Adaptive divergence despite strong genetic drift: genomic analysis of the evolutionary mechanisms causing genetic differentiation in the island fox (Urocyon littoralis)

The evolutionary mechanisms generating the tremendous biodiversity of islands have long fascinated evolutionary biologists. Genetic drift and divergent selection are predicted to be strong on islands and both could drive population divergence and speciation. Alternatively, strong genetic drift may preclude adaptation. We conducted a genomic analysis to test the roles of genetic drift and divergent selection in causing genetic differentiation among populations of the island fox (Urocyon littoralis). This species consists of six subspecies, each of which occupies a different California Channel Island. Analysis of 5293 SNP loci generated using Restriction‐site Associated DNA (RAD) sequencing found support for genetic drift as the dominant evolutionary mechanism driving population divergence among island fox populations. In particular, populations had exceptionally low genetic variation, small N_e (range = 2.1–89.7; median = 19.4), and significant genetic signatures of bottlenecks. Moreover, islands with the lowest genetic variation (and, by inference, the strongest historical genetic drift) were most genetically differentiated from mainland grey foxes, and vice versa, indicating genetic drift drives genome‐wide divergence. Nonetheless, outlier tests identified 3.6–6.6% of loci as high F_ST outliers, suggesting that despite strong genetic drift, divergent selection contributes to population divergence. Patterns of similarity among populations based on high F_ST outliers mirrored patterns based on morphology, providing additional evidence that outliers reflect adaptive divergence. Extremely low genetic variation and small N_e in some island fox populations, particularly on San Nicolas Island, suggest that they may be vulnerable to fixation of deleterious alleles, decreased fitness and reduced adaptive potential.     

Selection and geographic isolation influence hummingbird speciation: genetic, acoustic and morphological divergence in the wedge-tailed sabrewing (Campylopterus curvipennis)

Background  

Mesoamerica is one of the most threatened biodiversity hotspots in the world, yet we are far from understanding the geologic history and the processes driving population divergence and speciation for most endemic taxa. In species with highly differentiated populations selective and/or neutral factors can induce rapid changes to traits involved in mate choice, promoting reproductive isolation between allopatric populations that can eventually lead to speciation. We present the results of genetic differentiation, and explore drift and selection effects in promoting acoustic and morphological divergence among populations of Campylopterus curvipennis, a lekking hummingbird with an extraordinary vocal variability across Mesoamerica.     

ORIGINS AND CONSEQUENCES OF SERPENTINE ENDEMISM IN THE CALIFORNIA FLORA

Habitat specialization plays an important role in the creation and loss of biodiversity over ecological and evolutionary time scales. In California, serpentine soils have a distinctive flora, with 246 serpentine habitat specialists (i.e., endemics). Using molecular phylogenies for 23 genera containing 784 taxa and 51 endemics, we infer few transitions out of the endemic state, which is shown by an analysis of transition rates to simply reflect the low frequency of endemics (i.e., reversal rates were high). The finding of high reversal rates, but a low number of reversals, is consistent with the widely hypothesized trade‐off between serpentine tolerance and competitive ability, under which serpentine endemics are physiologically capable of growing in less‐stressful habitats but competitors lead to their extirpation. Endemism is also characterized by a decrease in speciation and extinction rates and a decrease in the overall diversification rate. We also find that tolerators (species with nonserpentine and serpentine populations) undergo speciation in serpentine habitats to give rise to new serpentine endemics but are several times more likely to lose serpentine populations to produce serpentine‐intolerant taxa. Finally, endemics were younger on average than nonendemics, but this alone does not explain their low diversification.     

CHARACTERIZING SELECTION ON PHENOTYPIC PLASTICITY IN RESPONSE TO NATURAL ENVIRONMENTAL HETEROGENEITY

Adaptive genetic differentiation and adaptive phenotypic plasticity can increase the fitness of plant lineages in heterogeneous environments. We examine the relative importance of genetic differentiation and plasticity in determining the fitness of the annual plant, Erodium cicutarium, in a serpentine grassland in California. Previous work demonstrated that the serpentine sites within this mosaic display stronger dispersal‐scale heterogeneity than nonserpentine sites. We conducted a reciprocal transplant experiment among six sites to characterize selection on plasticity expressed by 180 full‐sibling families in response to natural environmental heterogeneity across these sites. Multivariate axes of environmental variation were constructed using a principal components analysis of soil chemistry data collected at every experimental block. Simple linear regressions were used to characterize the intercept, and slope (linear and curvilinear) of reaction norms for each full‐sibling family in response to each axis of environmental variation. Multiple linear regression analyses revealed significant selection on trait means and slopes of reaction norms. Multivariate analyses of variance demonstrated genetic differentiation between serpentine and nonserpentine lineages in the expression of plasticity in response to three of the five axes of environmental variation considered. In all but one case, serpentine genotypes expressed a stronger adaptive plastic response than nonserpentine genotypes.     

Mortality dynamics of Daphnia in contrasting habitats and their role in ecological divergence

1. The Daphnia pulex‐pulicaria species complex has been proposed as an example of rapid ecological speciation, associated with divergence along the gradient of waterbody size from temporary ponds to deep, stratified lakes. However, this divergence is incomplete, and thus represents an opportunity to study ecological divergence as it is occurring. 2. Dynamics of twelve populations of Daphnia in the pulex‐pulicaria species complex were monitored over 1 year. Six temporary pond populations and six permanent lake populations were compared to evaluate demographic differences that may contribute to ecological divergence in this complex. 3. Pond populations experienced greater changes in density, which were reflected in more extreme growth rates, higher birth rates and higher mortality rates than those of lake populations. 4. Mitochondrial DNA was isolated from up to three clones from each population, the D‐loop of the control region was sequenced, and a phylogenetic tree was constructed. This tree revealed two strongly supported clades. The clades were not congruent with habitat type and nominal status, indicating that interhabitat gene flow occurs easily and that the nominal taxa are incompletely diverged. 5. Published reports of genetic life history differences in the D. pulex‐pulicaria complex are consistent with the demographic differences reported here. This suggests that ecological differences between the habitats are selectively maintaining trait differences despite the possibility for genetic exchange. Thus, these taxa may be at the inception of ecological speciation.     

DIFFERENTIAL RESPONSES TO MAGNESIUM AND CALCIUM BY NATIVE POPULATIONS OF AGROPYRON SPICATUM

Plants of Agropyron spicatum (Pursh) Scribn. and Smith. from populations native to serpentine and nonserpentine soils were grown at varying levels of magnesium and calcium in culture solutions. The yields of plants from the two populations were different. At high Mg levels (low Ca) the yield of the serpentine population was significantly higher than that of the nonserpentine population. At low Mg the yield of the serpentine population leveled off at a Mg: Ca ratio of 1:2, while the yield of the nonserpentine population increased up to a Mg:Ca ratio of 1:8 and showed no leveling off. Chemical analyses of tissue showed that the Ca uptake of plants from the serpentine population was significantly higher than that of the nonserpentine population. In addition, the serpentine population maintained a lower Mg concentration in the shoots than the nonserpentine population at comparable Mg substrate levels. The two populations showed differences in Ca and Mg uptake efficiency and Mg/Ca, Ca + Mg/K + Na, and Ca + Mg + K + Na in the shoots. The ecotypic differentiation with respect to Mg and Ca between native populations of serpentine and nonserpentine A. spicatum does not appear to be due to any single mechanism but, rather, a combination of several possible mechanisms, i.e., differences in root morphology, uptake mechanisms, translocation of nutrients, and interactions between cations.     

Geographic variation in advertisement calls of a Microhylid frog – testing the role of drift and ecology

Acoustic signals for mating are important traits that could drive population differentiation and speciation. Ecology may play a role in acoustic divergence through direct selection (e.g., local adaptation to abiotic environment), constraint of correlated traits (e.g., acoustic traits linked to another trait under selection), and/or interspecific competition (e.g., character displacement). However, genetic drift alone can also drive acoustic divergence. It is not always easy to differentiate the role of ecology versus drift in acoustic divergence. In this study, we tested the role of ecology and drift in shaping geographic variation in the advertisement calls of Microhyla fissipes. We examined three predictions based on ecological processes: (1) the correlation between temperature and call properties across M. fissipes populations; (2) the correlation between call properties and body size across M. fissipes populations; and (3) reproductive character displacement (RCD) in call properties between M. fissipes populations that are sympatric with and allopatric to a congener M. heymonsi. To test genetic drift, we examined correlations among call divergence, geographic distance, and genetic distance across M. fissipes populations. We recorded the advertisement calls from 11 populations of M. fissipes in Taiwan, five of which are sympatrically distributed with M. heymonsi. We found geographic variation in both temporal and spectral properties of the advertisement calls of M. fissipes. However, the call properties were not correlated with local temperature or the callers' body size. Furthermore, we did not detect RCD. By contrast, call divergence, geographic distance, and genetic distance between M. fissipes populations were all positively correlated. The comparisons between phenotypic Q_st (P_st) and F_st values did not show significant differences, suggesting a role of drift. We concluded that genetic drift, rather than ecological processes, is the more likely driver for the geographic variation in the advertisement calls of M. fissipes.     

Calls,colours, shape,and genes: a multi‐trait approach to the study of geographic variation in the Amazonian frog Allobates femoralis

Evolutionary divergence in behavioural traits related to mating may represent the initial stage of speciation. Direct selective forces are usually invoked to explain divergence in mate‐recognition traits, often neglecting a role for neutral processes or concomitant differentiation in ecological traits. We adopted a multi‐trait approach to obtain a deeper understanding of the mechanisms behind allopatric divergence in the Amazonian frog, Allobates femoralis. We tested the null hypothesis that geographic distance between populations correlates with genetic and phenotypic divergence, and compared divergence between mate‐recognition (acoustic) and ecological (coloration, body‐shape) traits. We quantified geographic variation in 39 phenotypic traits and a mitochondrial DNA marker among 125 individuals representing eight populations. Geographic variation in acoustic traits was pronounced and tracked the spatial genetic variation, which appeared to be neutral. Thus, the evolution of acoustic traits tracked the shared history of the populations, which is unexpected for pan‐Amazonian taxa or for mate‐recognition traits. Divergence in coloration appeared uncorrelated with genetic distance, and might be partly attributed to local selective pressures, and perhaps to Batesian mimicry. Divergence in body‐shape traits was low. The results obtained depict a complex evolutionary scenario and emphasize the importance of considering multiple traits when disentangling the forces behind allopatric divergence. ©2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 826–838.     

Genetic divergence is more tightly related to call variation than landscape features in the Amazonian frogs Physalaemus petersi and P. freibergi

Behavioural isolation from divergence in male advertisement calls and female preferences is hypothesized to cause genetic divergence and speciation in the Amazonian frogs Physalaemus petersi and P. freibergi, yet the importance of call variation and landscape features in genetic divergence is unresolved. We tested for correlations between genetic divergence at microsatellite loci and (1) call variables; and (2) landscape variables among 10 populations of these frogs. Genetic divergence was not correlated with geographical distance, rivers or elevation. There was a strong positive relationship, however, between genetic divergence and inter‐population differences in one call variable, whine dominant frequency. Effective population sizes varied among sites (range = 15–846) and were often small, suggesting that genetic drift could influence call evolution. Evidence for fine‐scale genetic structure within sites was also found. Our results support the hypothesis that behavioural isolation from divergence in male calls and female preferences causes genetic divergence and speciation.     

THE EFFECT OF SERPENTINE ON THE POPULATION STRUCTURE OF SILENE DIOICA (CARYOPHYLLACEAE)

Serpentine soils are rich in heavy metals and have a distinctive flora. Silene dioica is a member of the Scandinavian serpentine plant community but is also widespread outside serpentine soils. To study the population genetic consequences of serpentine stress and the origin and evolution of serpentine populations we analyzed the isozyme genetic structure of S. dioica. Seventeen populations located in the mountains of Västerbotten and Jämtland, central Sweden, were investigated by starch gel enzyme electrophoresis. About one half of the populations grow in serpentine soils and the rest on adjacent non-serpentine sites. Analyses of allele frequencies show that both serpentine and non-serpentine populations in the northern part of the studied area (Västerbotten) are genetically similar. Evidently serpentine does not exert strong selection acting upon isozyme loci. In the south (Jämtland), however, the serpentine populations exhibit genetic differentiation. This allozyme divergence is probably not due to direct selection but rather represents the effects of isolation and genetic drift. The results suggest that S. dioica has colonized serpentine repeatedly and that the tolerant populations have a multiple origin.     

Snails in the desert: Species diversification of Theba (Gastropoda: Helicidae) along the Atlantic coast of NW Africa

The spatial subdivision of species often plays a pivotal role in speciation. Across their entire range, species are rarely panmictic and crucial consequences of spatial subdivision are (1) random genetic drift including historical factors, (2) uniform selection, and (3) divergent selection. Each of these consequences may result in geographic variation and eventually reproductive isolation, but their relative importance in speciation is still unclear. In this study, we used a combination of genetic, morphological, and climatic data to obtain a comprehensive picture of differentiation among three closely related, parapatrically distributed taxa of the land snail genus Theba occurring along the Atlantic coasts of South Morocco and Western Sahara. We conducted Mantel and partial Mantel tests to relate phenotypic and genotypic variation of these species to geography and/or climate. As null hypothesis for an evolutionary scenario, we assumed nonadaptive speciation and expected a pattern of isolation by distance among taxa. Rejection of the null hypothesis would indicate isolation by environment due to adaptation. Generally, genetic drift plays an important role but is rarely considered as sole driver of speciation. It is the combination of drift and selection that predominantly drives speciation. This study, however, provides a potential example, in which nonadaptive speciation, that is, genetic drift, is apparently the main driver of shaping the diversity of Theba in NW Africa. Restriction of gene flow between populations caused by geographic isolation probably has played an important role. Climate oscillations during the Plio‐ and Pleistocene may have led to repeated ecological changes in NW Africa and disruptions of habitats promoting differentiation by geographic isolation. The inferred evolutionary scenario, however, did not fully explain the incongruence between the AFLP‐ and mtDNA‐tree topologies. This incongruence might indicate past hybridization among the studied Theba forms.     

ECOLOGICAL GENETICS OF SERPENTINE TOLERANCE IN THE MOSS,FUNARIA FLAVICANS: VARIATION WITHIN AND AMONG HAPLOID SIB FAMILIES

Many mosses occur both on and off serpentine soils, but experiments designed to test whether serpentine populations of widespread species display genetic adaptations for growth on serpentines have not been reported. Toward that end, two populations of the moss, Funaria flavicans, were grown on nutrient media varying in nickel and chromium concentration and in the ratio of magnesium to calcium. Three haploid siblings from each of five sporophytic families from the two populations were grown on five experimental media. There was no evidence that serpentine plants were more tolerant of nickel, chromium, high magnesium/calcium, or high nickel combined with high magnesium/calcium. In fact, plants from the nonserpentine population produced more protonemal growth than the serpentine plants on every medium except the control, on which plants from the two populations were indistinguishable. Large differences in nickel tolerance among haploid sib families (families of meiotic progeny derived from the same sporophyte) from the nonserpentine site provided evidence of genetic polymorphism in that population.     

Pinpointing the level of isolation between two cryptic species sharing the same microhabitat: a case study with a scarabaeid species complex

Understanding the processes of speciation is an important challenge in improving knowledge of the origin of biodiversity. One crucial point is to assess the causes of reproductive isolation, especially in the case of co‐occurring species. Differences in microscale spatial distribution in small organisms may blur the issue. We explored biological processes underlying speciation within dung beetles belonging to the vacca species complex (Scarabaeidae: Onthophagus). The two taxa of this complex, O. vacca and O. medius, not only are known to have a large overlapping Palearctic distribution range but also share the same cowpat with no physical barriers and no observed specific aggregated patterns in the local distribution. The present study aimed at determining the level of isolation between the two taxa and discusses the most likely scenario of the speciation (sympatry vs. allopatry) based on the Coyne & Orr's (2004) four criteria. We conducted a full study on populations sampled within the Mediterranean region integrating morphological analysis (digital image analysis of the elytral melanism pattern), two‐gene phylogenies, population genetic analyses on populations sampled from an area where both species occur and another one with O. vacca only, as well as intra‐ and interspecific mating and crossing bioassays. The variation in the elytral melanism pattern clearly followed a bimodal distribution, with O. medius being more melanic than O. vacca, with a very limited overlapping area. The two taxa are reproductively isolated, with a strong postzygotic incompatibility despite the absence of sexual isolation. Sequence analysis of both nuclear and mitochondrial markers revealed a deep divergence between the two taxa dating back to 8.7 Mya. All findings concurred with some phenological observations and the conclusion that the most likely scenario for speciation in the vacca complex was an allopatric speciation followed by secondary contact.     

Nuclear locus divergence at the early stages of speciation in the Orchard Oriole complex

As two lineages diverge from one another, mitochondrial DNA should evolve fixed differences more rapidly than nuclear DNA due to its smaller effective population size and faster mutation rate. As a consequence, molecular systematists have focused on the criteria of reciprocal monophyly in mitochondrial DNA for delimiting species boundaries. However, mitochondrial gene trees do not necessarily reflect the evolutionary history of the taxa in question, and even mitochondrial loci are not expected to be reciprocally monophyletic when the speciation event happened very recently. The goal of this study was to examine mitochondrial paraphyly within the Orchard Oriole complex, which is composed of Icterus spurius (Orchard Oriole) and Icterus fuertesi (Fuertes' Oriole). We increased the geographic sampling, added four nuclear loci, and used a range of population genetic and coalescent methods to examine the divergence between the taxa. With increased taxon sampling, we found evidence of clear structure between the taxa for mitochondrial DNA. However, nuclear loci showed little evidence of population structure, indicating a very recent divergence between I. spurius and I. fuertesi. Another goal was to examine the genetic variation within each taxon to look for evidence of a past founder event within the I. fuertesi lineage. Based on the high amounts of genetic variation for all nuclear loci, we found no evidence of such an event – thus, we found no support for the possible founding of I. fuertesi through a change in migratory behavior, followed by peripheral isolates speciation. Our results demonstrate that these two taxa are in the earliest stages of speciation, at a point when they have fixed differences in plumage color that are not reflected in monophyly of the mitochondrial or nuclear DNA markers in this study. This very recent divergence makes them ideal for continued studies of species boundaries and the earliest stages of speciation.     

Limited dispersal and local adaptation promote allopatric speciation in a biodiversity hotspot

Recently diverged or diverging populations can offer unobstructed insights into early barriers to gene flow during the initial stages of speciation. The current study utilised a novel insect system (order Mantophasmatodea) to shed light on the early drivers of speciation. The members of this group have limited dispersal abilities, small allopatric distributions and strong habitat associations in the Cape Floristic Region biodiversity hotspot in South Africa. Sister taxa from the diverse family Austrophasmatidae were chosen as focal species (Karoophasma biedouwense, K. botterkloofense). Population genetics and Generalized Dissimilarity Modelling (GDM) were used to characterise spatial patterns of genetic variation and evaluate the contribution of environmental factors to population divergence and speciation. Extensive sampling confirmed the suspected allopatry of these taxa. However, hybrids were identified in a narrow region occurring between the species' distributions. Strong population structure was found over short geographic distances; particularly in K. biedouwense in which geographic distance accounted for 32% of genetic variation over a scale of 50 km (r = .56, p < .001). GDM explained 42%–78% of the deviance in observed genetic dissimilarities. Geographic distance was consistently indicated to be important for between species and within population differentiation, suggesting that limited dispersal ability may be an important neutral driver of divergence. Temperature, altitude, precipitation and vegetation were also indicated as important factors, suggesting the possible role of adaptation to local environmental conditions for species divergence. The discovery of the hybrid-zone, and the multiple allopatric species pairs in Austrophasmatidae support the idea that this could be a promising group to further our understanding of speciation modes.     

GENETIC RELATIONSHIPS AND PATTERNS OF ALLOZYMIC DIVERGENCE IN THE IPOMOPSIS AGGREGATA COMPLEX AND RELATED SPECIES (POLEMONIACEAE)

The Ipomopsis aggregata complex consists of diploid, outcrossing, perennial herbs. The group is highly variable morphologically and is treated as three species: I. aggregata, I. tenuituba, and I. arizonica. Geographic races of I. aggregata and I. tenuituba are recognized as subspecies. Enzyme electrophoresis was used to examine genetic relationships among populations and taxa in the Ipomopsis aggregata complex and some related species. Genetic data for 23 allozyme loci from 60 populations were also used to determine how genetic variation is distributed geographically. Populations in the southwestern United States were more variable than those in the northwest: the center of genetic diversity corresponded to the center of species diversity. Allozymic data provided no evidence of loss of genetic variability associated with recent and rapid divergence. Genetic relationships based on Nei's genetic identity did not correspond to taxonomic relationships. For example, populations of both I. arizonica and I. tenuituba clustered within I. aggregata. Despite relatively high levels of genetic diversity among populations, diversity among taxa was low. Results indicated that floral divergence and concomitant speciation have occurred recently in the Ipomopsis aggregata complex. Allozymic patterns also reflected convergent evolution for floral morphology and possible introgression. Despite morphological differences among species, insufficient evolutionary time has elapsed for allelic fixation at neutral or near-neutral allozyme loci.     

An unusual barrier to gene flow: perpetually immature larvae from inter-population crosses in the flour beetle, Tribolium castaneum

We genetically characterize an unusual hybrid incompatibility phenotype manifest in F₁ offspring of crosses between two populations of Tribolium castaneum. Hybrid larvae cease development at the third larval instar, persisting as ‘perpetually immature larvae’ thereafter. Although unable to produce viable adult hybrid offspring with one another, each population produces abundant, fertile hybrids with other populations, indicating a recent origin of the incompatibility and facilitating genetic studies. We mapped the paternal component of the hybrid phenotype to a single region, which exhibits two characteristics common to hybrid incompatibility: marker transmission ratio distortion within crosses and elevated genetic divergence between populations. The incompatible variation and an elevation in between‐population genetic divergence is associated with a region containing the T. castaneum ecdysone receptor homologue, a major regulatory switch, controlling larval moults, pupation and metamorphosis. This contributes to understanding the genetics of speciation in the Coleoptera, one of the most speciose of all arthropod taxa.     

Comparisons of primary production and nutrients absorption by aMiscanthus sinensis community in different soils

Soil properties, primary production, nitrogen and phosphorus uptake in aMiscanthus sinensis community on serpentine gangue area were compared with that on nonserpentine area. Soil water content, soil pH and nitrogen content were quite different between the serpentine gangue area and nonserpentine area; but phosphorus content of the soil was similar between the two sites. The maximum above-ground net production in the serpentine gangue and nonserpentine areas was 4.5±0.2 kg m^–2 yr^–1 and 7.8±0.2 kg m^–2 yr^–1, respectively. The total maximum standing biomass in the serpentine gangue and nonserpentine areas was 8.5±0.8 kg m^–2 and 11.9±0.4 kg m^–2, respectively. Nitrogen uptake by plants in the nonserpentine area was 2.4 times greater than that in the serpentine gangue area. Phosphorus uptake by plants were similar for the two sites. The most probable reasons for the small biomass produced by theMiscanthus sinensis community in this serpentine gangue area are the low levels of nitrogen and water availability in the soil.     

Population genetic structure and a possible role for selection in driving phenotypic divergence in a rainbowfish (Melanotaeniidae)

Quantitative genetic divergence may be driven by drift or selection. The rainbowfish Melanotaenia australis exhibits phenotypic divergence among populations in Western Australia, although the mechanisms driving this divergence are unknown. We used microsatellites to assess neutral genetic divergence (F_ST), and found a hierarchical pattern of subdivision consistent with low divergence between upstream and downstream populations (within drainages), moderate divergence between drainages (within regions), and high divergence between regions. Using a common‐garden approach, we measured quantitative genetic divergence in phenotypic traits (Q_ST). By comparing this to expectations from neutral processes (F_ST), we concluded that the effect of selection varies depending on the spatial scale considered. Within drainages, selection may be causing divergence between upstream and downstream phenotypes but, between regions, selection appears to homogenize phenotypes. This highlights the importance of spatial scale in studies of this kind, and suggests that, because variance in selection pressures can drive speciation, polymorphism in M. australis may represent speciation in action. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 144–160.     

Molecular phylogenetics at the population/species interface in cave spiders of the southern Appalachians (Araneae:Nesticidae:Nesticus)

This paper focuses on the relationship between population genetic structure and speciation mechanisms in a monophyletic species group of Appalachian cave spiders (Nesticus). Using mtDNA sequence data gathered from 256 individuals, I analyzed patterns of genetic variation within and between populations for three pairs of closely related sister species. Each sister-pair comparison involves taxa with differing distributional and ecological attributes; if these ecological attributes are reflected in basic demographic differences, then speciation might proceed differently across these sister taxa comparisons. Both frequency-based and gene tree analyses reveal that the genetic structure of the Nesticus species studied is characterized by similar and essentially complete population subdivision, regardless of differences in general ecology. These findings contrast with results of prior genetic studies of cave-dwelling arthropods that have typically revealed variation in population structure corresponding to differences in general ecology. Species fragmentation through both extrinsic and intrinsic evolutionary forces has resulted in discrete, perhaps independent, populations within morphologically defined species. Large sequence divergence values observed between populations suggest that this independence may extend well into the past. These patterns of mtDNA genealogical structure and divergence imply that species as morphological lineages are currently more inclusive than basal evolutionary or phylogenetic units, a suggestion that has important implications for the study of speciation mechanisms.