Out of Florida: mtDNA reveals patterns of migration and Pleistocene range expansion of the Green Anole lizard (Anolis carolinensis)

Anolis carolinensis is an emerging model species and the sole member of its genus native to the United States. Considerable morphological and physiological variation has been described in the species, and the recent sequencing of its genome makes it an attractive system for studies of genome variation. To inform future studies of molecular and phenotypic variation within A. carolinensis, a rigorous account of intraspecific population structure and relatedness is needed. Here, we present the most extensive phylogeographic study of this species to date. Phylogenetic analyses of mitochondrial DNA sequence data support the previous hypothesis of a western Cuban origin of the species. We found five well‐supported, geographically distinct mitochondrial haplotype clades throughout the southeastern United States. Most Florida populations fall into one of three divergent clades, whereas the vast majority of populations outside Florida belong to a single, shallowly diverged clade. Genetic boundaries do not correspond to major rivers, but may reflect effects of Pleistocene glaciation events and the Appalachian Mountains on migration and expansion of the species. Phylogeographic signal should be examined using nuclear loci to complement these findings.     

The geography of speciation in narrow‐range endemics of the ‘Haenydra’ lineage (Coleoptera,Hydraenidae, Hydraena)

Aim We test whether species of western Mediterranean aquatic Coleoptera of the ‘Haenydra’ lineage (Hydraenidae, Hydraena) originated through: (1) successive periods of dispersal and speciation, (2) range fragmentation by random vicariance, or (3) range fragmentation by geographic isolation owing to a general reduction of population density. Location Europe. Methods To discriminate between scenarios we use contrasting predictions of the relationship between phylogenetic and geographic distance. The phylogeny was based on 3 kb of four mitochondrial and two nuclear gene fragments of about half of the known species of ‘Haenydra’, including most western Mediterranean taxa. Divergences were estimated using a molecular clock. The relationship between phylogenetic and geographic distance was tested using bivariate plots, Mantel tests and comparison of the observed phylogeny with the one minimizing geographic distances between species, as measured using Euclidean minimum spanning trees (EMSTs). Results The monophyly of ‘Haenydra’ was strongly supported, although its phylogenetic placement was not resolved. ‘Haenydra’ was estimated to be of late Miocene age, with most species originating during the Pleistocene. In two clades (Hydraena tatii and Hydraena emarginata clades) there was a significant association between geographic and phylogenetic distance, and the reconstructed phylogeny was identical to that obtained through the EMST, demonstrating a strong non‐randomness of the geographic distribution of the species. In two other clades (Hydraena iberica and Hydraena bitruncata clades) there was no association between geographic and phylogenetic distance, and the observed phylogeny was not the one minimizing geographic distances. In one of the clades this seems to be due to a secondary, recent range expansion of one species (H. iberica), which erased the geographic signal of their distributions. Main conclusions We show that it is possible to obtain strong evidence of stasis of the geographic ranges of narrow‐range endemic species through the study of their phylogenetic relationships and current distributions. In at least two of the studied clades, current species seem to have originated through the fragmentation of a more widely distributed species, without further range movements. A process of range expansion and fragmentation may have occurred repeatedly within the ‘Haenydra’ lineage, contributing to the accumulation of narrow‐range endemics in Mediterranean Pleistocene refugia.     

Climatic stability and genetic divergence in the tropical insular lizard Anolis krugi,the Puerto Rican ‘Lagartijo Jardinero de la Montaña’

Two factors that can lead to geographic structuring in conspecific populations are barriers to dispersal and climatic stability. Populations that occur in different physiographic regions may be restricted to those areas by physical and/or ecological barriers, which may facilitate the formation of phylogeographic clades. Long‐term climatic stability can also promote genetic diversification, because new clades are more likely to evolve in areas that experience lesser climatic shifts. We conducted a phylogeographic study of the Puerto Rican lizard Anolis krugi to assess whether populations of this anole show genetic discontinuities across the species’ range, and if they do, whether these breaks coincide with the boundaries of the five physiographic regions of Puerto Rico. We also assessed whether interpopulation genetic distances in A. krugi are positively correlated with relative climatic stability in the island. Anolis krugi exhibits genetic structuring, but the phylogroups do not correspond to the physiographic regions of Puerto Rico. We used climatic reconstructions of two environmental extremes of the Quaternary period, the present conditions and those during the last glacial maximum (LGM), to quantify the degree of climatic stability between sampling locations. We documented positive correlations between genetic distances and relative climatic stability, although these associations were not significant when corrected for autocorrelation. Principal component analyses indicated the existence of climatic niche differences between some phylogeographic clades of A. krugi. The approach that we employed to assess the relationship between climatic stability and the genetic architecture of A. krugi can also be used to investigate the impact of factors such as the spatial distribution of food sources, parasites, predators or competitors on the genetic landscape of a species.     

Molecular phylogeny and phylogeography of the Cuban cave-fishes of the genus Lucifuga: evidence for cryptic allopatric diversity

Underground environments are increasingly recognized as reservoirs of faunal diversity. Extreme environmental conditions and limited dispersal ability of underground organisms have been acknowledged as important factors promoting divergence between species and conspecific populations. However, in many instances, there is no correlation between genetic divergence and morphological differentiation. Lucifuga Poey is a stygobiotic fish genus that lives in Cuban and Bahamian caves. In Cuba, it offers a unique opportunity to study the influence of habitat fragmentation on the genetic divergence of stygobiotic species and populations. The genus includes four species and one morphological variant that have contrasting geographical distributions. In this study, we first performed a molecular phylogenetic analysis of the Lucifuga Cuban species using mitochondrial and nuclear markers. The mitochondrial phylogeny revealed three deeply divergent clades that were supported by nuclear and morphological characters. Within two of these main clades, we identified five lineages that are candidate cryptic species and a taxonomical synonymy between Lucifuga subterranea and Lucifuga teresinarum. Secondly, phylogeographic analysis using a fragment of the cytochrome b gene was performed for Lucifuga dentata, the most widely distributed species. We found strong geographical organization of the haplotype clades at different geographic scales that can be explained by episodes of dispersal and population expansion followed by population fragmentation and restricted gene flow. At a larger temporal scale, these processes could also explain the diversification and the distribution of the different species.     

Phylogenetic relationships and ecological niche conservatism in killifish (Profundulidae) in Mesoamerica

The family Profundulidae is a group of small-sized fish species distributed between southern Mexico and Honduras, where they are frequently the only fish representatives at higher elevations in the basins where they occur. We characterized their ecological niche using different methods and metrics drawn from niche modelling and by re-examining phylogenetic relationships of a recently published molecular phylogeny of this family to gain a better understanding of its biogeographic and evolutionary history. We assessed both lines of evidence from the perspective of niche conservatism to set a foundation for discussing hypotheses about the processes underlying the distribution and evolution of the group. In fish clades where the species composition is not clear, we examined whether niche classification could be informative to discriminate groups geographically and ecologically consistent with any of the different hypotheses of valid species. The characterization of the ecological niche was carried out using the Maxent algorithm under different parameterizations and the projection of the presence on the main components of the most relevant environmental coverage, and the niche comparison was calculated with two indices (D and I), both in environmental space and in that projected geographically. With the molecular data, a species tree was generated using the *BEAST method. The comparison of these data was calculated with an age-overlap correlation test. Based on the molecular phylogeny and on niche overlap analyses, we uncovered strong evidence to support the idea that ecologically similar species are not necessarily sister species. The correlation analysis for genetic distance and niche overlap was not significant (P > 0.05). In clades with taxonomic conflicts, we only identified Profundulus oaxacae as a geographically and ecologically distinct group from P. punctatus. All the evidence considered leads us to propose that Profundulidae do not show evidence of niche conservatism and that there are reasons to consider P. oaxacae as a valid species. Our study suggests that niche divergence is a driving evolutionary force that caused the diversification and speciation processes of the Profundulidae, along with the geological and climatic events that promoted the expansion or contraction of suitable environments.     

Phylogeny of Schisandraceae based on morphological data: evidence from modern plants and the fossil record

Schisandraceae are traditionally subdivided in two genera, Schisandra and Kadsura, based on differences in the organisation of the floral receptacle, the carpels, and the presence or absence of a ``pseudostigma'. Recently, phylogenetic analyses utilizing ITS sequence data and morphological data resulted in incongruent tree topologies, with the morphological trees suggesting monophyly of the two genera, whereas ITS trees did not resolve Schisandra and Kadsura as monophyletic clades. In the present paper we study seed morphology and leaf epidermal features of 22 species of Schisandraceae in order to provide additional data for a morphological data matrix. Seed morphological characters are highly homoplastic and do not yield further evidence for monophyly of the two genera. Instead, a number of characters appear to support sister group relationships between taxa within the genera, such as, for instance, for K. coccinea and K. scandens, both of which have large seeds along with a multi-layered mesotesta. Considering leaf epidermal characteristics, species of Kadsura were found to be consistently amphistomatic, whereas species of Schisandra are always hypostomatic. Phylogenetic analysis using the extended data matrix resulted in weakly supported Kadsura and Schisandra clades with five and four synapomorphies indicating monophyly of Kadsura and Schisandra, respectively. Fossils ascribed to Schisandraceae date back to the Late Cretaceous. These are tri-and hexacolpate pollen types displaying a combination of features found in modern Schisandraceae and partly also in Illiciaceae. Leaf remains from this period are poorly preserved and difficult to ascribe to Schisandraceae because of the lack of synapomorphies for the family. In the Early Cainozoic, leaf and seed remains from North America and Europe unambiguously belong to the family. Seeds from the Eocene of North America show some similarities to the modern Schisandra glabra from North America, while fossils from Europe show more similarities to modern Asian species.     

Polymorphism in a common Atlantic reef coral (Montastraea cavernosa) and its long-term evolutionary implications

Recent advances in morphometrics and genetics have led to the discovery of numerous cryptic species in coral reef ecosystems. A prime example is the Montastraea annularis scleractinian coral species complex, in which morphological, genetic, and reproductive data concur on species boundaries, allowing evaluation of long-term patterns of speciation and evolutionary innovation. Here we test for cryptic species in the Atlantic species, Montastraea cavernosa, long recognized as polymorphic. Our modern samples consist of 94 colonies collected at four locations (Belize, Panamá, Puerto Rico in the Caribbean; S?o Tomé in the Eastern Atlantic). Our fossil samples consist of 78 colonies from the Plio-Pleistocene of Costa Rica and Panamá. Landmark morphometric data were collected on thin sections of 46 modern and 78 fossil colonies. Mahalanobis distances between colonies were calculated using Bookstein coordinates, revealing two modern and four fossil morphotypes. The remaining 48 of the 94 modern colonies were assigned to morphotype using discriminant analysis of calical measurements. Cross-tabulation and multiple comparisons tests show no significant morphological differences among geographic locations or water depths. Patterns of variation within and among fossil morphotypes are similar to modern morphotypes. DNA sequence data were collected for two polymorphic nuclear loci (β-tub1 and β-tub2) on all 94 modern colonies. Haplotype networks show that both genes consist of two clades, but morphotypes are not associated with genetic clades. Genotype frequencies and two-locus genotype assignments indicate genetic exchange across clades, and ϕst values show no genetic differentiation between morphotypes at different locations. Taken together, our morphological and genetic results do not provide evidence for cryptic species in M. cavernosa, but indicate instead that this species has an unusually high degree of polymorphism over a wide geographic area and persisting for >25 million years (myr).     

Genetic and Physiological Diversity in the Diatom Nitzschia inconspicua

Nitzschia inconspicua is an ecologically important diatom species, which is believed to have a widespread distribution and to be tolerant to salinity and to organic or nutrient pollution. However, its identification is not straightforward and there is no information on genetic and ecophysiological diversity within the species. We used morphological, molecular (rbcL and LSU D1–D3), ecophysiological and reproductive data to investigate whether N. inconspicua constitutes a single species with a broad ecological tolerance or two or more cryptic species with shared or different ecological preferences. Molecular genetic data for clones from upstream and deltaic sites in the Ebro River basin (Catalonia, Spain) revealed seven N. inconspicua rbcL + LSU genotypes grouped into three major clades. Two of the clades were related to other Nitzschia and Denticula species, making N. inconspicua paraphyletic and suggesting the need for taxonomic revision. Most clones were observed to be automictic, exhibiting paedogamy, and so the biological species concept cannot be used to establish species boundaries. Although there were morphological differences among clones, we found no consistent differences among genotypes belonging to different clades, which are definable only through sequence data. Nevertheless, separating the genotypes could be important for ecological purposes because two different ecophysiological responses were encountered among them.     

Species boundaries in Malagasy snakes of the genus Madagascarophis (Serpentes: Colubridae sensu lato) assessed by nuclear and mitochondrial markers

We studied mitochondrial divergence in 27 individuals of colubrid snakes of the genus Madagascarophis Mertens from most of its distribution area in Madagascar. Combined analyses of 16S rRNA and cytochrome b sequences identified six major clades which only partly agreed with previously proposed classifications. Analysis of nuclear DNA sequences of the c-mos gene as well as of ISSR fingerprints revealed consistent differences only among three clades which we consider as distinct species: a widespread Madagascarophis colubrinus (Schlegel), with M. citrinus (Boettger) as a junior synonym, a southern M. meridionalis Domergue, and a presumably undescribed species from the extreme north of Madagascar. The species M. ocellatus Domergue was not available for our study. Within M. colubrinus there are two populations from the north-west, each showing two divergent haplotypes with pairwise divergences of up to 5.2% in the cytochrome b gene. Maximum divergence in this gene within M. colubrinus was 7.1%. These high values emphasise that caution needs to be applied before genetic distance values are used for species delimitation. Phylogeographically, most of the genetic variation in M. colubrinus is found in northern Madagascar, indicating that the species might have originated in this region. Later one haplotype clade colonised western and eastern Madagascar, with a putative secondary introgression into north-western populations.     

The blackburni/murchisona species complex in Australian Pseudotetracha (Coleoptera: Carabidae: Cicindelinae: Megacephalini): evaluating molecular and karyological evidence

Our phylogenetic analysis of three endemic species of the Australian tiger beetle genus Pseudotetracha (Fleutiaux, 1864) from South Australia used sequences of two fragments of the mitochondrial genes 16S rRNA and cytochrome oxidase III. A matrix for each gene and two combined matrices were constructed. We compared these three riparian species, together with data from nine taxa of this genus available in GenBank, using parsimony and Bayesian methods. These molecular results are in agreement with the phylogenetic hypothesis for the blackburni/murchisona species complex previously proposed based on morphology, whereas other recent molecular analyses have questioned the existence of this species complex. In all of our analyses, samples of P. blackburni divided into two statistically supported clades, one of which is more closely related to P. mendacia and P. pulchra than to the other P. blackburni clade. This suggests the existence of a cryptic new species. Additionally, we analysed chromosomes of the second metaphase cells of members of the two clades. The observations showed different karyotypes as blackburni‐1 has two types of second meiotic metaphase cells with 11 and 12 chromosomes, whereas in blackburni‐2, all cells have 12 chromosomes, adding evidence for the putative existence of two species.     

Tracing the radiation of Maniola (Nymphalidae) butterflies: new insights from phylogeography hint at one single incompletely differentiated species complex

The use of DNA sequence data often leads to the recognition of cryptic species within putatively well‐known taxa. The opposite case, detecting less diversity than originally described, has, however, far more rarely been documented. Maniola jurtina, the Meadow Brown butterfly, occurs all over Europe, whereas all other six species in the genus Maniola are restricted to the Mediterranean area. Among them, three are island endemics on Sardinia, Cyprus, and Chios, respectively. Maniola species are almost indistinguishable morphologically, and hybridization seems to occur occasionally. To clarify species boundaries and diversification history of the genus, we reconstructed the phylogeography and phylogeny of all seven species within Maniola analyzing 138 individuals from across its range using mitochondrial and nuclear genetic markers. Examination of variation in mitochondrial and nuclear DNA surprisingly revealed a case of taxonomic “oversplitting”. The topology of the recovered phylogenetic tree is not consistent with accepted taxonomy, but rather reveals haplotype clades that are incongruent with nominal species boundaries: instead of seven species, we recognized only two major, yet incompletely segregated, lineages. Our results are consistent with the hypothesis that Maniola originated in Africa. We suggest that one lineage dispersed over the Strait of Gibraltar and the Iberian Peninsula to the west of Europe, while the other lineage spreads eastward through Asia Minor and over the Bosporus to Eastern Europe.     

Phylogeography and post-glacial colonization patterns of the rainbow darter, Etheostoma caeruleum (Teleostei: Percidae)

Aim To examine the effects of historical climate change and drainage isolation on the distribution of mitochondrial DNA cytochrome b genetic variation within the rainbow darter, Etheostoma caeruleum (Percidae: Etheostomatinae). Location Eastern North American streams including tributaries to the Mississippi River, Great Lakes, Potomac River and Hudson Bay drainages. Methods Parsimony analyses, Bayesian analyses and haplotype networks of mitochondrial DNA sequences. Results Four major clades were recovered from sampled populations of E. caeruleum. Three of four clades are distributed in the western portion of the species’ range (primarily west of the Mississippi River). Samples from this region do not form a monophyletic group, and sequences often vary greatly between samples from adjacent stream systems (up to 7.2% divergence). A basal clade includes samples from the White River system in the Ozark Highlands. The northern Ozarks–upper Midwest clade includes samples from Missouri River tributaries and the upper Midwest (Hudson Bay, upper Mississippi River, and western Lake Michigan drainage). The eastern clade is composed of individuals from the Ohio River, Great Lakes and Potomac River. The Mississippi River corridor clade includes samples from middle and lower Mississippi River tributaries. Main conclusions The four major clades of E. caeruleum are deep allopatric lineages with well‐defined boundaries and have additional phylogeographical structure within each clade. The Ozark Highlands have the greatest levels of diversity relative to distributional area, with marked cytochrome b subdivisions between adjacent stream systems. Samples from previously glaciated areas do not have a subset of the cytochrome b diversity found in unglaciated areas, but four separate source areas are identified based on phylogenetic analyses. Dispersal into previously glaciated areas followed several known glacial outlets and, based on sequence divergence between populations, may have occurred during different glacial or interglacial stages. The disjunct distribution and cytochrome b pattern of E. caeruleum in the Mississippi River corridor clade is consistent with late Pleistocene and Recent changes in the course and characteristics of the middle and lower Mississippi River. Phylogeographical boundaries between clades of E. caeruleum correspond to independent sources of biogeographical information and provide insight into historical stream drainage relationships, post‐glacial colonization and drainage isolation patterns.     

ALTERNATE PATHWAYS OF BODY SHAPE EVOLUTION TRANSLATE INTO COMMON PATTERNS OF LOCOMOTOR EVOLUTION IN TWO CLADES OF LIZARDS

Body shape has a fundamental impact on organismal function, but it is unknown how functional morphology and locomotor performance and kinematics relate across a diverse array of body shapes. We showed that although patterns of body shape evolution differed considerably between lizards of the Phrynosomatinae and Lerista, patterns of locomotor evolution coincided between clades. Specifically, we found that the phrynosomatines evolved a stocky phenotype through body widening and limb shortening, whereas Lerista evolved elongation through body lengthening and limb shortening. In both clades, relative limb length played a key role in locomotor evolution and kinematic strategies, with long‐limbed species moving faster and taking longer strides. In Lerista, the body axis also influenced locomotor evolution. Similar patterns of locomotor evolution were likely due to constraints on how the body can move. However, these common patterns of locomotor evolution between the two clades resulted in different kinematic strategies and levels of performance among species because of their morphological differences. Furthermore, we found no evidence that distinct body shapes are adaptations to different substrates, as locomotor kinematics did not change on loose or solid substrates. Our findings illustrate the importance of studying kinematics to understand the mechanisms of locomotor evolution and phenotype‐function relationships.     

Phylogeny of Riama (Squamata: Gymnophthalmidae), impact of phenotypic evidence on molecular datasets,and the origin of the Sierra Nevada de Santa Marta endemic fauna

Riama is the most speciose genus of the Neotropical lizard family Gymnophthalmidae. Its more than 30 montane species occur throughout the northern Andes, the Cordillera de la Costa (CC) in Venezuela, and Trinidad. We present the most comprehensive phylogenetic analysis of Riama to date based on a total evidence (TE) approach and direct optimization of molecular and morphological evidence. Analyses use DNA sequences from four loci and 35 phenotypic characters. The dataset consists of 55 ingroup terminals representing 25 of the 30 currently recognized species of Riama plus five undescribed taxa, including an endemic species from the Sierra Nevada de Santa Marta (SNSM) in Colombia, and 66 outgroup terminals of 47 species. Analysis results in a well‐supported hypothesis in which Riama is polyphyletic, with its species falling into three clades. The Tepuian Anadia mcdiarmidi nests within one clade of Riama, and the recently resurrected Pantodactylus nests within Cercosaura. Accordingly, we propose a monophyletic taxonomy that reflects historical relationships. Analysis of character evolution indicates that the presence/absence of prefrontals—a cornerstone of the early genus‐level taxonomy of cercosaurines—is optimally explained as having been plesiomorphically present in the most recent common ancestor of Cercosaurinae and lost in that of the immediately less inclusive clade. Multiple independent reversals to present and subsequent returns to absent occur within this clade. To evaluate the impact of phenotypic evidence on our results, we compare our TE results with results obtained from analyses using only molecular data. Although phenotypic evidence comprises only 1.2% of the TE matrix, its inclusion alters both the topology and support values of the clades that do not differ. Finally, current phylogenetic evidence reveals a SNSM–CC–Trinidad–tepuis biogeographical link. We hypothesize that an ancient connection facilitated the exchange of species between the SNSM and the CC.     

Genetic variation of an endangered Malagasy frog, <Emphasis Type="Italic">Mantella cowani</Emphasis>, and its phylogeographic relationship to the widespread <Emphasis Type="Italic">M. baroni</Emphasis>

We investigated the degree and distribution of the genetic variation, and phylogeography, of two species of Malagasy poison frogs, Mantella cowani and M. baroni. The former is critically endangered due to its restricted distribution, habitat destruction and overcollection for the pet trade. Analysis of 526 bp of mtDNA (cytochrome b) resulted in separate haplotype networks for the two species, and discovered hybridization at a single locality. The two networks confirm the status of M. baroni and M. cowani as separate evolutionary species and units for conservation. Within both mitochondrial haplotype networks, specimens from different localities shared numerous identical haplotypes, even those from the most distant sample sites of M. baroni. Most populations were characterized by high haplotype diversity and no haplotype clades exclusive to geographical regions were observed. Protection of a few large populations of these species is therefore likely to conserve much of the mtDNA genetic diversity found in the entire species. While M. baroni is widespread and occurs in many nature reserves, we recommend efficient legal protection of some M. cowani habitats to protect this species against extinction.     

Haplotype mining panel for genetic dissection and breeding in Eucalyptus

To improve our understanding of genetic mechanisms underlying complex traits in plants, a comprehensive analysis of gene variants is required. Eucalyptus is an important forest plantation genus that is highly outbred. Trait dissection and molecular breeding in eucalypts currently relies on biallelic single-nucleotide polymorphism (SNP) markers. These markers fail to capture the large amount of haplotype diversity in these species, and thus multi-allelic markers are required. We aimed to develop a gene-based haplotype mining panel for Eucalyptus species. We generated 17 999 oligonucleotide probe sets for targeted sequencing of selected regions of 6293 genes implicated in growth and wood properties, pest and disease resistance, and abiotic stress responses. We identified and phased 195 834 SNPs using a read-based phasing approach to reveal SNP-based haplotypes. A total of 8915 target regions (at 4637 gene loci) passed tests for Mendelian inheritance. We evaluated the haplotype panel in four Eucalyptus species (E. grandis, E. urophylla, E. dunnii and E. nitens) to determine its ability to capture diversity across eucalypt species. This revealed an average of 3.13–4.52 haplotypes per target region in each species, and 33.36% of the identified haplotypes were shared by at least two species. This haplotype mining panel will enable the analysis of haplotype diversity within and between species, and provide multi-allelic markers that can be used for genome-wide association studies and gene-based breeding approaches.     

Molecular characterization of Gonatocerus tuberculifemur (Ogloblin) (Hymenoptera: Mymaridae), a prospective Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) biological control candidate agent from South America: divergent clades

We genetically characterized the prospective South American egg parasitoid candidate, Gonatocerus tuberculifemur, of the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, for a neoclassical biological control program in California. Two molecular methods, inter-simple sequence repeat-polymerase chain reaction DNA fingerprinting and a phylogeographic approach inferred from the mitochondrial cytochrome oxidase subunit I gene (COI), were utilized. Five geographic populations from South America were analyzed; in addition, a phylogenetic analysis was performed with several named and one unnamed Gonatocerus species using the COI gene. DNA fingerprinting demonstrated a fixed geographic banding pattern difference in the population from San Rafael, Mendoza Province, Argentina. The COI analysis uncovered haplotype or geographic structure in G. tuberculifemur. A neighbour-joining distance (NJ) and a single most parsimonious tree (MP) clustered the populations into two well-supported distinct clades with strong bootstrap values (97-99% and 92-99%, respectively) with populations from San Rafael clustering into clade 2 and the rest of the populations clustering into clade 1. No haplotype sharing was observed between individuals from the two clades. Phylogenetic analyses performed by NJ and MP methods with 15 Gonatocerus species confirmed species boundaries and again uncovered two distinct clades in G. tuberculifemur with strong bootstrap support (95-100% and 68-100%, respectively). However, the NJ tree supported the morphologically defined relationships better than the MP tree. The molecular evidence in the present study is suggestive of a species level divergence. Because G. tuberculifemur is under consideration as a potential biological control agent for GWSS in California, understanding cryptic variation in this species is critical.     

Different filtering strategies of genotyping‐by‐sequencing data provide complementary resolutions of species boundaries and relationships in a clade of sexually deceptive orchids

Ongoing hybridization and retained ancestral polymorphism in rapidly radiating lineages could mask recent cladogenetic events. This presents a challenge for the application of molecular phylogenetic methods to resolve differences between closely related taxa. We reanalyzed published genotyping‐by‐sequencing (GBS) data to infer the phylogeny of four species within the Ophrys sphegodes complex, a recently radiated clade of orchids. We used different data filtering approaches to detect different signals contained in the dataset generated by GBS and estimated their effects on maximum likelihood trees, global F_ST and bootstrap support values. We obtained a maximum likelihood tree with high bootstrap support, separating the species by using a large dataset based on loci shared by at least 30% of accessions. Bootstrap and F_ST values progressively decreased when filtering for loci shared by a higher number of accessions. However, when filtering more stringently to retain homozygous and organellar loci, we identified two main clades. These clades group individuals independently from their a priori species assignment, but were associated with two organellar haplotype clusters. We infer that a less stringent filtering preferentially selects for rapidly evolving lineage‐specific loci, which might better delimit lineages. In contrast, when using homozygous/organellar DNA loci the signature of a putative hybridization event in the lineage prevails over the most recent phylogenetic signal. These results show that using differing filtering strategies on GBS data could dissect the organellar and nuclear DNA phylogenetic signal and yield novel insights into relationships between closely related species.     

Connecting the dots: Bridging genetic and spatial differentiation of the genus Eumerus (Diptera: Syrphidae) in the Mediterranean Basin and Balkans

In this study, we explored intraspecific genetic differentiation of hoverfly species of the genus Eumerus with regard to landscape discontinuities (due to paleogeological events), isolation‐by‐distance, evolutionary processes, and Quaternary climatic oscillations. We unveil genetically diverging regions and discuss the potential driving forces that gave rise to these spatial genetic patterns. We generated mitochondrial DNA (mtDNA) barcodes for 274 individuals of nine Eumerus species, sampled from 58 localities in the Mediterranean and Balkans. Spatially explicit Bayesian clustering, correlation tests between geographic and genetic distances (presence of isolation‐by‐distance), median neighbor‐joining haplotype networks, and landscape shape interpolation analyses were employed to investigate spatial genetic patterns. Bayesian clustering generated one to three genetic clusters with high posterior probability values. We also observed high mtDNA haplotype diversity consisting of unique and shared haplotypes, as well as starlike mtDNA haplotype patterns. The mtDNA haplotype network was consistent with species distributions and Bayesian clustering for four tested species. The Mantel tests confirmed the absence of isolation‐by‐distance in seven species. We identified genetically diverging areas through our landscape shape interpolation analyses. Five species displayed neither spatial genetic patterns nor evidence of isolation‐by‐distance, indicative of relict taxa. Our study is the first broad‐ and large‐scale study of Eumerus species in the Mediterranean and Balkans; it reveals spatial genetic clusters in four species and identifies the potential factors driving those patterns.