Resolving species boundaries in the critically imperiled freshwater mussel species,Fusconaia mitchelli (Bivalvia: Unionidae)

Species are a fundamental unit of biology, and defining accurate species boundaries is integral to effective conservation and management of imperiled taxa. Freshwater mussels (Bivalvia: Unionidae) are among the most imperiled groups of organisms in North America, yet species boundaries remain uncertain for many taxa. The False Spike, Fusconaia mitchelli (Simpson in Dall, 1895), is a freshwater mussel considered to be endemic to central Texas (Brazos, Colorado, and Guadalupe drainages). Recent research revealed significant intraspecific genetic variation between geographically separated populations of F. mitchelli, which could be indicative of speciation; however, small sample sizes for several of the populations precluded formal taxonomic revision. Here, we increase taxon sampling and use multilocus DNA sequence data and traditional morphometrics to re‐evaluate species boundaries in F. mitchelli. We sequenced three loci: the protein‐coding mitochondrial DNA genes cytochrome c oxidase subunit 1 and NADH dehydrogenase 1, and the nuclear internal transcribed spacer 1. Phylogenetic analyses depicted deep genetic divergence between F. mitchelli in the Guadalupe and those in the Brazos and Colorado drainages, which was further supported by available biogeographic information. Morphometric analyses and coalescent‐based species delimitation models integrating both DNA sequence and morphological data provided strong support for the divergence observed between the two geographically isolated clades of F. mitchelli. Based on these results, we revise taxonomy accordingly by elevating the junior synonym Fusconaia iheringi (Wright, 1898) to represent the Brazos and Colorado populations and restrict the distribution of F. mitchelli to the Guadalupe River drainage. Our findings may impact pending management decisions to protect F. mitchelli under the U.S. Endangered Species Act.     

Microsatellite allele sizes alone are insufficient to delineate species boundaries in Symbiodinium

Symbiodinium are a diverse group of unicellular dinoflagellates that are important nutritional symbionts of reef‐building corals. Symbiodinium putative species (‘types’) are commonly identified with genetic markers, mostly nuclear and chloroplast encoded ribosomal DNA regions. Population genetic analyses using microsatellite loci have provided insights into Symbiodinium biogeography, connectivity and phenotypic plasticity, but are complicated by: (i) a lack of consensus criteria used to delineate inter‐ vs. intragenomic variation within species; and (ii) the high density of Symbiodinium in host tissues, which results in single samples comprising thousands of individuals. To address this problem, Wham & LaJeunesse (2016) present a method for identifying cryptic Symbiodinium species from microsatellite data based on correlations between allele size distributions and nongeographic genetic structure. Multilocus genotypes that potentially do not recombine in sympatry are interpreted as secondary ‘species’ to be discarded from downstream population genetic analyses. However, Symbiodinium species delineations should ideally incorporate multiple physiological, ecological and molecular criteria. This is because recombination tests may be a poor indicator of species boundaries in Symbiodinium due to their predominantly asexual mode of reproduction. Furthermore, discontinuous microsatellite allele sizes in sympatry may be explained by secondary contact between previously isolated populations and by mutations that occur in a nonstepwise manner. Limitations of using microsatellites alone to delineate species are highlighted in earlier studies that demonstrate occasional bimodal distributions of allele sizes within Symbiodinium species and considerable allele size sharing among Symbiodinium species. We outline these issues and discuss the validity of reinterpretations of our previously published microsatellite data from Symbiodinium populations on the Great Barrier Reef (Howells et al. 2013).     

A genomic assessment of species boundaries and hybridization in a group of highly polymorphic anoles (distichus species complex)

Delimiting young species is one of the great challenges of systematic biology, particularly when the species in question exhibit little morphological divergence. Anolis distichus, a trunk anole with more than a dozen subspecies that are defined primarily by dewlap color, may actually represent several independent evolutionary lineages. To test this, we utilized amplified fragment length polymorphisms (AFLP) genome scans and genetic clustering analyses in conjunction with a coalescent‐based species delimitation method. We examined a geographically widespread set of samples and two heavily sampled hybrid zones. We find that genetic divergence is associated with a major biogeographic barrier, the Hispaniolan paleo‐island boundary, but not with dewlap color. Additionally, we find support for hypotheses regarding colonization of two Hispaniolan satellite islands and the Bahamas from mainland Hispaniola. Our results show that A. distichus is composed of seven distinct evolutionary lineages still experiencing a limited degree of gene flow. We suggest that A. distichus merits taxonomic revision, but that dewlap color cannot be relied upon as the primary diagnostic character.     

Symbiodinium population genetics: testing for species boundaries and analysing samples with mixed genotypes

Population genetic markers are increasingly being used to study the diversity, ecology and evolution of Symbiodinium, a group of eukaryotic microbes that are often mutualistic with reef‐building corals. Population genetic markers can resolve individual clones, or strains, from samples of host tissue; however, samples may comprise different species that may confound interpretations of gene flow and genetic structure. Here, we propose a method for resolving species from population genetic data using tests for genetic recombination. Assigning individuals to genetically recombining populations prior to further analyses avoids critical errors in the interpretation of gene flow and dispersal. To demonstrate the effectiveness of the approach, we first apply this method to a simulated data set. We then use the method to resolve two species of host generalist Symbiodinium that commonly co‐occur in reef‐building corals collected from Indo‐West Pacific reefs. We demonstrate that the method is robust even when some hosts contain genotypes from two distinct species. Finally, we examine population genetic data sets from two recently published papers in Molecular Ecology. We show that each strongly supports a two species interpretation, which significantly changes the original conclusions presented in these studies. When combined with available phylogenetic and ecological evidence, the use of population genetic data offers a robust method for unambiguously delimiting morphologically cryptic species.     

Multivariate species boundaries and conservation of harlequin poison frogs

In this study, we present an iterative method for delimiting species under the general lineage concept (GLC) based on the multivariate clustering of morphological, ecological and genetic data. Our rationale is that distinct multivariate groups correspond to evolutionarily independent metapopulation lineages because they reflect the common signal of different secondary defining properties (environmental and genetic distinctiveness, phenotypic diagnosability, etc.) that imply the existence of barriers preventing or limiting gene exchange. We applied this method to study a group of endangered poison frogs, the Oophaga histrionica complex. In our study case, we used next‐generation targeted amplicon sequencing to obtain a robust genetic data set that we combined with patterns of morphological and ecological features. Our analyses revealed the existence of at least five different species in the histrionica complex (three, new to science), some of them, occurring in small isolated populations outside any protected areas. The lineage delimitation proposed here has important conservation implications as it revealed that some of the Oophaga species should be considered among the most vulnerable of the Neotropical frogs. More broadly, our study exemplifies how multiple‐amplicon and multivariate statistical techniques can be integrated to successfully identify species and their boundaries.     

Hybridization in natural sympatric populations of Dermacentor ticks in northwestern North America

Hybridization in ticks has been described in a handful of species and mostly as a result of laboratory experiments. We used 148 AFLP loci to describe putative hybridization events between D. andersoni and D. variabilis in sympatric populations from northwestern North America. Recently, D. variabilis has expanded its range westward into the natural range of D. andersoni. Using a sample of 235 D. andersoni and 62 D. variabilis, we identified 31 individuals as putative hybrids: four F₂ individuals and 27 backcrosses to D. andersoni (as defined by NewHybrids ). We found no evidence of hybrids backcrossing into D. variabilis. Furthermore, all hybrids presented 16S mtDNA signatures characteristic of D. andersoni, which indicates the directionality of the hybrid crosses: female D. andersoni × male D. variabilis. We also discovered 13 species‐specific AFLP fragments for D. andersoni. These loci were found to have a decreased occurrence in the putative hybrids and were absent altogether in D. variabilis samples. AFLP profiles were also used to determine the levels of genetic population structure and gene flow among nine populations of D. andersoni and three of D. variabilis. Genetic structure exists in both species (D. andersoni, Φ_ST = 0.110; D. variabilis, Φ_ST = 0.304) as well as significant estimates of isolation by distance (D. andersoni, ρ = 0.066, P = 0.001; D. variabilis, ρ = 0.729, P = 0.001).     

Asymmetrical thermal constraints on the parapatric species boundaries of two widespread generalist butterflies

Abstract.  1. The sibling species Papilio glaucus and Papilio canadensis meet in a narrow hybrid zone believed to be maintained by temperature thresholds acting independently on both species. The present study tests if this assertion is true for the cold-adapted species, P. canadensis , which is presumed to be limited by the effect of high temperatures in late summer and/or autumn on pupal survival.
2. Three experiments were conducted examining the effects of: (i) short periods of high temperature stress in autumn, (ii) prolonged warm temperatures in autumn, and (iii) temperatures simulating warmer winters/longer springs upon the survival of P. canadensis and P. glaucus.
3. Results indicated that short periods of high temperatures did not induce the high mortality rates required to be the key factor limiting the range of P. canadensis . However, P. canadensis did exhibit a considerably lower tolerance to high temperature extremes, prolonged warm temperatures in autumn, and conditions simulating shorter/warmer winters than P. glaucus .
4. Differences in temperature tolerance throughout the pupal stage are likely to be a significant factor in maintaining the southern range limit of P. canadensis . Further warming as may occur during climate change, particularly in winter and spring, will likely affect the dynamics of southerly populations of P. canadensis.     

Hybridization between two parapatric ranid frog species in the northern Sierra Nevada,California, USA

Contact zones between species provide a unique opportunity to test whether taxa can hybridize or not. Cross‐breeding or hybridization between closely related taxa can promote gene flow (introgression) between species, adaptation, or even speciation. Though hybridization events may be short‐lived and difficult to detect in the field, genetic data can provide information about the level of introgression between closely related taxa. Hybridization can promote introgression between species, which may be an important evolutionary mechanism for either homogenization (reversing initial divergence between species) or reproductive isolation (potentially leading to speciation). Here, we used thousands of genetic markers from nuclear DNA to detect hybridization between two parapatric frog species (Rana boylii and Rana sierrae) in the Sierra Nevada of California. Based on principal components analysis, admixture, and analysis of heterozygosity at species diagnostic SNPs, we detected two F1 hybrid individuals in the Feather River basin, as well as a weak signal of introgression and gene flow between the frog species compared with frog populations from two other adjacent watersheds. This study provides the first documentation of hybridization and introgression between these two species, which are of conservation concern.     

Behavioral modifications lead to disparate demographic consequences in two sympatric species

Life‐history theory suggests species that typically have a large number of offspring and high adult mortality may make decisions that benefit offspring survival in exchange for increased adult risks. Such behavioral adaptations are essential to understanding how demographic performance is linked to habitat selection during this important life‐history stage. Though studies have illustrated negative fitness consequences to attendant adults or potential fitness benefits to associated offspring because of adaptive habitat selection during brood rearing, equivocal relationships could arise if both aspects of this reproductive trade‐off are not assessed simultaneously. To better understand how adaptive habitat selection during brood rearing influences demographics, we studied the brood survival, attendant parental survival, and space use of two sympatric ground‐nesting bird species, the northern bobwhite (hereafter: “bobwhite”; Colinus virgininanus) and scaled quail (Callipepla squamata). During the 2013–2014 breeding seasons, we estimated habitat suitability across two grains (2 m and 30 m) for both species and determined how adult space use of these areas influenced individual chick survival and parental risk. We found the proportion of a brood's home range containing highly suitable areas significantly increased bobwhite chick survival (β = 0.02, SE = 0.006). Additionally, adult weekly survival for bobwhite was greater for individuals not actively brooding offspring (0.9716, SE = 0.0054) as compared to brooding adults (0.8928, SE = 0.0006). Conversely, brood habitat suitability did not influence scaled quail chick survival during our study, nor did we detect a survival cost for adults that were actively brooding offspring. Our research illustrates the importance of understanding life‐history strategies and how they might influence relationships between adaptive habitat selection and demographic parameters.     

Formation of a recent hybrid zone offers insight into the geographic puzzle and maintenance of species boundaries in musk turtles

Speciation is the result of an accumulation of reproductive barriers between populations, but pinpointing the factors involved is often difficult. However, hybrid zones can form when these barriers are not complete, especially when lineages come into contact in intermediate or modified habitats. We examine a hybrid zone between two closely related riverine turtle species, Sternotherus depressus and S. peltifer, and use dual‐digest RAD sequencing to understand how this hybrid zone formed and elucidate genomic patterns of reproductive isolation. First, the geographical extent and timing of formation of the hybrid zone is established to provide context for understanding the role of extrinsic and intrinsic reproductive isolating mechanisms in this system. The strength of selection on taxon‐specific contributions to maintenance of the hybrid zone is then inferred using a Bayesian genomic cline model. These analyses identify a role for selection inhibiting introgression in some genomic regions at one end of the hybrid zone and promoting introgression in many loci at the other. When selective pressures necessary to generate outliers to the genomic cline are considered with the geographical and temporal context of this hybrid zone, we conclude that habitat‐specific selection probably limits introgression from S. depressus to S. peltifer in the direction of river flow. However, selection is mediating rapid, unidirectional introgression from S. peltifer to S. depressus, which is probably facilitated by anthropogenic habitat alteration. These findings indicate a potentially imminent threat of population‐level genomic extinction for an already imperiled species due to ongoing human‐caused habitat alteration.     

Genetic variability and taxonomic revision of the genus Auxenochlorella (Shihira et Krauss) Kalina et Puncocharova (Trebouxiophyceae,Chlorophyta)

The monotypic genus Auxenochlorella with its type species A. protothecoides is so far only known from specific habitats such as the sap of several tree species. Several varieties were described according to physiological performances in culture on different organic substrates. However, two strains designated as Auxenochlorella were isolated from other habitats (an endosymbiont of Hydra viridis and an aquatic strain from an acidic volcano stream). We studied those isolates and compared them with six strains of Auxenochlorella belonging to different varieties. The integrative approach used in this study revealed that all strains showed similar morphology but differed in their SSU and ITS rDNA sequences. The Hydra endosymbiont formed a sister taxon to A. protothecoides, which included the varieties protothecoides, galactophila, and communis. The variety acidicola is not closely related to Auxenochlorella and represented its own lineage within the Trebouxiophyceae. In view of these results, we propose a new species of Auxenochlorella, A. symbiontica, for the Hydra symbiont, and a new genus Pumiliosphaera, with its type species, P. acidophila, for acidophilic strain. These results are supported by several compensatory base changes in the conserved region of ITS‐2 and ITS‐2 DNA barcodes.     

Genetic and morphological discrimination of three species of ninespined stickleback Pungitius spp. (Teleostei,Gasterosteidae) in France with the revalidation of Pungitius vulgaris (Mauduyt, 1848)

The taxonomy of French ninespined sticklebacks (Pungitius spp.) has long been controversial. To clarify the taxonomy in this group, we use mitochondrial (COI) and nuclear (RNF213) sequence markers, as well as morphological data. In France, both genetic markers discriminate three evolutionary lineages. Morphological analysis on fresh and type specimens supports the different lineages and the existence of three species in France. Pungitius pungitius, occurring in the North of France and Rhone basin, is characterized by specimens longer than 35 mm SL, by a flat head with a straight or slightly concave snout, typically 9–10 dorsal spines, 10–11 dorsal soft rays, 9–10 anal soft rays, 0–12 scutes on the caudal peduncle with a keel reaching the last anal‐fin ray, longer pelvic fin, post‐dorsal and caudal peduncle lengths, and a slim caudal peduncle (caudal peduncle depth/length 11.8%–21.9%). Pungitius laevis, occurring in France, in the English Channel basins and Loire drainage, differs from the other species by a head rounded with concave snout in specimens longer than 35 mm SL, accentuating the impression of fleshy lips, 0–4 scutes on the caudal peduncle and a higher caudal peduncle depth/length ratio (15.7%–34.5%). Finally, Pungitius vulgaris, endemic to the Vienne River and rivers of south‐western France as far north as the Garonne estuary, is differentiated by a rounded head with a straight or slightly convex snout, the absence of scutes on the caudal peduncle and by having 11 pectoral‐fin rays. Our data confirm the existence of a hybridization zone in the North of France between P. pungitius and P. laevis. As a result, Pungitius lotharingus is invalid, as it was described based on hybrid specimens. A lectotype for P. laevis was designated because the syntypes included hybrids. This revision provides new perspectives for evolutionary biology studies and will have consequences for Pungitius conservation in France.     

Ecological niche comparison and molecular phylogeny segregate the invasive moss species Campylopus introflexus (Leucobryaceae,Bryophyta) from its closest relatives

The delimitation of the invasive moss species Campylopus introflexus from its closest relative, Campylopus pilifer, has been long debated based on morphology. Previous molecular phylogenetic reconstructions based on the nuclear ribosomal internal transcribed spacers (ITS) 1 and 2 showed that C. pilifer is split into an Old World and a New World lineage, but remained partly inconclusive concerning the relationships between these two clades and C. introflexus. Analyses of an extended ITS dataset displayed statistically supported incongruence between ITS1 and ITS2. ITS1 separates the New World clade of C. pilifer from a clade comprising C. introflexus and the Old World C. pilifer. Ancestral state reconstruction showed that this topology is morphologically supported by differences in the height of the dorsal costal lamellae in leaf cross‐section (despite some overlap). ITS2, in contrast, supports the current morphological species concept, i.e., separating C. introflexus from C. pilifer, which is morphologically supported by the orientation of the hyaline hair point at leaf apex as well as costal lamellae height. Re‐analysis of published and newly generated plastid atpB‐rbcL spacer sequences supported the three ITS lineages. Ecological niche modeling proved a useful approach and showed that all three molecular lineages occupy distinct environmental spaces that are similar, but undoubtedly not equivalent. In line with the ITS1 topology, the C. pilifer lineage from the New World occupies the most distinct environmental niche, whereas the niches of Old World C. pilifer and C. introflexus are very similar. Taking the inferences from ecological niche comparisons, phylogenetics, and morphology together, we conclude that all three molecular lineages represent different taxa that should be recognized as independent species, viz. C. introflexus, C. pilifer (Old World clade), and the reinstated C. lamellatus Mont. (New World clade).     

Mitogenomics reveals high synteny and long evolutionary histories of sympatric cryptic nematode species

Species with seemingly identical morphology but with distinct genetic differences are abundant in the marine environment and frequently co‐occur in the same habitat. Such cryptic species are typically delineated using a limited number of mitochondrial and/or nuclear marker genes, which do not yield information on gene order and gene content of the genomes under consideration. We used next‐generation sequencing to study the composition of the mitochondrial genomes of four sympatrically distributed cryptic species of the Litoditis marina species complex (PmI, PmII, PmIII, and PmIV). The ecology, biology, and natural occurrence of these four species are well known, but the evolutionary processes behind this cryptic speciation remain largely unknown. The gene order of the mitochondrial genomes of the four species was conserved, but differences in genome length, gene length, and codon usage were observed. The atp8 gene was lacking in all four species. Phylogenetic analyses confirm that PmI and PmIV are sister species and that PmIII diverged earliest. The most recent common ancestor of the four cryptic species was estimated to have diverged 16 MYA. Synonymous mutations outnumbered nonsynonymous changes in all protein‐encoding genes, with the Complex IV genes (coxI‐III) experiencing the strongest purifying selection. Our mitogenomic results show that morphologically similar species can have long evolutionary histories and that PmIII has several differences in genetic makeup compared to the three other species, which may explain why it is better adapted to higher temperatures than the other species.     

Existence of species complex largely reduced barcoding success for invasive species of Tephritidae: a case study in Bactrocera spp.

Fruit flies in the family Tephritidae are the economically important pests that have many species complexes. DNA barcoding has gradually been verified as an effective tool for identifying species in a wide range of taxonomic groups, and there are several publications on rapid and accurate identification of fruit flies based on this technique; however, comprehensive analyses of large and new taxa for the effectiveness of DNA barcoding for fruit flies identification have been rare. In this study, we evaluated the COI barcode sequences for the diagnosis of fruit flies using 1426 sequences for 73 species of Bactrocera distributed worldwide. Tree‐based [neighbour‐joining (NJ)]; distance‐based, such as Best Match (BM), Best Close Match (BCM) and Minimum Distance (MD); and character‐based methods were used to evaluate the barcoding success rates obtained with maintaining the species complex in the data set, treating a species complex as a single taxon unit, and removing the species complex. Our results indicate that the average divergence between species was 14.04% (0.00–25.16%), whereas within a species this was 0.81% (0.00–9.71%); the existence of species complexes largely reduced the barcoding success for Tephritidae, for example relatively low success rates (74.4% based on BM and BCM and 84.8% based on MD) were obtained when the sequences from species complexes were included in the analysis, whereas significantly higher success rates were achieved if the species complexes were treated as a single taxon or removed from the data set – BM (98.9%), BCM (98.5%) and MD (97.5%), or BM (98.1%), BCM (97.4%) and MD (98.2%).     

Delimitation of sympatric Palaemon (Decapoda,Palaemonidae) species of the Laguna Madre,Mexico

Grass shrimps Palaemon mundusnovus, P. pugio and P. vulgaris sympatrically inhabit in the Laguna Madre, Mexico. They exhibit very close morphological similarity and overlap in their diagnostic characteristics, which has hindered certainty in their identification and has raised doubt regarding their taxonomic validity. In this work, we analyse intra‐ and interspecific morphometric and meristic variability through a multivariate analysis, and we determine the molecular variation using mitochondrial sequences of the 12S, 16S and COX1 genes to confirm the validity of the three taxa as having distinct lineages or to recognize a smaller number of species with phenotypic plasticity. Our results corroborate the taxonomic validity of the three species; however, there is intraspecific plasticity, interspecific overlap of characteristics and greater morphological and molecular similarity between the species P. mundusnovus and P. pugio, whereas P. vulgaris was better delimited. These species form a monophyletic group but the phylogenetic relationship obtained is discussed. Telson length was the primary variable in the principal components analysis, whereas the length of the propodus of the second pereiopod was the best discriminant. The range of variation reported in the characteristics linked to the rostrum is extended, and its influence on the separation of these species is recognized. The joint application of multivariate analysis from morphological variables and molecular tools is recommended to clarify the taxonomic status of species featuring close morphological similarity and sympatric distribution.