Taxonomy as a hypothesis: testing the status of the Bermuda buckeye butterfly Junonia coenia bergi (Lepidoptera: Nymphalidae)

Species determination and definition in eukaryotes have traditionally been based on morphology, with little focus on genetic differentiation. Molecular methods allow for the independent assessment of morphology‐based taxonomic hypotheses. Three criteria used to define a full species for taxonomic purposes are morphological distinction, formation of a monophyletic lineage, and reproductive isolation. Junonia butterflies (Nymphalidae) are becoming an important experimental model system, but the taxonomy of many New World Junonia species is unclear. One of these species is J. coenia, which contains the subspecies J. coenia coenia, J. coenia grisea and J. coenia bergi. Previous studies suggest that J. coenia grisea may meet the criteria for full species status. Therefore, we evaluated the geographically isolated and rarely studied Bermuda buckeye butterfly J. coenia bergi to determine if it was similarly distinct. Physical examination of specimens and phylogenetic and population genetic analyses of mitochondrial cytochrome c oxidase subunit I, nuclear wingless, and complete mitochondrial genome sequences suggest that while J. coenia bergi is smaller in body size than many Junonia and has distinctive ventral hindwing colouration, it does not form a monophyletic lineage and shows indications of continued gene flow with North American mainland J. coenia coenia populations. Thus, J. coenia bergi does not meet the criteria for full species designation, but geographic isolation, morphological distinctiveness, and cultural importance suggest that it remain recognized as a subspecies of J. coenia. Similar analyses will be useful for addressing further taxonomic questions in Junonia and other taxa, especially where morphology‐based taxonomic determinations are ambiguous.     

Cryptic Speciation Patterns in Iranian Rock Lizards Uncovered by Integrative Taxonomy

While traditionally species recognition has been based solely on morphological differences either typological or quantitative, several newly developed methods can be used for a more objective and integrative approach on species delimitation. This may be especially relevant when dealing with cryptic species or species complexes, where high overall resemblance between species is coupled with comparatively high morphological variation within populations. Rock lizards, genus Darevskia, are such an example, as many of its members offer few diagnostic morphological features. Herein, we use a combination of genetic, morphological and ecological criteria to delimit cryptic species within two species complexes, D. chlorogaster and D. defilippii, both distributed in northern Iran. Our analyses are based on molecular information from two nuclear and two mitochondrial genes, morphological data (15 morphometric, 16 meristic and four categorical characters) and eleven newly calculated spatial environmental predictors. The phylogeny inferred for Darevskia confirmed monophyly of each species complex, with each of them comprising several highly divergent clades, especially when compared to other congeners. We identified seven candidate species within each complex, of which three and four species were supported by Bayesian species delimitation within D. chlorogaster and D. defilippii, respectively. Trained with genetically determined clades, Ecological Niche Modeling provided additional support for these cryptic species. Especially those within the D. defilippii-complex exhibit well-differentiated niches. Due to overall morphological resemblance, in a first approach PCA with mixed variables only showed the separation between the two complexes. However, MANCOVA and subsequent Discriminant Analysis performed separately for both complexes allowed for distinction of the species when sample size was large enough, namely within the D. chlorogaster-complex. In conclusion, the results support four new species, which are described herein.     

On nine-spined sticklebacks of the genus Pungitius (Gasterosteiformes) of Lake Bol’shoe (Bol’shoi Shantar Island, Shantar Islands)

Specific features of morphology and biology of two species of nine-spined sticklebacks??Pungitius sinensis and P. pungitius??dwelling in brackish Lake Bol??shoe (Bol??shoi Shantar Island) have been studied. Differences in the number of dorsal spines, vertebrae, and coloration, as well as the absence of specimens with an intermediate phenotype indicate the reproductive isolation of two species supported by the temporal separation of spawning periods. Species P. pungitius was found in Lake Bol??shoe for the first time.     

It’s not what it looks like: molecular data fails to substantiate morphological differences in two sea hares (Mollusca,Heterobranchia, Aplysiidae) from southern Brazil

Species of sea hares have been recognized traditionally based on morphological traits, mainly the radula, external coloration, and reproductive anatomy. However, recent studies have shown substantial color variation in some sea slug species. Molecular data have been successfully used to differentiate morphologically similar species of “opisthobranchs” and resolve questions on the taxonomic value of color. The objective of this paper is to use molecular data in an attempt to elucidate whether specimens of Aplysia brasiliana with distinct colorations and morphologies are actually the same species. To this end, DNA from 14 specimens of A. brasiliana was extracted, including five specimens identified as a distinct morphotype from typical A. brasiliana. Although the two morphotypes have consistent differences in their external morphology and radula, the molecular data confirmed that there are no significant genetic differences between them. This is another example of the need to re-evaluate taxonomic decisions based on morphology in light of molecular evidence.     

Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data

Cryptic species may cause biological invasions to be overlooked leading to underestimation of the potential impacts of invaders on the new ecosystems. Identification of freshwater snails is challenging because of the scarcity of discriminative morphological characters and the limited taxonomic knowledge of some taxa. Here, molecular and morphological analyses were performed to investigate the identity of viable populations of the genus Physa in aquatic ecosystems of different basins in northern and central Chile, including habitats where the native species Physa chilensis and Physa nodulosa have been traditionally recognized. Molecular analyses based on mitochondrial DNA sequences from the small subunit 16S rDNA and cytochrome c oxidase subunit 1 identified all specimens sequenced as belonging to the globally invasive species Physa acuta. Microscopic examination of the radula and morphological observations of the reproductive system were congruent with these findings. Highly divergent haplotypes found in El Salto suggest multiple introductions of different lineages of the invader in this locality. It is clear that for future management planning a more reliable assessment of the status of P. chilensis and P. nodulosa is required.     

Phylogenetic Analysis Reveals a Cryptic Species Blastomyces gilchristii,sp. nov. within the Human Pathogenic Fungus Blastomyces dermatitidis

Background

Analysis of the population genetic structure of microbial species is of fundamental importance to many scientific disciplines because it can identify cryptic species, reveal reproductive mode, and elucidate processes that contribute to pathogen evolution. Here, we examined the population genetic structure and geographic differentiation of the sexual, dimorphic fungus Blastomyces dermatitidis, the causative agent of blastomycosis.

Methodology/Principal Findings

Criteria for Genealogical Concordance Phylogenetic Species Recognition (GCPSR) applied to seven nuclear loci (arf6, chs2, drk1, fads, pyrF, tub1, and its-2) from 78 clinical and environmental isolates identified two previously unrecognized phylogenetic species. Four of seven single gene phylogenies examined (chs2, drk1, pyrF, and its-2) supported the separation of Phylogenetic Species 1 (PS1) and Phylogenetic Species 2 (PS2) which were also well differentiated in the concatenated chs2-drk1-fads-pyrF-tub1-arf6-its2 genealogy with all isolates falling into one of two evolutionarily independent lineages. Phylogenetic species were genetically distinct with interspecific divergence 4-fold greater than intraspecific divergence and a high Fst value (0.772, P<0.001) indicative of restricted gene flow between PS1 and PS2. Whereas panmixia expected of a single freely recombining population was not observed, recombination was detected when PS1 and PS2 were assessed separately, suggesting reproductive isolation. Random mating among PS1 isolates, which were distributed across North America, was only detected after partitioning isolates into six geographic regions. The PS2 population, found predominantly in the hyper-endemic regions of northwestern Ontario, Wisconsin, and Minnesota, contained a substantial clonal component with random mating detected only among unique genotypes in the population.

Conclusions/Significance

These analyses provide evidence for a genetically divergent clade within Blastomyces dermatitidis, which we use to describe a novel species, Blastomyces gilchristii sp. nov. In addition, we discuss the value of population genetic and phylogenetic analyses as a foundation for disease surveillance, understanding pathogen evolution, and discerning phenotypic differences between phylogenetic species.     

Molecular phylogenetics of slit‐faced bats (Chiroptera: Nycteridae) reveal deeply divergent African lineages

The bat family Nycteridae contains only the genus Nycteris, which comprises 13 currently recognized species from Africa and the Arabian Peninsula, one species from Madagascar, and two species restricted to Malaysia and Indonesia in South‐East Asia. We investigated genetic variation, clade membership, and phylogenetic relationships in Nycteridae with broad sampling across Africa for most clades. We sequenced mitochondrial cytochrome b (cytb) and four independent nuclear introns (2,166 bp) from 253 individuals. Although our samples did not include all recognized species, we recovered at least 16 deeply divergent monophyletic lineages using independent mitochondrial and multilocus nuclear datasets in both gene tree and species tree analyses. Mean pairwise uncorrected genetic distances among species‐ranked Nycteris clades (17% for cytb and 4% for concatenated introns) suggest high levels of phylogenetic diversity in Nycteridae. We found a large number of designated clades whose members are distributed wholly or partly in East Africa (10 of 16 clades), indicating that Nycteris diversity has been historically underestimated and raising the possibility that additional unsampled and/or undescribed Nycteris species occur in more poorly sampled Central and West Africa. Well‐resolved mitochondrial, concatenated nuclear, and species trees strongly supported African ancestry for SE Asian species. Species tree analyses strongly support two deeply diverged subclades that have not previously been recognized, and these clades may warrant recognition as subgenera. Our analyses also strongly support four traditionally recognized species groups of Nycteris. Mitonuclear discordance regarding geographic population structure in Nycteris thebaica appears to result from male‐biased dispersal in this species. Our analyses, almost wholly based on museum voucher specimens, serve to identify species‐rank clades that can be tested with independent datasets, such as morphology, vocalizations, distributions, and ectoparasites. Our analyses highlight the need for a comprehensive revision of Nycteridae.     

Rhizobium etli and Rhizobium gallicum Nodulate Common Bean (Phaseolus vulgaris) in a Traditionally Managed Milpa Plot in Mexico: Population Genetics and Biogeographic Implications

The stability of the genetic structure of rhizobial populations nodulating Phaseolus vulgaris cultivated in a traditionally managed milpa plot in Mexico was studied over three consecutive years. The set of molecular markers analyzed (including partial rrs, glnII, nifH, and nodB sequences), along with host range experiments, placed the isolates examined in Rhizobium etli bv. phaseoli and Rhizobium gallicum bv. gallicum. Cluster analysis of multilocus enzyme electrophoresis and plasmid profile data separated the two species and identified numerically dominant clones within each of them. Population genetic analyses showed that there was high genetic differentiation between the two species and that there was low intrapopulation differentiation of the species over the 3 years. The results of linkage disequilibrium analyses are consistent with an epidemic genetic structure for both species, with frequent genetic exchange taking place within conspecific populations but not between the R. etli and R. gallicum populations. A subsample of isolates was selected and used for 16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, nifH copy number determination, and host range experiments. Plasmid profiles and nifH hybridization patterns also revealed the occurrence of lateral plasmid transfer among distinct multilocus genotypes within species but not between species. Both species were recovered from nodules of the same plants, indicating that mechanisms other than host, spatial, or temporal isolation may account for the genetic barrier between the species. The biogeographic implications of finding an R. gallicum bv. gallicum population nodulating common bean in America are discussed.     

Intraspecific variation in male mating strategies in an African ground squirrel (Xerus inauris)

Male mating strategies respond to female availability such that variation in resources that affect spatial distribution can also alter cost–benefit tradeoffs within a population. In arid‐adapted species, rainfall alters reproduction, behavior, morphology, and population density such that populations differing in resource availability may also differ in successful reproductive strategies. Here, we compare two populations of Cape ground squirrels (Xerus inauris), a sub‐Saharan species with year‐round breeding and intense mating competition. Unlike most mammals where males resort to aggressive interactions over females, male X. inauris are tolerant of one another, relying instead on other nonaggressive pre‐ and postcopulatory strategies to determine reproductive success. Our findings suggest that differences in resource availability affect female distribution, which ultimately leads to intraspecific variation in male reproductive tactics and sexual morphology. Sperm competition, assessed by reproductive morphometrics, was more pronounced in our high resource site where females were distributed evenly across the landscape, whereas dominance seemed to be an important determinant of success in our low resource site where females were more aggregated. Both sites had similar mating intensities, and most males did not sire any offspring. However, our low resource site had a higher variance in fertilization success with fewer males siring multiple offspring compared with our high resource site where more individuals were successful. Our results lend support to resource models where variations in female spatial distribution attributed to environmental resources ultimately impact male reproductive behaviors and morphology.     

Integrating a Numerical Taxonomic Method and Molecular Phylogeny for Species Delimitation of Melampsora Species (Melampsoraceae,Pucciniales) on Willows in China

The species in genus Melampsora are the causal agents of leaf rust diseases on willows in natural habitats and plantations. However, the classification and recognition of species diversity are challenging because morphological characteristics are scant and morphological variation in Melampsora on willows has not been thoroughly evaluated. Thus, the taxonomy of Melampsora species on willows remains confused, especially in China where 31 species were reported based on either European or Japanese taxonomic systems. To clarify the species boundaries of Melampsora species on willows in China, we tested two approaches for species delimitation inferred from morphological and molecular variations. Morphological species boundaries were determined based on numerical taxonomic analyses of morphological characteristics in the uredinial and telial stages by cluster analysis and one-way analysis of variance. Phylogenetic species boundaries were delineated based on the generalized mixed Yule-coalescent (GMYC) model analysis of the sequences of the internal transcribed spacer (ITS1 and ITS2) regions including the 5.8S and D1/D2 regions of the large nuclear subunit of the ribosomal RNA gene. Numerical taxonomic analyses of 14 morphological characteristics recognized in the uredinial-telial stages revealed 22 morphological species, whereas the GMYC results recovered 29 phylogenetic species. In total, 17 morphological species were in concordance with the phylogenetic species and 5 morphological species were in concordance with 12 phylogenetic species. Both the morphological and molecular data supported 14 morphological characteristics, including 5 newly recognized characteristics and 9 traditionally emphasized characteristics, as effective for the differentiation of Melampsora species on willows in China. Based on the concordance and discordance of the two species delimitation approaches, we concluded that integrative taxonomy by using both morphological and molecular variations was an effective approach for delimitating Melampsora species on willows in China.     

Intraspecific divergence in sperm morphology of the green sea urchin, <Emphasis Type="Italic">Strongylocentrotus droebachiensis</Emphasis>: implications for selection in broadcast spawners

Background  

Sperm morphology can be highly variable among species, but less is known about patterns of population differentiation within species. Most studies of sperm morphometric variation are done in species with internal fertilization, where sexual selection can be mediated by complex mating behavior and the environment of the female reproductive tract. Far less is known about patterns of sperm evolution in broadcast spawners, where reproductive dynamics are largely carried out at the gametic level. We investigated variation in sperm morphology of a broadcast spawner, the green sea urchin (Strongylocentrotus droebachiensis), within and among spawnings of an individual, among individuals within a population, and among populations. We also examined population-level variation between two reproductive seasons for one population. We then compared among-population quantitative genetic divergence (Q _ST) for sperm characters to divergence at neutral microsatellite markers (F _ST).     

Riding across the selection landscape: fitness consequences of annual variation in reproductive characteristics

Evolutionary models estimating phenotypic selection in character size usually assume that the character is invariant across reproductive bouts. We show that variation in the size of reproductive traits may be large over multiple events and can influence fitness in organisms where these traits are produced anew each season. With data from populations of two orchid species, Caladenia valida and Tolumnia variegata, we used Bayesian statistics to investigate the effect on the distribution in fitness of individuals when the fitness landscape is not flat and when characters vary across reproductive bouts. Inconsistency in character size across reproductive periods within an individual increases the uncertainty of mean fitness and, consequently, the uncertainty in individual fitness. The trajectory of selection is likely to be muddled as a consequence of variation in morphology of individuals across reproductive bouts. The frequency and amplitude of such changes will certainly affect the dynamics between selection and genetic drift.     

MOLECULAR EVOLUTIONARY DIVERGENCE AMONG NORTH AMERICAN CAVE CRICKETS. I. ALLOZYME VARIATION

Forty-nine populations of nine species of North American cave crickets (genera Euhadenoecus and Hadenoecus) have been studied for genetic variation at 41 loci by electrophoresis. Wright's F_ST, Slatkin's Nm^* gene-flow estimator, and Nei's genetic distances (D) have been used to compare closely related species that have different ecological requirements (cave vs. forest species), distribution patterns, and/or different degrees of geographic isolation among populations. Cave and epigean (noncave) species differ greatly in their levels of genetic differentiation. Cave species have lower rates of gene exchange (low Nm, high D, and F_ST) than epigean species. Within cave species the degree of genetic differentiation among populations is correlated with the limestone structure of the area where the species occur. Species or groups of populations inhabiting areas where the limestone is continuous and highly fissured (e.g., H. subterraneus populations in the Mammoth Cave region) are genetically less differentiated than are populations occurring in regions where the limestone distribution is more fragmented, such as the Appalachian Ridge where E. fragilis occurs; this effect is more extreme in Central Tennessee where genetically differentiated E. insolitus populations occur only a few kilometers apart. This suggests that epigean dispersal through forest habitat in cave-dwelling species is negligable. For forest species, the data indicate relatively recent radiation with ongoing gene exchange among populations. For cave species, the distribution of protein polymorphisms is apparently more a function of historical patterns of gene exchange rather than current gene exchange. Phylogenetic relationships were studied using cluster analyses (UPGMA and Wagner algorithms) of Nei's and Edwards' genetic distances and multivariate analysis (correspondence analysis) of the raw allele frequencies. Different algorithms result in branching patterns that are similar but not entirely concordant with one another or with the phylogeny based on morphology.     

The Detection,Rate and Manifestation of Residual Sexuality in Apomictic Populations of Pilosella (Asteraceae,Lactuceae)

The effect of maternal, facultatively apomictic plants on population diversity was evaluated in seven hybridizing polyploid Pilosella populations, where apomictic (P. bauhini or P. aurantiaca) and sexual (P. officinarum) biotypes coexist. The ploidy level, reproductive system, morphology, clonal structure and chloroplast DNA haplotypes were used to characterize these plants and their hybrids. The reproductive origins of the progeny were assessed through either a flow cytometric seed screen and/or a comparison between the ploidy level of progeny embryos/seedlings and the maternal ploidy level. The cultivated progeny derived from residual sexuality in maternal apomicts were also identified based on their morphology and reproductive behaviour. The progeny different from their maternal parents (0.6?92.3 % of progeny embryos and 0?100 % of progeny seedlings) originated either sexually or via haploid parthenogenesis. Comparing the facultatively apomictic and sexual mothers, the progeny arrays generated in the field showed that apomictic mothers produce progeny that is more variable in ploidy level. This effect was demonstrated at both the embryonic and seedling stages of progeny development. Residual sexuality in apomicts was also effective in experimental crosses, generating progeny similar to spontaneous hybrids in the field. The 2n + n hybrids produced from an apomictic and a sexual parent displayed similar reproductive behaviour, producing polyhaploid, sexual and apomictic progeny in variable ratios. Repeated hybridizations between parental species and/or multi-step crosses can result in hybrid swarms rich in cytotypes and morphotypes. The variation recorded in these populations suggests prevailing introgressive hybridization towards the sexual species P. officinarum.     

Parahellenia,a new genus segregated from Hellenia (Costaceae) based on phylogenetic and morphological evidence

Previous studies recognized three major lineages of the family Costaceae: a South American clade, an Asian clade and a Costus clade. However, the genus Hellenia within the Asian clade has been shown to be non-monophyletic and its morphology has not been studied carefully. Therefore, the complete plastid genomes of Hellenia species were obtained and the monophyly of Hellenia was tested through four different datasets in this study. Plastid phylogenomic analyses of Costaceae revealed that Hellenia is strongly supported as paraphyletic. Two major clades are recovered, namely the Hellenia s.s. subclade and the Parahellenia subclade. Phylogenetic analyses based on an enlarged taxon sampling of the Asian clade using a two chloroplast markers dataset (trnK intron and trnL-F spacer) confirmed the paraphyly of Hellenia. Meanwhile, morphological analyses suggested that members of the Parahellenia subclade differ from the remaining Hellenia species in many characters including inflorescences, bracts, stigma, axillary buds, floral tubes and labellum. According to the present molecular and morphological evidence, the latter subclade is recognized as a new genus, Parahellenia. Two new species are described, four new combinations are made, and identification keys are also provided.     

Phylogeny and biogeography of the genus Cephalenchus (Tylenchomorpha,Nematoda)

The phylogenetic position of Cephalenchus is enigmatic with respect to other tylench nematodes. In this study, Cephalenchus populations representing 11 nominal species were sampled worldwide for molecular and morphological characterization. Species identification was based on light microscopy (LM) and scanning electron microscopy (SEM). Molecular analyses were based on the genes (i.e. 18S, 28S, 5.8S) and internal transcribed spacers (ITS‐1 and ITS‐2) of the ribosomal RNA (rRNA). Phylogenetic analyses (i.e. full and reduced alignments) of either concatenated or single genes always supported the monophyly of Cephalenchus. A sister relationship between Cephalenchus and Eutylenchus excretorius was recovered by most analyses, although branch support varies depending on the dataset used. The position of Cephalenchus + E. excretorius within Tylenchomorpha nevertheless remains ambiguous, thus highlighting the importance of sampling additional genes as well as taxa. Placement of Cephalenchus + E. excretorius as sister of Tylenchinae or Boleodorinae could not be rejected on the basis of 18S and 28S rRNA genes. Within Cephalenchus, amphidial opening morphology shows congruence with molecular‐based phylogenetic relationships, whereas the number of lines in the lateral field is likely to be a convergent trait. Morphometric analyses clearly distinguished short tail from medium–long tail species, and SEM observations seem to suggest a relation between tail length and amphidial opening. In addition, molecular phylogenies support the non‐monophyly of Cephalenchus cephalodiscus, Cephalenchus cylindricus, Cephalenchus daisuce and Cephalenchus leptus. The known extent of Cephalenchus diversity is increased with the inclusion of two new species, and the biogeography of the genus is discussed.     

Phylogenetic species recognition and species concepts in fungi

The operational species concept, i.e., the one used to recognize species, is contrasted to the theoretical species concept. A phylogenetic approach to recognize fungal species based on concordance of multiple gene genealogies is compared to those based on morphology and reproductive behavior. Examples where Phylogenetic Species Recognition has been applied to fungi are reviewed and concerns regarding Phylogenetic Species Recognition are discussed.