Empirical and philosophical problems with the subspecies rank

Species‐level taxonomy derives from empirical sources (data and techniques) that assess the existence of spatiotemporal evolutionary lineages via various species “concepts.” These concepts determine if observed lineages are independent given a particular methodology and ontology, which relates the metaphysical species concept to what “kind” of thing a species is in reality. Often, species concepts fail to link epistemology back to ontology. This lack of coherence is in part responsible for the persistence of the subspecies rank, which in modern usage often functions as a placeholder between the evolutionary events of divergence or collapse of incipient species. Thus, prospective events like lineages merging or diverging require information from unknowable future information. This is also conditioned on evidence that the lineage already has a detectably distinct evolutionary history. Ranking these lineages as subspecies can seem attractive given that many lineages do not exhibit intrinsic reproductive isolation. We argue that using subspecies is indefensible on philosophical and empirical grounds. Ontologically, the rank of subspecies is either identical to that of species or undefined in the context of evolutionary lineages representing spatiotemporally defined individuals. Some species concepts more inclined to consider subspecies, like the Biological Species Concept, are disconnected from evolutionary ontology and do not consider genealogy. Even if ontology is ignored, methods addressing reproductive isolation are often indirect and fail to capture the range of scenarios linking gene flow to species identity over space and time. The use of subspecies and reliance on reproductive isolation as a basis for an operational species concept can also conflict with ethical issues governing the protection of species. We provide a way forward for recognizing and naming species that links theoretical and operational species concepts regardless of the magnitude of reproductive isolation.     

Intricate evolutionary histories in montane species: a phylogenetic window into craniodental discrimination in the Peromyscus mexicanus species group (Mammalia: Rodentia: Cricetidae)

Mountain‐associated species, which exhibit allopatric distributions associated with elevation, endemisms and complex evolutionary histories, pose challenging evolutionary scenarios in which to discern the diversification of species. The Peromyscus mexicanus mice group, distributed along mountains in southern Mexico and Central America, is morphometrically variable, a key rationale for the ongoing controversy regarding its species delimitation. Based on the recognized 15 mitochondrial lineages for the group, we analysed external and craniodental morphometric variables to test whether lineages can be differentiated morphometrically and allow for the delimitation of species. We also aimed to test the prediction that the phylogenetic structure of the morphometric data is concordant with that of the molecular information. Based on 19 craniodental measurements from 521 specimens, multivariate and discriminant analyses showed that lineages are morphometrically discernible, representing distinct phenotypes, and that overall size and mandible measurements are significant features that discriminate lineages, supporting hypotheses about differences in feeding habits between species. Also, a pattern of increasing size with elevation was observed, further supported by specific morphological differences exhibited between highland and lowland lineages inhabiting the same mountain. Our results demonstrate that P. mexicanus is both genetically and morphometrically variable, where most highland montane species are differentiated from lowland species; also, a significant correlation between mitochondrial and morphometric information is indicative of phenetic concordance, altogether in agreement with a recent taxonomic proposal for the group. We suggest that the group's intricate diversification responds to ecological diversification and adaptation to a variety of mountain habitats and Pleistocene biogeographic climatic dynamics.     

Defining reproductively isolated units in a cryptic and syntopic species complex using mitochondrial and nuclear markers: the brooding brittle star, Amphipholis squamata (Ophiuroidea)

At a time when biodiversity is threatened, we are still discovering new species, and particularly in the marine realm. Delimiting species boundaries is the first step to get a precise idea of diversity. For sympatric species which are morphologically undistinguishable, using a combination of independent molecular markers is a necessary step to define separate species. Amphipholis squamata , a cosmopolitan brittle star, includes several very divergent mitochondrial lineages. These lineages appear totally intermixed in the field and studies on morphology and colour polymorphism failed to find any diagnostic character. Therefore, these mitochondrial lineages may be totally interbreeding presently. To test this hypothesis, we characterized the genetic structure of the complex in the French Mediterranean coast using sequences of mitochondrial DNA (16S) and for the first time, several nuclear DNA markers (introns and microsatellites). The data revealed six phylogenetic lineages corresponding to at least four biological species. These sibling species seem to live in syntopy. However, they seem to display contrasted levels of genetic diversity, suggesting they have distinct demographic histories and/or life-history traits. Genetic differentiation and isolation-by-distance within the French Mediterranean coasts are revealed in three lineages, as expected for a species without a free larval phase. Finally, although recombinant nuclear genotypes are common within mitochondrial lineages, the data set displays a total lack of heterozygotes, suggesting a very high selfing rate, a feature likely to have favoured the formation of the species complex.     

Seven new species within western Atlantic Starksia atlantica, S. lepicoelia, and S. sluiteri (Teleostei, Labrisomidae), with comments on congruence of DNA barcodes and species

Specimens of Starksia were collected throughout the western Atlantic, and a 650-bp portion of the mitochondrial gene cytochrome oxidase-c subunit I (COl) was sequenced as part of a re-analysis of species diversity of western Central Atlantic shorefishes. A neighbor-joining tree constructed from the sequence data suggests the existence of several cryptic species. Voucher specimens from each genetically distinct lineage and color photographs of vouchers taken prior to dissection and preservation were examined for diagnostic morphological characters. The results suggest that Starksia atlantica, Starksia lepicoelia, and Starksia sluiteri are species complexes, and each comprises three or more species. Seven new species are described. DNA data usually support morphological features, but some incongruence between genetic and morphological data exists. Genetic lineages are only recognized as species if supported by morphology. Genetic lineages within western Atlantic Starksia generally correspond to geography, such that members of each species complex have a very restricted geographical distribution. Increasing geographical coverage of sampling locations will almost certainly increase the number of Starksia species and species complexes recognized in the western Atlantic. Combining molecular and morphological investigations is bringing clarity to the taxonomy of many genera of morphologically similar fishes and increasing the number of currently recognized species. Future phylogenetic studies should help resolve species relationships and shed light on patterns of speciation in western Atlantic Starksia.     

The wheat curl mite Aceria tosichella (Acari: Eriophyoidea) is a complex of cryptic lineages with divergent host ranges: evidence from molecular and plant bioassay data

Aceria tosichella (the wheat curl mite, WCM) is a global pest of wheat and other cereals, causing losses by direct damage, as well as the transmission of plant viruses. The mite is considered to have an unusually wide host range for an eriophyoid species. The present study tested the commonly held assumption that WCM is a single, highly polyphagous species by assessing the host range of genetically distinct lineages of WCM occurring in Poland on different host plants. Genotyping was performed by analyzing nucleotide sequence data from fragments of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear D2 region of 28S rDNA. Mean between‐lineage distance estimated using COI data was found to be one order of magnitude greater than the within‐clade lineage and, in some cases, comparable to distances between WCM lineages and a congeneric outgroup species. Host acceptance was tested by quantifying population growth for different WCM mitochondrial (mt)DNA lineages when transferred from source host plants to test plants. These experiments revealed significant differences in host colonization ability between mtDNA lineages, ranging from highly polyphagous to more host‐specific. The present study reveals that WCM is composed of several discrete genetic lineages with divergent host‐acceptance and specificity traits. Genetic variation for host acceptance within A. tosichella s.l. may act as a reproductive barrier between these lineages, most of which had narrow host ranges. Two lineages appear to have high pest potential on cereals, whereas several others appear to specialize on wild grass species. We conclude that WCM is not a homogeneous species comprising polyphagous panmictic populations rather it is a complex of genetically distinct lineages with variable host ranges and therefore variable pest potential. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 165–180.     

Considering gene flow when using coalescent methods to delimit lineages of North American pitvipers of the genus Agkistrodon

Examining species diversity and mechanisms of speciation using coalescent models provides a framework for how regional diversity is accrued, even in well‐studied areas such as the Nearctic. It is likely, that gene flow among closely‐related species with adjacent distributions may be common. However, the absence of gene flow is a primary assumption of many phylogeographical methods that produce species trees and delimit species using Bayesian or likelihood functions in a coalescent framework. In the present study, we examine delimitation when gene flow between species is present using empirical datasets from two species of North American pitvipers of the genus Agkistrodon. We also use niche modelling to determine whether these young lineages occur in distinct environmental niches. To manage the problem of gene flow between species, we first identify admixed individuals, demonstrate that gene flow has occurred, and then identify the impact of alternative population assignments of admixed individuals on delimitation posterior probabilities. In addition, we examine the influence of mitochondrial genes relative to other loci combined in coalescent analyses that delimit species. Here, we find that the copperheads (Agkistrodon contortrix) and the cottonmouths (Agkistrodon piscivorus) are each composed of two distinct species, with each occupying different niches. Importantly, we find that species can be delimited when the amount of gene flow between lineages is low, although the methods are acutely sensitive to population assignment of individuals. © 2014 The Linnean Society of London     

Using phylogeographic analyses of gene trees to test species status and processes

A gene tree is an evolutionary reconstruction of the genealogical history of the genetic variation found in a sample of homologous genes or DNA regions that have experienced little or no recombination. Gene trees have the potential of straddling the interface between intra- and interspecific evolution. It is precisely at this interface that the process of speciation occurs, and gene trees can therefore be used as a powerful tool to probe this interface. One application is to infer species status. The cohesion species is defined as an evolutionary lineage or set of lineages with genetic exchangeability and/or ecological interchangeability. This species concept can be phrased in terms of null hypotheses that can be tested rigorously and objectively by using gene trees. First, an overlay of geography upon the gene tree is used to test the null hypothesis that the sample is from a single evolutionary lineage. This phase of testing can indicate that the sampled organisms are indeed from a single lineage and therefore a single cohesion species. In other cases, this null hypothesis is not rejected due to a lack of power or inadequate sampling. Alternatively, this null hypothesis can be rejected because two or more lineages are in the sample. The test can identify lineages even when hybridization and lineage sorting occur. Only when this null hypothesis is rejected is there the potential for more than one cohesion species. Although all cohesion species are evolutionary lineages, not all evolutionary lineages are cohesion species. Therefore, if the first null hypothesis is rejected, a second null hypothesis is tested that all lineages are genetically exchangeable and/or ecologically interchangeable. This second test is accomplished by direct contrasts of previously identified lineages or by overlaying reproductive and/or ecological data upon the gene tree and testing for significant transitions that are concordant with the previously identified lineages. Only when this second null hypothesis is rejected is a lineage elevated to the status of cohesion species. By using gene trees in this manner, species can be identified with objective, a priori criteria with an inference procedure that automatically yields much insight into the process of speciation. When one or more of the null hypotheses cannot be rejected, this procedure also provides specific guidance for future work that will be needed to judge species status.     

A paradigm shift in our view of species drives current trends in biological classification

Discontent about changes in species classifications has grown in recent years. Many of these changes are seen as arbitrary, stemming from unjustified conceptual and methodological grounds, or leading to species that are less distinct than those recognised in the past. We argue that current trends in species classification are the result of a paradigm shift toward which systematics and population genetics have converged and that regards species as the phylogenetic lineages that form the branches of the Tree of Life. Species delimitation now consists of determining which populations belong to which individual phylogenetic lineage. This requires inferences on the process of lineage splitting and divergence, a process to which we have only partial access through incidental evidence and assumptions that are themselves subject to refutation. This approach is not free of problems, as horizontal gene transfer, introgression, hybridisation, incorrect assumptions, sampling and methodological biases can mislead inferences of phylogenetic lineages. Increasing precision is demanded through the identification of both sister relationships and processes blurring or mimicking phylogeny, which has triggered, on the one hand, the development of methods that explicitly address such processes and, on the other hand, an increase in geographical and character data sampling necessary to infer/test such processes. Although our resolving power has increased, our knowledge of sister relationships – what we designate as species resolution – remains poor for many taxa and areas, which biases species limits and perceptions about how divergent species are or ought to be. We attribute to this conceptual shift the demise of trinominal nomenclature we are witnessing with the rise of subspecies to species or their rejection altogether; subspecies are raised to species if they are found to correspond to phylogenetic lineages, while they are rejected as fabricated taxa if they reflect arbitrary partitions of continuous or non-hereditary variation. Conservation strategies, if based on taxa, should emphasise species and reduce the use of subspecies to avoid preserving arbitrary partitions of continuous variation; local variation is best preserved by focusing on biological processes generating ecosystem resilience and diversity rather than by formally naming diagnosable units of any kind. Since many binomials still designate complexes of species rather than individual species, many species have been discovered but not named, geographical sampling is sparse, gene lineages have been mistaken for species, plenty of species limits remain untested, and many groups and areas lack adequate species resolution, we cannot avoid frequent changes to classifications as we address these problems. Changes will not only affect neglected taxa or areas, but also popular ones and regions where taxonomic research remained dormant for decades and old classifications were taken for granted.     

An integrative taxonomic review of the Natal mountain catfish,Amphilius natalensis Boulenger 1917 (Siluriformes,Amphiliidae), with description of four new species

Species delimitation is a permanent issue in systematics. The increasing recognition of geographically isolated populations as independent lineages allowed by new methods of analysis has inflated the species-populations dilemma, which involves deciding whether to consider separate lineages as different species or structured genetic populations. This is commonly observed between fishes of adjacent river basins, with some lineages being considered allopatric sister species and others considered isolated populations or variants of the same species. Pseudocorynopoma doriae is a characid diagnosed from its single congener by the number of anal-fin rays and sexually dimorphic characters of males, including distinct fin colouration. The authors found variation in the colour pattern between isolated populations previously identified as P. doriae but no variation in scale or fin-ray counts. They analysed molecular evidence at the population level and morphological differences related to life history (e.g., colour dimorphism related to inseminating behaviour). The results provide compelling evidence for the recognition of a new species of Pseudocorynopoma despite the lack of discrete differences in meristic data. The recognition of the new species is consistent with biogeographical evidence for the long-term isolation of the respective river drainages and with differences between the ichthyofaunal communities of these rivers.     

Gall morpho-type corresponds to separate species of gall-inducing thrips (Thysanoptera: Phlaeothripidae)

Kladothrips rugosus Froggatt has previously been considered a single polyphagous species that, in Australia, induces galls on several species of Acacia , with the gall structure varying both within and between hosts. On Acacia papyrocarpa , two types of gall are induced by this species, one with the surface ridged but the other with the surface smooth. Using sequence data from cytochrome oxidase subunit I (CO I ) and elongation factor-1 alpha gene fragments, we show that the thrips inducing these two gall-types are genetically distinct and comprise separate lineages. Uncorrected ' p ' distances calculated from CO I gene fragments were 0.000 and 0.006 within lineages and 0.074 and 0.078 between lineages. The between-lineage distances are comparable with distances between morphologically distinct species of other Acacia gall-thrips. Re-examination of adult thrips from the two gall types revealed consistent differences in body colour, as well as in body sculpture. Together with observations on gall founding behaviour, these data indicate that the thrips populations in the two gall types on A. papyrocarpa are reproductively isolated and should be considered as separate species. The form from smooth galls on A. papyrocarpa is therefore described as Kladothrips nicolsoni sp. nov. , although the form from ridged galls can be considered only as ' K. rugosus agg.'. These inconsistencies in the taxonomic status of the various units within the K. rugosus species complex are discussed, although most of them cannot be distinguished morphologically at present.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 555–563.     

Cryptic diversity in the pen shell Atrina pectinata (Bivalvia: Pinnidae): high divergence and hybridization revealed by molecular and morphological data

Cryptic species have been increasingly revealed in the marine realm through an analytical approach incorporating multiple lines of evidence (e.g., mtDNA, nuclear genes and morphology). Illustrations of cryptic taxa improve our understanding of species diversity and evolutionary histories within marine animals. The pen shell Atrina pectinata is known to exhibit extensive morphological variations that may harbour cryptic diversity. In this study, we investigated A. pectinata populations along the coast of China and one from Japan to explore possible cryptic diversity and hybridization using a combination of mitochondrial (cytochrome c oxidase subunit I, mtCOI) and nuclear (ribosomal internal transcribed spacer, nrITS) genes as well as morphology. Phylogenetic analyses of mtCOI ‘DNA barcoding gene’ sequences resolved six divergent lineages with intralineage divergences between 0.4% and 0.8%. Interlineage sequence differences ranged from 4.3% to 22.0%, suggesting that six candidate cryptic species are present. The nrITS gene revealed five deep lineages with Kimura 2‐parameter distances of 3.7–30.3%. The five nuclear lineages generally corresponded to mtCOI lineages 1–4 and (5 + 6), suggestive of five distinct evolutionary lineages. Multiple nrITS sequences of significant variance were found within an individual, clearly implying recent hybridization events between/among the evolutionary lineages, which contributed to cytonuclear discordance. Morphologically, five morphotypes matched the five genetic lineages, although the intermediates may well blur the boundaries of different morphotypes. This study demonstrates the importance of combining multiple lines of evidence to explore species cryptic diversity and past evolutionary histories.     

The cladistic solution to the species problem

The correct explanation of why species, in evolutionary theory, are individuals and not classes is the cladistic species concept. The cladistic species concept defines species as the group of organisms between two speciation events, or between one speciation event and one extinction event, or (for living species) that are descended from a speciation event. It is a theoretical concept, and therefore has the virtue of distinguishing clearly the theoretical nature of species from the practical criteria by which species may be recognized at any one time. Ecological or biological (reproductive) criteria may help in the practical recognition of species. Ecological and biological species concepts are also needed to explain why cladistic species exist as distinct lineages, and to explain what exactly takes place during a speciation event. The ecological and biological species concepts work only as sub-theories of the cladistic species concept and if taken by themselves independently of cladism they are liable to blunder. The biological species concept neither provides a better explanation of species indivudualism than the ecological species concept, nor, taken by itself, can the biological species concept even be reconciled with species individualism. Taking the individuality of species seriously requires subordinating the biological, to the cladistic, species concept.     

Genomic data reveal two distinct species from the widespread alpine ginger Roscoea tibetica Batalin (Zingiberaceae)

Species delimitation is a key foundation for exploring biodiversity. However, the existence of continuous phenotypic variation in widespread species challenges accurate species delimitation based on classical taxonomy. In this study, we investigated the cryptic diversity of a widespread herb (Roscoea tibetica Batalin) in a biodiversity hotspot (the Hengduan Mountains, China) using genotyping by sequencing, examining morphological traits, developing species distribution models, and simulating demographic history. Phylogenomic reconstruction, principal component analysis, and genetic structure inferences indicated that previously reported R. tibetica comprised two monophyletic lineages with a deep divergence. Several morphological diagnostic characteristics were discovered from field and common garden that corresponded to these independent evolutionary lineages. Species distribution models illustrated significant ecological divergence between both lineages. All evidence strongly supported that R. tibetica, as described in previous taxonomy, actually comprises two distinct species. Model test of gene flow and effective population size changes in fastsimcoal2, and a negative Tajima's D-value suggested that recent contact likely occurred between the two lineages. Our results proposed that cryptic diversity in previously reported R. tibetica was possibly associated with phenotypic plasticity in heterogeneous environments and morphological convergence in similar habitats. This study suggests that caution should be exercised when attempting to gain biological insight into species with large-scale morphological variation, and species delimitation should be done in advance.     

Environmental factors determining the phylogenetic structure of C4 grass communities

Aim To determine how the distribution of species richness is associated with environmental factors for the four major C₄ grass lineages in South Africa, as a means to explore the mechanisms responsible. Location South Africa, Lesotho and Swaziland. Methods The geographical distributions of species richness for four major C₄ grass lineages (Aristidoideae, Chloridoideae, Andropogoneae and Paniceae) were sourced from a recently published flora that divided the study region into different vegetation types. Mean values of potential environmental correlates were calculated for each vegetation type, and the relative importances of these were determined using single‐ and multiple‐predictor generalized linear models, with and without control for spatial autocorrelation. Model selection of the multiple‐predictor generalized linear models was conducted using an Akaike’s information criterion–information theoretic approach. Association with wet, intermediate or dry, shady or open, and disturbed or undisturbed habitats was also determined for each C₄ grass clade using habitat data for all the grass species, and analysed using chi‐square tests of independence. Results Andropogoneae and Paniceae are most species‐rich in areas of high precipitation and in mesic habitats. Andropogoneae are associated with high fire frequencies. Species richness in Andropogoneae decreases and in Paniceae increases in relation to livestock density. Chloridoideae species richness is relatively constant across South Africa, but is highest where there are infrequent fires, high temperatures and basic soils, and in mesic and disturbed habitats. Aristidoideae are most species‐rich in arid regions and in habitats with high temperatures, and are associated with disturbed habitats. Main conclusions Environmental variables other than precipitation, including temperature, fire frequency and grazing pressure, are strongly associated with the contrasting distributions of species richness for the various C₄ grass clades in South Africa. Our results suggest that ecological sorting is an important determinant of phylogenetic patterns in the species richness of these C₄ grass lineages.     

Spatial patterns of phylogenetic diversity and endemism in the Western Ghats,India: A case study using ancient predatory arthropods

The Western Ghats (WG) mountain chain in peninsular India is a global biodiversity hotspot, one in which patterns of phylogenetic diversity and endemism remain to be documented across taxa. We used a well‐characterized community of ancient soil predatory arthropods from the WG to understand diversity gradients, identify hotspots of endemism and conservation importance, and highlight poorly studied areas with unique biodiversity. We compiled an occurrence dataset for 19 species of scolopendrid centipedes, which was used to predict areas of habitat suitability using bioclimatic and geomorphological variables in Maxent. We used predicted distributions and a time‐calibrated species phylogeny to calculate taxonomic and phylogenetic indices of diversity, endemism, and turnover. We observed a decreasing latitudinal gradient in taxonomic and phylogenetic diversity in the WG, which supports expectations from the latitudinal diversity gradient. The southern WG had the highest phylogenetic diversity and endemism, and was represented by lineages with long branch lengths as observed from relative phylogenetic diversity/endemism. These results indicate the persistence of lineages over evolutionary time in the southern WG and are consistent with predictions from the southern WG refuge hypothesis. The northern WG, despite having low phylogenetic diversity, had high values of phylogenetic endemism represented by distinct lineages as inferred from relative phylogenetic endemism. The distinct endemic lineages in this subregion might be adapted to life in lateritic plateaus characterized by poor soil conditions and high seasonality. Sites across an important biogeographic break, the Palghat Gap, broadly grouped separately in comparisons of species turnover along the WG. The southern WG and Nilgiris, adjoining the Palghat Gap, harbor unique centipede communities, where the causal role of climate or dispersal barriers in shaping diversity remains to be investigated. Our results highlight the need to use phylogeny and distribution data while assessing diversity and endemism patterns in the WG.     

Evolutionary Phycology: Toward a Macroalgal Species Conceptual Framework

Species concepts formalize evolutionary and ecological processes, but often conflict with one another when considering the mechanisms that ultimately lead to species delimitation. Evolutionary biologists are, however, recognizing that the conceptualization of a species is separate and distinct from the delimitation of species. Indeed, if species are generally defined as separately evolving metapopulation lineages, then characteristics, such as reproductive isolation or monophyly, can be used as evidence of lineage separation and no longer conflict with the conceptualization of a species. However, little of this discussion has addressed the formalization of this evolutionary conceptual framework for macroalgal species. This may be due to the complexity and variation found in macroalgal life cycles. While macroalgal mating system variation and patterns of hybridization and introgression have been identified, complex algal life cycles generate unique eco-evolutionary consequences. Moreover, the discovery of frequent macroalgal cryptic speciation has not been accompanied by the study of the evolutionary ecology of those lineages, and, thus, an understanding of the mechanisms underlying such rampant speciation remain elusive. In this perspective, we aim to further the discussion and interest in species concepts and speciation processes in macroalgae. We propose a conceptual framework to enable phycological researchers and students alike to portray these processes in a manner consistent with dialogue at the forefront of evolutionary biology. We define a macroalgal species as an independently evolving metapopulation lineage, whereby we can test for reproductive isolation or the occupation of distinct adaptive zones, among other mechanisms, as secondary lines of supporting evidence.     

Evolutionary systematics of the Australian Eocyzicus fauna (Crustacea: Branchiopoda: Spinicaudata) reveals hidden diversity and phylogeographic structure

Although ‘large branchiopods’ are an important faunal element of the temporary water bodies in Australia's vast (semi)arid regions, knowledge of their diversity, distribution and ecology is still poor. Here, on the basis of one mitochondrial [cytochrome oxidase subunit I (COI)] and three nuclear (EF1α, ITS2 and 28S) markers, we present new data relating to the diversity and phylogeography of eastern and central Australian Eocyzicus (Spinicaudata) fauna. Using a combination of phylogenetic, haplotype network and DNA barcoding analyses of COI, 312 individuals were grouped into eleven main lineages. To infer whether these lineages are reproductively isolated from each other (the prerequisite for species delineation according to the Biological or Hennigian Species Concepts), separate analyses of each nuclear marker were performed on a subset of specimens. Although some lineages are non‐monophyletic in the analysis of one nuclear marker, this is mostly attributed to processes such as incomplete lineage sorting rather than ongoing reproduction. The eleven lineages translate into at least seven species whose reproductive isolation is additionally indicated by sympatry, including both Australian Eocyzicus species previously described. Another three lineages may constitute further species, but their clear allopatric distribution rendered the test for reproductive isolation inapplicable. One lineage appears not to be reproductively isolated and is therefore considered a genetically distinct lineage within one of the other species, and one divergent lineage within E. argillaquus may constitute an additional species. Although sympatry is very common – six species occur in the central Paroo River catchment in eastern Australia, for instance – syntopic occurrence is rare. It is possible that a combination of differing habitat preferences and priority effects inhibits the presence of more than one Eocyzicus species per water body. There is little to no genetic differentiation between certain populations of the species found in eastern and central Australia (e.g. the Murray–Darling Basin, the Bulloo River catchment and the eastern and northern Lake Eyre Basin; LEB), suggesting high dispersal rates within this large area. Between the central Australian populations themselves, however (e.g. those inhabiting the central and western LEB), genetic differentiation is pronounced, probably as a result of the lack of abundance of important dispersal vectors (aquatic birds) and the lower diversity and density of suitable habitats in the area. The most prominent biogeographical break exists towards north‐eastern Australia (north‐east LEB), which does not share species with any other region studied.     

Nuclear DNA recapitulates the cryptic mitochondrial lineages of Lumbricus rubellus and suggests the existence of cryptic species in an ecotoxological soil sentinel

Mitochondrial DNA analysis has revealed two distinct phylogenetic lineages within the ecotoxological sentinel earthworm model Lumbricus rubellus Hoffmeister, 1843. The existence of these lineages could complicate ecotoxicological studies that use the species as a sentinel for soil contamination testing, as they may respond differently to contamination; however, as mitochondrial haplotypes are not always expected to segregate in the same way as chromosomal DNA in natural populations, we further investigated this issue by using nuclear DNA markers (microsatellites) to measure genetic diversity, differentiation, and gene flow in sympatric populations of the two L. rubellus lineages at two sites in South Wales. Our results show that sympatric populations of the two lineages are more genetically differentiated than geographically distant populations of the same lineage, and Bayesian clustering analysis revealed no evidence of gene flow between the lineages at either site. Additionally, DNA sequencing of these microsatellite loci uncovered substantial differentiation between lineages at homologous flanking regions. Overall our findings indicate a high degree of nuclear genetic differentiation between the two lineages of L. rubellus, implying reproductive isolation at the two study sites and therefore the potential existence of cryptic species. The existence of two cryptic taxa has major implications for the application of L. rubellus as an ecotoxicological sentinel. It may therefore be necessary to consider the lineages as separate taxa during future ecotoxicological studies. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 780–795.     

Population analysis using the nuclear white gene detects Pliocene/Pleistocene lineage divergence within Anopheles nuneztovari in South America

Anopheles (Nyssorhynchus) nuneztovari Gabaldón (Diptera: Culicidae), a locally important malaria vector in some regions of South America, has been hypothesized to consist of at least two cryptic incipient species. We investigated its phylogeographic structure in several South American localities to determine the number of lineages and levels of divergence using the nuclear white gene, a marker that detected two recently diverged genotypes in the primary Neotropical malaria vector Anopheles darlingi Root. In An. nuneztovari, five distinct lineages (1-5) were elucidated: (1) populations from northeastern and central Amazonia; (2) populations from Venezuela east and west of the Andes; (3) populations from Colombia and Venezuela west of the Andes; (4) southeastern and western Amazonian Brazil populations, and (5) southeastern and western Amazonian Brazil and Bolivian populations. There was a large amount of genetic differentiation among these lineages. The deepest and earliest divergence was found between lineage 3 and lineages 1, 2 and 4, which probably accounts for the detection of lineage 3 in some earlier studies. The multiple lineages within Amazonia are partially congruent with previous mtDNA and ITS2 data, but were undetected in many earlier studies, probably because of their recent (Pleistocene) divergence and the differential mutation rates of the markers. The estimates for the five lineages, interpreted as recently evolved or incipient species, date to the Pleistocene and Pliocene. We hypothesize that the diversification in An. nuneztovari is the result of an interaction between the Miocene/Pliocene marine incursion and Pleistocene climatic changes leading to refugial isolation. The identification of cryptic lineages in An. nuneztovari could have a significant impact on local vector control measures.