Fine-scale Population Research Articles

Mechanisms of peripheral phylogeographic divergence in the indo-Pacific: lessons from the spiny lobster Panulirus homarus

BackgroundThere is increasing recognition of the concordance between marine biogeographic and phylogeographic boundaries. However, it is still unclear how population-level divergence translates into species-level divergence, and what are the principal factors that first initiate that divergence, and then maintain reproductive isolation. This study examines the likely forces driving population and lineage divergences in the broadly-distributed Indo-Pacific spiny lobster Panulirus homarus, which has peripheral divergent lineages in the west and east. The study focuses particularly on the West Indian Ocean, which is emerging as a region of unexpected diversity. Mitochondrial control region (mtCR) and COI sequences as well as genotypes of 9 microsatellite loci were examined in 410 individuals from 17 locations grouped into 7 regions from South Africa in the west, and eastward across to Taiwan and the Marquesas Islands. Phylogenetic and population-level analyses were used to test the significance and timing of divergences and describe the genetic relationships among populations.ResultsAnalyses of the mtCR revealed high levels of divergence among the seven regions (ФST = 0.594, P < 0.001). Microsatellite analyses also revealed significant divergence among regions, but at a much lower level (FST = 0.066, P < 0.001). The results reveal different patterns of mtCR v. nDNA divergence between the two distinct peripheral lineages: a subspecies in South Africa and Madagascar, and a phylogeographically diverged population in the Marquesas. The results also expose a number of other more fine-scale population divergences, particularly in the Indian Ocean.ConclusionsThe divergence of peripheral lineages in the west and east of the species’ range appear to have been initiated and maintained by very different processes. The pattern of mitochondrial and nuclear divergence of the western lineage, implicates processes of parapatric isolation, secondary contact and introgression, and suggests possible maintenance through adaptation and behavioural reproductive isolation. In contrast, the eastern lineage appears to have diverged through a rare colonisation event, maintained through long-term isolation, and matches expectations of the core-periphery hypothesis. The process of active peripheral speciation may be a common force in the Indo-Pacific that helps drive some of the regions’ recognized biogeographic boundaries.

Genetic legacy of tertiary climatic change: a case study of two freshwater loaches, Schistura fasciolata and Pseudogastromyzon myersi, in Hong Kong.

The high biodiversity and strong population structure of freshwater fauna has often been attributed to historical geological and climatic alterations. The impact of these historical changes on obligate freshwater species on a small geographical scale has not been well understood due to the lack of fine-scale comparative phylogeographic studies. Strong population structure has been reported in a goby and a caridean shrimp in Hong Kong, a small but highly developed city in South China, but the common drivers of population differentiation in freshwater fauna in this region remain unclear. This study examined the fine-scale phylogeographic patterns of two freshwater loaches, Schistura fasciolata and Pseudogastromyzon myersi in Hong Kong, using sequence data of mitochondrial control region and two nuclear markers (interphotoreceptor retinoid binding protein gene 2 and ribosomal protein S13 gene). Results show that they exhibit pronounced population structure as supported by high and significant ΦST. Phylogenetic analyses based on the control region reveal six and three distinct lineages in S. fasciolata and P. myersi, respectively. Phylogeographic structure of both species generally follows the paleodrainage pattern, though P. myersi shows a shallower structure on the Mainland, perhaps due to their higher mobility. Most of these lineages diverged during the Pliocene and Late Pleistocene, a period with marked sea-level fluctuations. In a broader context, this suggests that sea-level fluctuation played an important role in shaping even the fine-scale population structure of freshwater fish in South China, implying that the genetic diversity of this fauna may be higher than expected.

Restricted dispersal determines fine-scale spatial genetic structure of Mongolian gerbils.

Restricted gene flow may cause positive spatial genetic autocorrelation of animal populations at fine spatial scales. The Mongolian gerbil Meriones unguiculatus is a territorial, social rodent. Territoriality may create social fences to restrict dispersal or gene flow of Mongolian gerbils to a short distance. Restricted dispersal may differentiate fine-scale spatial genetic structure of populations with increasing distances (i.e., isolation by distance [IBD]). Competition for mates and inbreeding avoidance may result in equal dispersal propensity and subsequently similar spatial genetic autocorrelation between males and females of monogamous gerbils. We genotyped 327 gerbils, live captured from 26 burrow systems on a 9-ha plot in northcentral Inner Mongolia, China, using seven microsatellite loci. Spatial genetic autocorrelation was positive within 80 m and became negative from 80 m to 200 m, suggesting restricted gene flow. Inter-group genetic and geographic distances were related positively, supporting the IBD model. Live trapping data demonstrated equal dispersal propensities of male and female gerbils. Restricted dispersal and social organization may determine fine-scale spatial population genetic structure of social rodents.

Fine-scale population structure of common bottlenose dolphins (Tursiops truncatus) in offshore and coastal waters of the US Gulf of Mexico

Patterns of dispersal and population structure of highly mobile marine fauna in the open ocean are not well understood. Much insight, however, can be gained from using common bottlenose dolphins (Tursiops truncatus), one of the most abundant marine mammals in the northern Gulf of Mexico (GOMx), as a model to better understand these aspects of the marine realm. Populations of common bottlenose dolphins in estuarine environments show evidence of strong population structure; however, whether this species similarly partitions the open ocean to the point that genetically differentiated populations are present is unknown. Such information is essential to accurately assess natural and anthropogenic impacts and is crucial for effective conservation and management. We used mitochondrial DNA control region sequence data, 19 microsatellite and 39 single nucleotide polymorphism loci to identify population structure of T. truncatus in coastal and offshore waters of the northern GOMx. Bayesian clustering and a multivariate method of discriminant analysis identified seven genetically distinct populations and levels of genetic differentiation were in line with other recognized populations of T. truncatus. The newly identified populations do not correspond to the currently delineated management stocks, suggesting revision may be necessary to establish accurate representation and effective management of the existing biological populations of T. truncatus in these waters. The finding of significant population structure in dolphins living in open-ocean waters augments the growing body of evidence indicating pelagic waters are not homogeneous even for highly mobile species.

Fine scale population density data and its application in risk assessment

ABSTRACTPopulation density is one of the key parameters for assessing the magnitude of population exposed to risk, and the better quality data we have, the better the assessment of risk. The aim of this study is to elaborate a high-resolution spatially distributed population density grid, which estimates population at the commune scale with a reliability of over 90%. The novelty of the approach is population density estimation in a regular European grid, based on buildings vector data collected in the national topographic database. Using abductive reasoning in combination with statistics and spatial analysis, the authors extract approximate information about a population from the large-scale topographic data. Moreover, linking the obtained population data with the cadastral data – by unique building identifier – allows for regular, quick and census survey-independent updates of the population surface. A shortcoming of the approach is the issue of the possible existence of two houses per family, which leads to an overestimation of population. However, in the study area it affected only two of the total 14 communes by 7%–9%.

Whole-genome patterns of linkage disequilibrium across flycatcher populations clarify the causes and consequences of fine-scale recombination rate variation in birds.

Recombination rate is heterogeneous across the genome of various species and so are genetic diversity and differentiation as a consequence of linked selection. However, we still lack a clear picture of the underlying mechanisms for regulating recombination. Here we estimated fine-scale population recombination rate based on the patterns of linkage disequilibrium across the genomes of multiple populations of two closely related flycatcher species (Ficedula albicollis and F. hypoleuca). This revealed an overall conservation of the recombination landscape between these species at the scale of 200kb, but we also identified differences in the local rate of recombination despite their recent divergence (<1 million years). Genetic diversity and differentiation were associated with recombination rate in a lineage-specific manner, indicating differences in the extent of linked selection between species. We detected 400-3,085 recombination hotspots per population. Location of hotspots was conserved between species, but the intensity of hotspot activity varied between species. Recombination hotspots were primarily associated with CpG islands (CGIs), regardless of whether CGIs were at promoter regions or away from genes. Recombination hotspots were also associated with specific transposable elements (TEs), but this association appears indirect due to shared preferences of the transposition machinery and the recombination machinery for accessible open chromatin regions. Our results suggest that CGIs are a major determinant of the localization of recombination hotspots, and we propose that both the distribution of TEs and fine-scale variation in recombination rate may be associated with the evolution of the epigenetic landscape.

Implications of fidelity and philopatry for the population structure of female black-tailed deer

Lay SummaryReturning to previously used areas (site fidelity) or place of birth (philopatry) are important behavioral adaptations in many species, with known fitness benefits. The combined population level consequences of site fidelity and philopatry, however, have received little attention. Based on GPS location data and mitochondrial DNA we demonstrate how high levels of fidelity and philopatric behavior in the matrilineage of black-tailed deer can lead to fine-scale population structure and discuss possible implications for population dynamics.

Identifying the Relevant Local Population for Environmental Impact Assessments of Mobile Marine Fauna

Environmental impact assessments must be addressed at an appropriate scale of biological organization for the species affected. It can be challenging to identify the relevant local wildlife population for impact assessment for those species that are continuously distributed and highly mobile. Here, we document the existence of local communities of Indo-Pacific bottlenose dolphins (Tursiops aduncus) inhabiting coastal and estuarine waters of Perth, Western Australia, where major coastal developments have been undertaken or are proposed. Using sighting histories from a four-year photo-identification study, we investigated fine-scale, social community structure of dolphins based on measures of social affinity and network (Half-Weight Index - HWI, preferred dyadic association tests, and Lagged Association Rates - LAR), home ranges, residency patterns (Lagged Identification Rates - LIR), and genetic relatedness. Analyses revealed four socially and spatially distinct, mixed-sex communities. The four communities had distinctive social patterns varying in strength, site fidelity and residency patterns. Overlap in home ranges and relatedness explained little to none of the association patterns between individuals, suggesting complex local social structures. The study demonstrated that environmental impact assessments for mobile, continuously distributed must evaluate impacts in light of local population structure, especially where proposed developments may affect core habitats of resident communities or sub-populations. Here, the risk of local extinction is particularly significant for an estuarine community because of its small size, limited connectivity with adjacent communities, and use of areas subject to intensive human use. In the absence of information about fine-scale population structure, impact assessments may fail to consider the appropriate biological context.

Fine-scale population genetic structure of the Bengal tiger (Panthera tigris tigris) in a human-dominated western Terai Arc Landscape, India.

Despite massive global conservation strategies, tiger populations continued to decline until recently, mainly due to habitat loss, human-animal conflicts, and poaching. These factors are known to affect the genetic characteristics of tiger populations and decrease local effective population sizes. The Terai Arc Landscape (TAL) at the foothills of the Himalaya is one of the 42 source sites of tigers around the globe. Therefore, information on how landscape features and anthropogenic factors affect the fine-scale spatial genetic structure and variation of tigers in TAL is needed to develop proper management strategies for achieving long-term conservation goals. We document, for the first time, the genetic characteristics of this tiger population by genotyping 71 tiger samples using 13 microsatellite markers from the western region of TAL (WTAL) of 1800 km2. Specifically, we aimed to estimate the genetic variability, population structure, and gene flow. The microsatellite markers indicated that the levels of allelic diversity (MNA = 6.6) and genetic variation (Ho = 0.50, HE = 0.64) were slightly lower than those reported previously in other Bengal tiger populations. We observed moderate gene flow and significant genetic differentiation (FST= 0.060) and identified the presence of cryptic genetic structure using Bayesian and non-Bayesian approaches. There was low and significantly asymmetric migration between the two main subpopulations of the Rajaji Tiger Reserve and the Corbett Tiger Reserve in WTAL. Sibship relationships indicated that the functionality of the corridor between these subpopulations may be retained if the quality of the habitat does not deteriorate. However, we found that gene flow is not adequate in view of changing land use matrices. We discuss the need to maintain connectivity by implementing the measures that have been suggested previously to minimize the level of human disturbance, including relocation of villages and industries, prevention of encroachment, and banning sand and boulder mining in the corridors.

Tight species cohesion among sympatric insular wild gingers (Asarum spp. Aristolochiaceae) on continental islands: Highly differentiated floral characteristics versus undifferentiated genotypes.

The Amami Island group of the Ryukyu Archipelago, Japan, harbors extensive species diversity of Asarum in a small landmass. The fine-scale population genetic structure and diversity of nine insular endemic Asarum species were examined using nuclear DNA microsatellite loci and ITS sequences. High population genetic diversity (HS = 0.45–0.79) was estimated based on the microsatellites, implying outcrossing of Asarum species within populations accompanied by inbreeding. Bayesian clustering analyses revealed that species were divided into three robust genetic clusters and that the species within each cluster had a homogeneous genetic structure, indicating incomplete lineage sorting. This conclusion was supported by an ITS phylogeny. The degree of genetic differentiation among species was very low both within and between clusters (FST = 0.096–0.193, and 0.096–0.266, respectively). Although species can be crossed artificially to produce fertile hybrids, our results indicate that there is very little evidence of hybridization or introgression occurring among species in the wild, even within stands composed of multiple sympatric species. The highly differentiated floral morphology of the studied species is likely to impose reproductive isolation between them and maintain their integrity in the wild. A lack of genetic differentiation between sympatric species suggests that speciation within this group occurred rapidly and recently.

Population genetics of reef coral endosymbionts (Symbiodinium, Dinophyceae).

Symbiodinium is a diverse genus of unicellular dinoflagellate symbionts associating with various marine protists and invertebrates. Although the broadscale diversity and phylogenetics of the Symbiodinium complex is well established, there have been surprisingly few data on fine-scale population structure and biogeography of these dinoflagellates. Yet population-level processes contribute strongly to the biology of Symbiodinium, including how anthropogenic-driven global climate change impacts these symbionts and their host associations. Here, we present a synthesis of population-level characteristics for Symbiodinium, with an emphasis on how phylogenetic affinities, dynamics within and among host individuals, and a propensity towards clonality shape patterns on and across reefs. Major inferences include the following: (i) Symbiodinium populations within individual hosts are comprised mainly of cells belonging to a single or few genetic clones. (ii) Symbiont populations exhibit a mixed mode of reproduction, wherein at least one sexual recombination event occurs in the genealogy between most genotypes, but clonal propagation predominates overall. (iii) Mutualistic Symbiodinium do not perpetually persist outside their hosts, instead undergoing turnover and replacement via the continuous shedding of viable clonal cells from host individuals. (iv) Symbiont populations living in the same host, but on different reefs, are often genetically subdivided, suggesting low connectivity, adaptation to local conditions, or prolific asexual reproduction and low effective population sizes leading to disproportionate success within and among hosts. Overall, this synthesis forms a basis for future investigations of coral symbiosis ecology and evolution as well as delimitation of species boundaries in Symbiodinium and other eukaryotic microorganisms.

Large and fine scale population structure in European hake (Merluccius merluccius) in the Northeast Atlantic

Recently, there have been reports of increased abundance and landings of European hake in the northern part of the species range. Biological studies are however scarce and information about finer scale population structure important for stock assessments and fishery management is largely lacking. Here, we report on a population genetic study using neutral and outlier SNP loci assessing population structure in hake in the north-eastern parts of its range in the Atlantic. Hake samples from localities along the west coast of Norway, the Kattegat, the northern North Sea, and one locality in the Bay of Biscay were analysed using 53 SNPs, six of which were outliers potentially influenced by natural selection. We detected small-scale structure among northern samples, all of which were also distinct from Bay of Biscay hake, with the exception of a few individuals from the North Sea and the coast of Norway who clustered genetically together with Bay of Biscay hake. Our findings suggest that the present management unit of a single northern stock of hake is not biologically correct, and that there is more detail in the fine-scale population structure indicating that independent population dynamics could be expected in response to fishing patterns or changing environmental conditions.

Mapping fine-scale population distributions at the building level by integrating multisource geospatial big data

ABSTRACTFine-scale population distribution data at the building level play an essential role in numerous fields, for example urban planning and disaster prevention. The rapid technological development of remote sensing (RS) and geographical information system (GIS) in recent decades has benefited numerous population distribution mapping studies. However, most of these studies focused on global population and environmental changes; few considered fine-scale population mapping at the local scale, largely because of a lack of reliable data and models. As geospatial big data booms, Internet-collected volunteered geographic information (VGI) can now be used to solve this problem. This article establishes a novel framework to map urban population distributions at the building scale by integrating multisource geospatial big data, which is essential for the fine-scale mapping of population distributions. First, Baidu points-of-interest (POIs) and real-time Tencent user densities (RTUD) are analyzed by using a random forest algorithm to down-scale the street-level population distribution to the grid level. Then, we design an effective iterative building-population gravity model to map population distributions at the building level. Meanwhile, we introduce a densely inhabited index (DII), generated by the proposed gravity model, which can be used to estimate the degree of residential crowding. According to a comparison with official community-level census data and the results of previous population mapping methods, our method exhibits the best accuracy (Pearson R = .8615, RMSE = 663.3250, p < .0001). The produced fine-scale population map can offer a more thorough understanding of inner city population distributions, which can thus help policy makers optimize the allocation of resources.

Clustering of 770,000 genomes reveals post-colonial population structure of North America

Despite strides in characterizing human history from genetic polymorphism data, progress in identifying genetic signatures of recent demography has been limited. Here we identify very recent fine-scale population structure in North America from a network of over 500 million genetic (identity-by-descent, IBD) connections among 770,000 genotyped individuals of US origin. We detect densely connected clusters within the network and annotate these clusters using a database of over 20 million genealogical records. Recent population patterns captured by IBD clustering include immigrants such as Scandinavians and French Canadians; groups with continental admixture such as Puerto Ricans; settlers such as the Amish and Appalachians who experienced geographic or cultural isolation; and broad historical trends, including reduced north-south gene flow. Our results yield a detailed historical portrait of North America after European settlement and support substantial genetic heterogeneity in the United States beyond that uncovered by previous studies.

RAD sequencing resolves fine-scale population structure in a benthic invertebrate: implications for understanding phenotypic plasticity.

The field of molecular ecology is transitioning from the use of small panels of classical genetic markers such as microsatellites to much larger panels of single nucleotide polymorphisms (SNPs) generated by approaches like RAD sequencing. However, few empirical studies have directly compared the ability of these methods to resolve population structure. This could have implications for understanding phenotypic plasticity, as many previous studies of natural populations may have lacked the power to detect genetic differences, especially over micro-geographic scales. We therefore compared the ability of microsatellites and RAD sequencing to resolve fine-scale population structure in a commercially important benthic invertebrate by genotyping great scallops (Pecten maximus) from nine populations around Northern Ireland at 13 microsatellites and 10 539 SNPs. The shells were then subjected to morphometric and colour analysis in order to compare patterns of phenotypic and genetic variation. We found that RAD sequencing was superior at resolving population structure, yielding higher Fst values and support for two distinct genetic clusters, whereas only one cluster could be detected in a Bayesian analysis of the microsatellite dataset. Furthermore, appreciable phenotypic variation was observed in size-independent shell shape and coloration, including among localities that could not be distinguished from one another genetically, providing support for the notion that these traits are phenotypically plastic. Taken together, our results suggest that RAD sequencing is a powerful approach for studying population structure and phenotypic plasticity in natural populations.

Genetic variation and fine-scale population structure in American pikas across a human-modified landscape

Natural resource extraction can represent a major human modification to the landscape. Habitat reclamation is becoming an increasingly important strategy for abating the loss of biodiversity associated with these developments; however, the demographic and genetic consequences of colonizing artificial habitat remain unknown in many species. Here, we investigated the genetic consequences of landscape modifications for the American pika (Ochotona princeps) relative to two major developments in British Columbia, Canada: a large open-pit copper mine (Highland Valley Copper) under partial reclamation and a bisecting major highway (97C). We assessed microsatellite genotypic data for 109 individuals across 15 sites located either within the mine on artificial habitat or on adjacent natural habitat both north and south of the highway. There were no significant differences in levels of heterozygosity, allelic richness or inbreeding between natural (n = 7) and artificial sites (n = 8). However, pikas residing on artificial habitat exhibited significantly higher relatedness estimates. Bayesian clustering analyses revealed two distinct genetic units corresponding to north and south of the highway, with further substructure detected in the south. Likewise, high genetic friction was detected in the central region of the area, largely corresponding to the highway and modified landscape associated with the mine. At a finer scale, pairwise estimates of differentiation and migration rates suggest little gene flow may be occurring among sites across the sampling area, with some evidence for directional migration from artificial to natural sites. Overall, artificial habitat has been successful in promoting occupancy for American pikas, however, barriers to gene flow likely associated with resource extraction and road construction limit connectivity across the landscape.

Genetic structure in the Sherpa and neighboring Nepalese populations

BackgroundWe set out to describe the fine-scale population structure across the Eastern region of Nepal. To date there is relatively little known about the genetic structure of the Sherpa residing in Nepal and their genetic relationship with the Nepalese. We assembled dense genotype data from a total of 1245 individuals representing Nepal and a variety of different populations resident across the greater Himalayan region including Tibet, China, India, Pakistan, Kazakhstan, Uzbekistan, Tajikistan and Kirghizstan. We performed analysis of principal components, admixture and homozygosity.ResultsWe identified clear substructure across populations resident in the Himalayan arc, with genetic structure broadly mirroring geographical features of the region. Ethnic subgroups within Nepal show distinct genetic structure, on both admixture and principal component analysis. We detected differential proportions of ancestry from northern Himalayan populations across Nepalese subgroups, with the Nepalese Rai, Magar and Tamang carrying the greatest proportions of Tibetan ancestry.ConclusionsWe show that populations dwelling on the Himalayan plateau have had a clear impact on the Northern Indian gene pool. We illustrate how the Sherpa are a remarkably isolated population, with little gene flow from surrounding Nepalese populations.

English

A wide range of life-history tactics can be found within salmonid fish. The genetic basis for these adaptations remains largely unknown, but we have sought to investigate any large scale genetic changes associated with a non-anadromous life cycle. After the most recent ice age (approximately 9,500 years ago), some populations of Atlantic salmon Salmo salar L., were trapped in fresh water and developed into isolated landlocked populations that managed to complete a full life cycle without ever reaching the marine environment. To explore whether this transition was accompanied by gene-loss events, high-throughput sequencing of a non-migratory Namsblank (‘smablank’), an Atlantic salmon from the river Namsen in Norway, was performed. There were no indications of loss of coding regions and a phylogenetic analysis based on the mitochondrial genome revealed a close genetic relationship between anadromous Atlantic salmon and Namsblank. Lack of large-scale genomic changes suggests that fine-scale (epi)genetic alterations and population genetics processes underlie adaptation to the landlocked life-style. Key words: Landlocked, gene-loss, salmon, Illumina.

Fine-Scale Population Estimation by 3D Reconstruction of Urban Residential Buildings.

Fine-scale population estimation is essential in emergency response and epidemiological applications as well as urban planning and management. However, representing populations in heterogeneous urban regions with a finer resolution is a challenge. This study aims to obtain fine-scale population distribution based on 3D reconstruction of urban residential buildings with morphological operations using optical high-resolution (HR) images from the Chinese No. 3 Resources Satellite (ZY-3). Specifically, the research area was first divided into three categories when dasymetric mapping was taken into consideration. The results demonstrate that the morphological building index (MBI) yielded better results than built-up presence index (PanTex) in building detection, and the morphological shadow index (MSI) outperformed color invariant indices (CIIT) in shadow extraction and height retrieval. Building extraction and height retrieval were then combined to reconstruct 3D models and to estimate population. Final results show that this approach is effective in fine-scale population estimation, with a mean relative error of 16.46% and an overall Relative Total Absolute Error (RATE) of 0.158. This study gives significant insights into fine-scale population estimation in complicated urban landscapes, when detailed 3D information of buildings is unavailable.

Population genomic data delineate conservation units in mottled ducks (Anas fulvigula)

Large multi-locus data sets provide an unprecedented opportunity for detecting fine-scale population structure and identifying populations that warrant independent conservation and management. In this study, we used 3161 polymorphic loci obtained from double-digest restriction-site associated DNA sequencing (ddRAD-seq) to examine genetic differentiation between populations of mottled ducks (Anas fulvigula) and their close relative, the mallard (A. platyrhynchos). Florida (FL) and western gulf coast (WGC) mottled ducks group into discrete populations in ordination analyses and can be assigned unambiguously to their population of origin based on multi-locus genotypes. Moreover, we find limited evidence of recent admixture between populations, and the range gap separating them is a significant barrier to gene flow. We found no evidence of diversifying selection in the sample of loci we analyzed; instead, the genome-wide pattern of differentiation between mottled duck populations is consistent with historical isolation and divergence by neutral genetic drift. Thus, our results indicate that FL and WGC mottled ducks are discrete populations on separate evolutionary pathways and should be managed as independent conservation units, and perhaps recognized as distinct subspecies given observed differences in plumage and morphology. In contrast, the recently established South Atlantic coastal zone population is indistinguishable from the WGC population but shows evidence of a small amount of admixture from FL mottled ducks. Our study demonstrates a quantitative, operational approach for delineating discrete populations for conservation using ddRAD-seq data; multi-locus genotypes allow for a robust test of historical isolation and evolutionary independence on much shorter evolutionary timescales than the overly conservative standard of reciprocal monophyly in mtDNA.