Recognized Species Boundaries Research Articles

Challenges and Advances in the Taxonomy of Deep-Sea Peracarida: From Traditional to Modern Methods

As one of the oldest branches of biology, taxonomy deals with the identification, classification and naming of living organisms, using a variety of tools to explore traits at the morphological and molecular level. In the deep sea, particular challenges are posed to the taxonomic differentiation of species. Relatively limited sampling effort coupled with apparent high diversity, compared to many other marine environments, means that many species sampled are undescribed, and few specimens are available for each putative species. The resulting scarce knowledge of intraspecific variation makes it difficult to recognize species boundaries and thus to assess the actual diversity and distribution of species. In this review article, we highlight some of these challenges in deep-sea taxonomy using the example of peracarid crustaceans. Specifically, we offer a detailed overview of traditional as well as modern methods that are used in the taxonomic analysis of deep-sea Peracarida. Furthermore, methods are presented that have not yet been used in peracarid taxonomy, but have potential for the analysis of internal and external structures in the future. The focus of this compilation is on morphological methods for the identification, delimitation and description of species, with references to molecular analysis included where relevant, as these methods are an indispensable part of an integrative taxonomic approach. The taxonomic impediment, i.e. the shortage of taxonomists in view of a high undescribed biodiversity, is discussed in the context of the existing large taxonomic knowledge gaps in connection with the increasing threat to deep-sea ecosystems. Whilst peracarid crustaceans are used here as an exemplary taxon, the methodology described has broad relevance to many other deep-sea taxa, and thus will support broader research into deep-sea biodiversity and ecology more widely.

Molecular Tools for Detection and Identification of Paracoccidioides Species: Current Status and Future Perspectives.

Paracoccidioidomycosis (PCM) is a mycotic disease caused by the Paracoccidioides species, a group of thermally dimorphic fungi that grow in mycelial form at 25 °C and as budding yeasts when cultured at 37 °C or when parasitizing the host tissues. PCM occurs in a large area of Latin America, and the most critical regions of endemicity are in Brazil, Colombia, and Venezuela. The clinical diagnosis of PCM needs to be confirmed through laboratory tests. Although classical laboratory techniques provide valuable information due to the presence of pathognomonic forms of Paracoccidioides spp., nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratory practice. Recently, taxonomic changes driven by whole-genomic sequencing of Paracoccidioides have highlighted the need to recognize species boundaries, which could better ascertain Paracoccidioides taxonomy. In this scenario, classical laboratory techniques do not have significant discriminatory power over cryptic agents. On the other hand, several PCR-based methods can detect polymorphisms in Paracoccidioides DNA and thus support species identification. This review is focused on the recent achievements in molecular diagnostics of paracoccidioidomycosis, including the main advantages and pitfalls related to each technique. We discuss these breakthroughs in light of taxonomic changes in the Paracoccidioides genus.

Molecular and morphological recognition of species boundaries in the neglected ant genus Brachymyrmex (Hymenoptera: Formicidae): toward a taxonomic revision

Brachymyrmex is a neglected genus of Formicinae because of its small body size, soft mesosoma, and superficially monotonous external morphology. These features have complicated the documentation of morphological variation, resulting in poorly defined and incompletely described species. Consequently, the taxonomy of the genus is complex and problematic, which has impeded research and conservation efforts. Here, we integrate molecular and morphological data to recognize species boundaries in Brachymyrmex and to guide its long-overdue revision. Specifically, we (1) redefine the limits of all described species, subspecies, and varieties based on intra- and interspecific morphological variation in workers; (2) document this variation quantitatively by constructing morphospace occupation and statistically analyzing measurements; (3) synthesize our findings on diagnostic traits in a dichotomous, illustrated identification key; and (4) examine the significance of our morphological identification system with molecular evidence from four gene fragments (EF1aEF1, EF1aEF2, WG, and COI). We recognize 40 species, of which four are new to science: Brachymyrmex bahamensis, Brachymyrmex bicolor, Brachymyrmex iridescens, and Brachymyrmex sosai. Furthermore, Brachymyrmex attenuatus and Brachymyrmex bonariensis are raised to species, and we propose 25 new synonyms. Morphometrics indicated that even poorly distinguishable species pairs show statistically significant differences in some traits, and that taxonomically problematic cases relate to taxa that demonstrate large intraspecific trait variance. Our molecular analysis supports the monophyly of the genus based on increased taxon sampling, and of the 19 species that were included 18 were retrieved as monophyletic. The single case of incongruence was also flagged in morphological analyses and requires extended geographic sampling before it can be resolved. In conclusion, the molecular work corroborates the morphologically recognized species boundaries. We also document the presence of worker dimorphism and putative worker-queen intercastes in several Brachymyrmex species, which indicates that the genus may present a promising study system to understand caste evolution in ants.

Introgression Threatens the Genetic Diversity of Quercus austrocochinchinensis (Fagaceae), an Endangered Oak: A Case Inferred by Molecular Markers.

Natural introgression can cause negative effects where rare species experience genetic assimilation and invade by their abundant congeners. Quercus austrocochinchinensis and Q. kerrii (subgenus Cyclobalanopsis) are a pair of closely related species in the Indo-China area. Morphological intermediates of the two species have been reported in this region. In this study, we used AFLP, SSR and two key leaf morphological diagnostic traits to study the two Q. austrocochinchinensis populations, two pure Q. kerrii and two putative hybrid populations in China. Rates of individual admixture were examined using the Bayesian clustering programs STRUCTURE and NewHybrids, with no a priori species assignment. In total, we obtained 151 SSR alleles and 781 polymorphic loci of AFLP markers. Population differentiation inferred by SSR and AFLP was incoherent with recognized species boundaries. Bayesian admixture analyses and principal coordinate analysis identified more hybrids and backcrossed individuals than morphological intermediates in the populations. SSR inferred a wide genetic assimilation in Q. austrocochinchinensis, except for subpopulation D2 in the core area of Xi-Shuang-Ban-Na Nature Reserve (XSBN). However, AFLP recognized more Q. austrocochinchinensis purebreds than SSR. Analysis using NewHybrids on AFLP data indicated that these hybridized individuals were few F2 and predominantly backcrosses with both parental species. All these evidences indicate the formation of a hybrid swarm at XSBN where the two species co-exist. Both AFLP and SSR recognized that the core protected area of XSBN (D2) has a high percentage of Q. austrocochinchinensis purebreds and a unique germplasm. The Hainan population and the other subpopulations of XSBN of the species might have lost their genetic integrity. Our results revealed a clear genetic differentiation in the populations and subpopulations of Q. austrocochinchinensis and ongoing introgression between Q. austrocochinchinensis and Q. kerrii at the disturbed contact areas. Combining the results from genetic and morphological analyses, the conservation of subpopulation D2 should be prioritized. Conservation and restoration of the integrity of tropical ravine rainforest is an important long-term goal for the successful conservation of Q. austrocochinchinensis. The fine-scale landscape might play an essential role in shaping the spatial patterns of hybridization. Further studies are needed to evaluate these patterns and dynamics.

Mountains and refuges: Genetic structure and evolutionary history in closely related, endemic Centaurea in continental Greece

Mountains of continental Greece are one of the main Mediterranean biodiversity hotspots, very rich in endemic species. The speciation in this area might have resulted from two main factors: a complex orography and its role as a refugium during past glaciations. We have investigated genetic diversity and population structure for a group of narrow endemics of Centaurea subsect. Phalolepis, with three main goals: to investigate population structure of these narrow endemics, to check whether patterns of genetic variation are in agreement with recognized species boundaries, and to get insights into the process of diversification within this group. Fifteen populations belonging to seven species were genotyped using cpDNA (rpl32-trnL region) sequences and nuclear microsatellites (eight loci). SSR were used to assess genetic variability, to analyse molecular variance, to identify genetic barriers, to estimate recent and historical gene flow, and to carry out a model-based Bayesian clustering. Analysis of cpDNA was used to construct a haplotype network. Despite being narrow endemics, all the studied species show moderate to high SSR genetic diversity. Genetic isolation of populations is very high, with no current gene flow among them. Patterns of genetic structure indicate that there are more genetic clusters than there are currently recognized taxa. Genetic data suggest that isolation in mountain ranges and subsequent allopatric speciation would be the main driver of diversification in the group; the refugial nature of the mountains of continental Greece has allowed the maintenance of high within-population genetic diversity.

Sporothrix chilensis sp. nov. (Ascomycota: Ophiostomatales), a soil-borne agent of human sporotrichosis with mild-pathogenic potential to mammals

A combination of phylogeny, evolution, morphologies and ecologies has enabled major advances in understanding the taxonomy of Sporothrix species, including members exhibiting distinct lifestyles such as saprobes, human/animal pathogens, and insect symbionts. Phylogenetic analyses of ITS1/2 + 5.8s sequences split Sporothrix genus in two well-defined groups with dissimilar ecologies. Species embedded in the Sporothrix schenckii complex are frequently agents of human and animal sporotrichosis, and some of these are responsible for large sapronoses and zoonoses around the warmer temperate regions of the world. At the other extreme, basal saprophytic species evolved in association with decaying wood and soil, and are rarely found to cause human disease. We propose to create a new taxa, Sporothrix chilensis sp. nov., to accommodate strains collected from a clinical case of onychomycosis as well as from environmental origins in Chile. Multigene analyses based on ITS1/2 + 5.8s region, beta-tubulin, calmodulin and translation elongation factor 1α revealed that S. chilensis is a member of the Sporothrix pallida complex, and the nearest taxon is Sporothrix mexicana, a rare soil-borne species, non-pathogenic to humans. The ITS region serves as a primary barcode marker, while each one of the protein-coding loci easily recognized species boundaries providing sufficient information for species identification. A disseminated model of murine sporotrichosis revealed a mild-pathogenic potential, with lung invasion. Although S. chilensis is not a primary pathogen, accidental infection may have an impact in the immunosuppressed population. With the introduction of distinct species with similar routes of transmission but different virulence, identification of Sporothrix agents at the species level is mandatory.

Nuclear microsatellite variation in Malagasy baobabs (Adansonia, Bombacoideae, Malvaceae) reveals past hybridization and introgression

Adansonia comprises nine species, six of which are endemic to Madagascar. Genetic relationships between the Malagasy species remain unresolved due to conflicting results between nuclear and plastid DNA variation. Morphologically intermediate individuals between distinct species have been identified, indicative of interspecific hybridization. In this paper, microsatellite data are used to identify potential cases of hybridization and to provide insights into the evolutionary history of the genus on Madagascar. Eleven microsatellites amplified with new primers developed for Adansonia rubrostipa were used to analyse 672 individuals collected at 27 sites for the six Malagasy species and morphologically intermediate individuals. Rates of individual admixture were examined using three Bayesian clustering programs, STRUCTURE, BAPS and NewHybrids, with no a priori species assignment. Population differentiation was coherent, with recognized species boundaries. In the four Malagasy species of section Longitubae, 8·0, 9·0 and 9·5 % of individuals with mixed genotypes were identified by BAPS, NewHybrids and STRUCTURE, respectively. At sites with sympatric populations of A. rubrostipa and A. za, NewHybrids indicated these individuals to be F2 and, predominantly, backcrosses with both parental species. In northern Madagascar, two populations of trees combining A. za and A. perrieri morphology and microsatellite alleles were identified in the current absence of the parental species. The clear genetic differentiation observed between the six species may reflect their adaptation to different assortments of climate regimes and habitats during the colonization of the island. Microsatellite variation reveals that hybridization probably occurred in secondary contact between species of section Longitubae. This type of hybridization may also have been involved in the differentiation of a local new stabilized entity showing specific microsatellite alleles and morphological characters, suggesting a potential role of hybridization in the recent history of diversification on Madagascar.

Niche differentiation and spatial partitioning in the evolution of two Australian monsoon tropical tree species

AbstractAimGeographical and climatic barriers to organismal dispersal and distribution play a major role in speciation. We use a sister‐pair of widespread savanna trees (Melaleuca argentea and M. fluviatilis) to test the influence of putative barriers on divergence within and between species across an otherwise continuous landscape.LocationThe Australian monsoon tropics (AMT).MethodsChloroplast and nuclear DNA sequences were used to estimate variation between and within species. Hypotheses invoking vicariance and ecological speciation as the mechanisms of divergence between species were explicitly tested using ecological niche modelling.ResultsWe found little evidence for divergence across the Carpentaria Basin, although some chloroplast DNA haplotypes were restricted to regions to the east or west. Pilbara populations were distinct from those to the east across the Great Sandy Desert, including those from the Kimberley. There was a complex pattern of genetic divergence and niche differentiation among M. argentea and M. fluviatilis within a region of secondary range overlap coincident with currently recognized species boundaries across the Great Dividing Range.Main conclusionsThe two morphospecies are ecologically and genetically distinct, and maintain those differences in sympatry. Speciation might have occurred in allopatry in separate drainage basins that later came into contact. The Pilbara population appears to be distinct but requires further investigation.

Hybridization and introgression in New World red mangroves,Rhizophora(Rhizophoraceae)

Hybridization is common in both animals and plants and can lead to a diverse array of outcomes ranging from the generation of new ecotypes or species to the breakdown of morphological differences. Here, we explore the extent of hybridization in the three currently recognized New World Rhizophora species-R. mangle, R. racemosa, and the putative hybrid species R. harrisonii. • We assayed variation across the three recognized Rhizophora species using two noncoding chloroplast (cpDNA), two flanking microsatellite regions (FMRs), and six microsatellite loci. • Gene genealogies of cpDNA and FMRs showed a strong phylogeographic break across the Central American Isthmus, but little relationship to recognized species boundaries. Instead, individuals collected in the same ocean basin and classified as R. mangle and R. racemosa by morphological characteristics were more closely related to each other than with similar looking individuals collected in the other ocean basin. Nonetheless, there were low, yet significant differences at microsatellite loci among co-occurring populations of R. mangle and R. racemosa in both ocean basins, suggesting that two taxonomic groups coexist. However, we found no genetic evidence that R. harrisonii was a hybrid species. Rather, R. harrisonii appears to represent a morphotype produced by ongoing hybridization and backcrossing between R. mangle and R. racemosa. • Our data support ancient and persistent introgressive hybridization among new world Rhizophora and argue for a full revision of the systematic relationships of the group based on much finer morphological, ecological, and genetic analyses.

Using patterns of genetic structure based on microsatellite loci to test hypotheses of current hybridization, ancient hybridization and incomplete lineage sorting in Conradina (Lamiaceae)

Hybridization and/or incomplete sorting of ancestral polymorphism are commonly implicated to explain discordant phylogenetic analyses of closely related species complexes. One genus in which these phenomena have been suggested to have played major roles based on phylogenetic data is Conradina, a genus of mints (Lamiaceae) endemic to the southeastern USA containing several endangered species. The goals of this study were to use microsatellite data to better understand patterns of genetic structure in Conradina, to test hypotheses of recent or ancient hybridization and incomplete lineage sorting, and to clarify species boundaries. Individuals from 55 populations representing all Conradina species were genotyped using 10 microsatellite loci. Analyses of the patterns of genetic structure in Conradina revealed a clear differentiation of populations following recognized species boundaries, indicating that species have diverged from one another genetically and interspecific hybridization has not occurred recently. Neither ancient hybridization nor incomplete lineage sorting is supported as the sole cause of species nonmonophyly, suggesting that both may have contributed to patterns found in phylogenetic trees; however, analyses of other types of data may be more appropriate to distinguish between these two hypotheses. Because all described species appear to be valid entities, the current listing status of most endangered species of Conradina is appropriate; however, populations of Conradina canescens are genetically differentiated into three groups, each of which may merit species status, and several recently discovered populations of Conradina in Dunn's Creek State Park in Florida are highly differentiated genetically and also appear to represent a new species.

GENETIC ARCHITECTURE OF THE CRYPTIC SPECIES COMPLEX OF ACANTHOCYCLOPS VERNALIS (CRUSTACEA: COPEPODA). II. CROSSBREEDING EXPERIMENTS, CYTOGENETICS, AND A MODEL OF CHROMOSOMAL EVOLOUTION

Collectively, populations of Acanthocyclops vernalis, a species complex of freshwater copepods, are remarkably similar as to morphology and DNA content, despite variability in chromosome number. Reproductive isolation had been reported among some populations, but with each new investigation the species boundaries and factors that may influence them appeared less clear. To clarify the pattern of biological species within this group of populations, we adopted a comprehensive approach and examined patterns of reproductive isolation in populations for which morphology, chromosome number, DNA content, and 18S rDNA sequences are known. In this study we established nine isofemale lines from four sites in Wisconsin and performed 266 crosses. Crosses within and among these lines were used to relate the degree of reproductive isolation to chromosome differences and to construct a model to explain the origin and maintenance of chromosome number variability. Different gametic and somatic chromosome numbers were observed among specimens within some isofemale lines. In a few cases, gametes with different haploid numbers were produced by a single female. Matings within isofemale lines always produced at least some reproductively successful replicate crosses (produced viable, fertile offspring). Crosses between lines from the same site showed reduced success relative to within-line crosses. Crosses between populations from distant sites showed limited genetic compatibility, producing viable, fertile F1 offspring but infertile F2 adults. One cross between lines with different chromosome numbers (one with 2n = 8 and one with 2n = 10) produced fertile viable offspring, which reproduced for at least 60 generations. These hybrids had either eight or nine chromosomes in the third generation of inbreeding, and eight chromosomes after 20 generations. These hybrids also had reduced nuclear DNA contents at the third generation, a level that persisted through the 20th generation. Successful backcrosses between some hybrids and their parental lines further demonstrated the potential for genetic compatibility among forms with different chromosome numbers. We propose a model in which alterations due to Robertsonian fusions, translocations, and/or loss of chromosomal fragments generate heritable variation, only some of which leads to reproductive isolation. Hence, some of the criteria traditionally used to recognize species boundaries in animals (morphology, DNA content, chromosome number) may not apply to this species complex.

GENETIC ARCHITECTURE OF THE CRYPTIC SPECIES COMPLEX OF ACANTHOCYCLOPS VERNALIS (CRUSTACEA: COPEPODA). II. CROSSBREEDING EXPERIMENTS, CYTOGENETICS, AND A MODEL OF CHROMOSOMAL EVOLUTION

Collectively, populations of Acanthocyclops vernalis, a species complex of freshwater copepods, are remarkably similar as to morphology and DNA content, despite variability in chromosome number. Reproductive isolation had been reported among some populations, but with each new investigation the species boundaries and factors that may influence them appeared less clear. To clarify the pattern of biological species within this group of populations, we adopted a comprehensive approach and examined patterns of reproductive isolation in populations for which morphology, chromosome number, DNA content, and 18S rDNA sequences are known. In this study we established nine isofemale lines from four sites in Wisconsin and performed 266 crosses. Crosses within and among these lines were used to relate the degree of reproductive isolation to chromosome differences and to construct a model to explain the origin and maintenance of chromosome number variability. Different gametic and somatic chromosome numbers were observed among specimens within some isofemale lines. In a few cases, gametes with different haploid numbers were produced by a single female. Matings within isofemale lines always produced at least some reproductively successful replicate crosses (produced viable, fertile offspring). Crosses between lines from the same site showed reduced success relative to within-line crosses. Crosses between populations from distant sites showed limited genetic compatibility, producing viable, fertile F1 offspring but infertile F2 adults. One cross between lines with different chromosome numbers (one with 2n = 8 and one with 2n = 10) produced fertile viable offspring, which reproduced for at least 60 generations. These hybrids had either eight or nine chromosomes in the third generation of inbreeding, and eight chromosomes after 20 generations. These hybrids also had reduced nuclear DNA contents at the third generation, a level that persisted through the 20th generation. Successful backcrosses between some hybrids and their parental lines further demonstrated the potential for genetic compatibility among forms with different chromosome numbers. We propose a model in which alterations due to Robertsonian fusions, translocations, and/or loss of chromosomal fragments generate heritable variation, only some of which leads to reproductive isolation. Hence, some of the criteria traditionally used to recognize species boundaries in animals (morphology, DNA content, chromosome number) may not apply to this species complex.

A Gene Genealogical Approach to Recognize Phylogenetic Species Boundaries in the Lichenized Fungus Letharia

Letharia, a genus of lichenized fungi described as a pair of sympatric species, one making abundant sexual structures and the other making few, was investigated as a model system in which to recognize species boundaries. Gene genealogies of 6 and 12 loci were used to estimate the evolutionary history of Letharia, based on the principles of lineage sorting of alleles in divergent lineages after genetic isolation. Instead of a species pair consisting of a putative clonal species derived from a progenitor sexual species, Letharia comprises at least six phylogenetic species. Judging by the presence of perennial apothecia and clonal reproductive structures, one species is exclusively sexual, three species are sexual and isidiate, and two species are sorediate and rarely sexual. Not all of these species would have been detected with a single gene genealogy, demonstrating the need for multiple independent loci in phylogenetic analysis to recognize recent speciation events. The results are concordant with aspects of both biological and phylogenetic species recognition. However, only phylogenetic species recognition can be applied to fungi like Letharia species that are difficult to cultivate and mate in the lab.

A gene genealogical approach to recognize phylogenetic species boundaries in the lichenized fungus Letharia

Letharia, a genus of lichenized fungi described as a pair of sympatric species, one making abundant sexual structures and the other making few, was investigated as a model system in which to recognize species boundaries. Gene genealogies of 6 and 12 loci were used to estimate the evolutionary history of Letharia, based on the principles of lineage sorting of alleles in divergent lineages after genetic isolation. Instead of a species pair consisting of a putative clonal species derived from a progenitor sexual species, Letharia comprises at least six phylogenetic species. Judging by the presence of perennial apothecia and clonal reproductive structures, one species is exclusively sexual, three species are sexual and isidiate, and two species are sorediate and rarely sexual. Not all of these species would have been detected with a single gene genealogy, demonstrating the need for multiple independent loci in phylogenetic analysis to recognize recent speciation events. The results are concordant with aspects of both biological and phylogenetic species recognition. However, only phylogenetic species recognition can be applied to fungi like Letharia species that are difficult to cultivate and mate in the lab.