitat differences, and comparative phylogeography of Southeast Asian seahorses (Syngnathidae: Hippocampus)

Four distinct phylogeographical patterns across Southeast Asia were observed for four species of seahorse (genus Hippocampus) with differing ecologies. For all species, genetic differentiation (based on cytochrome b sequence comparisons) was significantly associated with sample site (Phi(ST) = 0.190-0.810, P < 0.0001) and with geographical distance (Mantel's r = 0.37-0.59, P < 0.019). Geographic locations of genetic breaks were inconsistent across species in 7/10 comparisons, although some similarities across species were also observed. The two shallow-water species (Hippocampus barbouri and Hippocampus kuda) have colonized the Sunda Shelf to a lesser degree than the two deeper-water species (Hippocampus spinosissimus and Hippocampus trimaculatus). In all species the presence of geographically restricted haplotypes in the Philippines could indicate past population fragmentation and/or long-distance colonization. A nested clade analysis (NCA) revealed that long-distance colonization and/or fragmentation were likely the dominant forces that structure populations of the two shallow-water species, whereas range expansion and restricted dispersal with isolation by distance were proportionally more important in the history of the two deeper-water species. H. trimaculatus has the most widespread haplotypes [average clade distance (D(c)) of nonsingleton haplotypes = 1169 km], indicating potentially high dispersal capabilities, whereas H. barbouri has the least widespread haplotypes (average D(c) = 67 km) indicating potentially lower dispersal capabilities. Pleistocene separation of marine basins and postglacial flooding of the Sunda Shelf are extrinsic factors likely to have contributed to the phylogeographical structure observed, whereas differences among the species appear to reflect their individual ecologies.     

Genetic structure and phylogeography of a relict tree fern,Sphaeropteris brunoniana (Cyatheaceae) from China and Laos inferred from cpDNA sequence variations:Implications for conservation

In this study, we analyzed the genetic structure and phylogeography of Sphaeropteris brunoniana from China and Laos. Combining cpDNA trnL-trnF and atpB-rbcL sequence variations, five haplotypes were identi- fied from the 10 investigated populations. Moderate haplotype diversity (h = 0.66580) and low nucleotide diver- sity (π = 0.23 × 10 ?3 ) were detected. The S. brunoniana in Yunnan region had much higher genetic diversity (h = 0.60195, π = 0.35 × 10 ?3 ) than that of Hainan–Laos (h = 0.00000, π = 0.00). A...     

Genetic structure and phylogeography of a relict tree fern,Sphaeropteris brunoniana (Cyatheaceae) from China and Laos inferred from cpDNA sequence variations: Implications for conservation

In this study, we analyzed the genetic structure and phylogeography of Sphaeropteris brunoniana from China and Laos. Combining cpDNA trnL-trnF and atpB-rbcL sequence variations, five haplotypes were identified from the 10 investigated populations. Moderate haplotype diversity (h= 0.66580) and low nucleotide diversity (π= 0.23 × 10⁻³) were detected. The S. brunoniana in Yunnan region had much higher genetic diversity (h= 0.60195, π= 0.35 × 10⁻³) than that of Hainan–Laos (h= 0.00000, π= 0.00). A high level of genetic differentiation (94.74%) between the two regions was revealed by amova. Nested clade analysis identified two major clusters of the five haplotypes, one clade in the Yunnan region and the other in Hainan–Laos. The analysis indicated that restricted gene flow with isolation by distance and allopatric fragmentation were likely the major processes that shaped the spatial distribution of the haplotypes. The isolated distribution of clades implied the emergence of independent refugia of this species in each region during Quaternary glaciations. The Yunnan populations frequently contained an ancestral haplotype, and most of them harbored other descendent haplotypes. Based on the distribution pattern of haplotypes and the nested clade analysis results, the Yunnan region potentially had several refugia of this species during glacial periods, whereas the Hainan populations were probable new colonizations.     

Phylogeography of an Island endemic, the Puerto Rican Freshwater Crab (Epilobocera sinuatifrons)

The endemic Puerto Rican crab, Epilobocera sinuatifrons (Pseudothelphusidae), has a freshwater-dependant life-history strategy, although the species has some capabilities for terrestrial movement as adults. In contrast to all other freshwater decapods on the island (e.g., caridean shrimp), E. sinuatifrons does not undertake amphidromous migration, and is restricted to purely freshwater habitats and adjacent riparian zones. As Puerto Rico has a dynamic geologic history, we predicted that both the life history of E. sinuatifrons and the geological history of the island would be important determinants of phylogeographic structuring in the species. Using a fragment of the cytochrome c oxidase subunit 1 mtDNA (mitochondrial DNA) gene, we tested for deviations from panmixia among and within rivers draining Puerto Rico and used statistical phylogeography to explore processes that may explain extant patterns of genetic variation in the species. While populations of E. sinuatifrons were significantly differentiated among rivers, they were likely to be recently derived because nested clade analysis (NCA) indicated evolutionarily recent restricted gene flow with isolation by distance (IBD) and contiguous range expansion at various spatial scales. Ongoing drainage rearrangements associated with faulting and land slippage were invoked as processes involved in sporadic gene flow among rivers throughout the Pleistocene. Patterns of genetic differentiation conformed to IBD and population demographic statistics were nonsignificant, indicating that although recently derived, populations from different rivers were in drift-mutation equilibrium. A shallow (0.6 million years ago), paraphyletic split was observed in the haplotype network, which NCA indicated arose via allopatric fragmentation. This split coincides with an area of high relief in central Puerto Rico that may have experienced relatively little drainage rearrangements. Shallow but significant genetic isolation of populations of E. sinuatifrons among Puerto Rican rivers suggests phylogeographic patterns that are intermediate to terrestrial habitat specialists (highly divergent populations) and other freshwater biota, such as amphidromous species and insects with aerial adult dispersal (highly connected populations).     

The impact of habitat fragmentation on genetic structure of the Japanese sika deer (<Emphasis Type="Italic">Cervus nippon</Emphasis>) in southern Kantoh,revealed by mitochondrial D-loop sequences

In southern Kantoh, Japanese sika deer (Cervus nippon) are distributed discontinuously due to large urban areas and developed road networks. To assess the impact of habitat fragmentation on sika deer subpopulations, we examined mitochondrial D-loop sequences from 435 individuals throughout southern Kantoh. About 13 haplotypes were detected, and their distributions revealed spatial genetic structure. Significant genetic differentiation was observed among seven of eight subpopulations. We found no significant correlation between pairwise F _ST and geographical distance among subpopulations. Genetic diversity indices suggested that seven of eight subpopulations had probably experienced population bottlenecks in the recent past. Therefore, and in the light of the results of a nested clade analysis of these haplotypes, we conclude that recent fluctuations in population size and the interruption of gene flow due to past and present habitat fragmentation have played major roles influencing the spatial genetic structure of the sika deer population. This is the first evidence of spatial genetic population structure in the highly fragmented sika deer population in Honshu, Japan.     

Potential refugium on the Qinghai–Tibet Plateau revealed by the chloroplast DNA phylogeography of the alpine species Metagentiana striata (Gentianaceae)

Metagentiana striata is an alpine annual herbaceous plant endemic to the east of the Qinghai–Tibet (Q–T) Plateau and adjacent areas. The phylogeography of M. striata was studied by sequencing the chloroplast DNA (cpDNA) trn S– trn G intergenic spacer. Ten haplotypes were identified from an investigation of 232 individuals of M. striata from 14 populations covering the entire geographical range of this species. The level of differentiation amongst populations was very high ( G _ST = 0.746; N _ST = 0.774) and a significant phylogeographical structure was observed ( P  < 0.05). An analysis of molecular variance found a high variation amongst populations (76%), with F _ST = 0.762 (highly significant, P  < 0.001), indicating that little gene flow occurred amongst the different regions; this was explained by the isolation of populations by high mountains along the Q–T Plateau and adjacent areas ( N _m = 0.156). Only one ancestral haplotype (A) was common and widespread throughout the distributional range of M. striata. The populations of the Hengduan Mountains region of the south-eastern Q–T Plateau showed high diversity and uniqueness of haplotypes. It is suggested that this region was the potential refugium of M. striata during the Quaternary glaciation, and that interglacial and postglacial range expansion occurred from this refugium. This scenario was in good agreement with the results of nested clade analysis, which inferred that the current spatial distribution of cpDNA haplotypes and populations resulted from range expansion, together with past allopatric fragmentation events.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 125–140.     

Phylogeography and conservation genetics of the Columbia spotted frog (Rana luteiventris; Amphibia, Ranidae)

The Columbia spotted frog (Rana luteiventris) has a widespread distribution in western Canada and the western US, although the southern reach of its range is highly fragmented into several isolated populations. Threats from various factors have raised concerns regarding the long-term survival of many small, isolated populations. Here, we report a study designed to determine the phylogeographic and conservation genetic parameters of R. luteiventris in the western US. Mitochondrial DNA (mtDNA) sequences were examined for phylogeographic structuring using phylogenetic reconstruction methods, coupled with networking and nested clade analyses. These methods permitted a distinction to be made between historic and demographic forces acting to generate geographical patterning of genetic variation. Phylogenetic analysis revealed four geographically correlated monophyletic clades. Three of these clades correspond to well-defined, nonoverlapping geographical locations in the fragmented portion of the range. The other is comprised of all samples collected from the contiguous range and includes one isolate from northern Wyoming. Networking and nested clade analyses confirmed these results and revealed that historical processes, such as range expansion and vicariance, rather than recurrent gene flow are likely responsible for observed patterns of genetic variation. A measure of genetic variation (theta = 4N(e)mu) revealed that R. luteiventris populations in Utah have a relatively low amount of genetic variation compared with populations in the continuous portion of the range.