Species delimitation: A case study in a problematic ant taxon

Species delimitation has been invigorated as a discipline in systematics by an influx of new character sets, analytical methods, and conceptual advances. We use genetic data from 68 markers, combined with distributional, bioclimatic, and coloration information, to hypothesize boundaries of evolutionarily independent lineages (species) within the widespread and highly variable nominal fire ant species Solenopsis saevissima, a member of a species group containing invasive pests as well as species that are models for ecological and evolutionary research. Our integrated approach uses diverse methods of analysis to sequentially test whether populations meet specific operational criteria (contingent properties) for candidacy as morphologically cryptic species, including genetic clustering, monophyly, reproductive isolation, and occupation of distinctive niche space. We hypothesize that nominal S. saevissima comprises at least 4-6 previously unrecognized species, including several pairs whose parapatric distributions implicate the development of intrinsic premating or postmating barriers to gene flow. Our genetic data further suggest that regional genetic differentiation in S. saevissima has been influenced by hybridization with other nominal species occurring in sympatry or parapatry, including the quite distantly related Solenopsis geminata. The results of this study illustrate the importance of employing different classes of genetic data (coding and noncoding regions and nuclear and mitochondrial DNA [mtDNA] markers), different methods of genetic data analysis (tree-based and non-tree based methods), and different sources of data (genetic, morphological, and ecological data) to explicitly test various operational criteria for species boundaries in clades of recently diverged lineages, while warning against over reliance on any single data type (e.g., mtDNA sequence variation) when drawing inferences.

A species delimitation approach in the Trochulus sericeus/hispidus complex reveals two cryptic species within a sharp contact zone.

BACKGROUND: Mitochondrial DNA sequencing increasingly results in the recognition of genetically divergent, but morphologically cryptic lineages. Species delimitation approaches that rely on multiple lines of evidence in areas of co-occurrence are particularly powerful to infer their specific status. We investigated the species boundaries of two cryptic lineages of the land snail genus Trochulus in a contact zone, using mitochondrial and nuclear DNA marker as well as shell morphometrics. RESULTS: Both mitochondrial lineages have a distinct geographical distribution with a small zone of co-occurrence. In the same area, we detected two nuclear genotype clusters, each being highly significantly associated to one mitochondrial lineage. This association however had exceptions: a small number of individuals in the contact zone showed intermediate genotypes (4%) or cytonuclear disequilibrium (12%). Both mitochondrial lineage and nuclear cluster were statistically significant predictors for the shell shape indicating morphological divergence. Nevertheless, the lineage morphospaces largely overlapped (low posterior classification success rate of 69% and 78%, respectively): the two lineages are truly cryptic. CONCLUSION: The integrative approach using multiple lines of evidence supported the hypothesis that the investigated Trochulus lineages are reproductively isolated species. In the small contact area, however, the lineages hybridise to a limited extent. This detection of a hybrid zone adds an instance to the rare reported cases of hybridisation in land snails.

Friends or relatives? Phylogenetics and species delimitation in the controversial European orchid Genus ophrys

Background and Aims Highly variable, yet possibly convergent, morphology and lack of sequence variation have severely hindered production of a robust phylogenetic framework for the genus Ophrys. The aim of this study is to produce this framework as a basis for more rigorous species delimitation and conservation recommendations. Methods Nuclear and plastid DNA sequencing and amplified fragment length polymorphism (AFLP) were performed on 85 accessions of Ophrys, spanning the full range of species aggregates currently recognized. Data were analysed using a combination of parsimony and Bayesian tree-building techniques and by principal coordinates analysis. Key Results Complementary phylogenetic analyses and ordinations using nuclear, plastid and AFLP datasets identify ten genetically distinct groups (six robust) within the genus that may in turn be grouped into three sections (treated as subgenera by some authors). Additionally, genetic evidence is provided for a close relationship between the O. tenthredinifera, O. bombyliflora and O. speculum groups. The combination of these analytical techniques provides new insights into Ophrys systematics, notably recognition of the novel O. umbilicata group. Conclusions Heterogeneous copies of the nuclear ITS region show that some putative Ophrys species arose through hybridization rather than divergent speciation. The supposedly highly specific pseudocopulatory pollination syndrome of Ophrys is demonstrably 'leaky', suggesting that the genus has been substantially over-divided at the species level.

Molecular species delimitation of a symbiotic fig-pollinating wasp species complex reveals extreme deviation from reciprocal partner specificity

Background: Symbiotic relationships have contributed to major evolutionary innovations, the maintenance of fundamental ecosystem functions, and the generation and maintenance of biodiversity. However, the exact nature of host/symbiont associations, which has important consequences for their dynamics, is often poorly known due to limited understanding of symbiont taxonomy and species diversity. Among classical symbioses, figs and their pollinating wasps constitute a highly diverse keystone resource in tropical forest and savannah environments. Historically, they were considered to exemplify extreme reciprocal partner specificity (one-to-one host-symbiont species relationships), but recent work has revealed several more complex cases. However, there is a striking lack of studies with the specific aims of assessing symbiont diversity and how this varies across the geographic range of the host. Results: Here, we use molecular methods to investigate cryptic diversity in the pollinating wasps of a widespread Australian fig species. Standard barcoding genes and methods were not conclusive, but incorporation of phylogenetic analyses and a recently developed nuclear barcoding gene (ITS2), gave strong support for five pollinator species. Each pollinator species was most common in a different geographic region, emphasising the importance of wide geographic sampling to uncover diversity, and the scope for divergence in coevolutionary trajectories across the host plant range. In addition, most regions had multiple coexisting pollinators, raising the question of how they coexist in apparently similar or identical resource niches. Conclusion: Our study offers a striking example of extreme deviation from reciprocal partner specificity over the full geographical range of a fig-wasp system. It also suggests that superficially identical species may be able to co-exist in a mutualistic setting albeit at different frequencies in relation to their fig host’s range. We show that comprehensive sampling and molecular taxonomic techniques may be required to uncover the true structure of cryptic biodiversity underpinning intimate ecological interactions.

Species delimitation in sharpnose sharks (genus Rhizoprionodon) in the western Atlantic Ocean using mitochondrial DNA

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Species delimitation, patterns of diversification and historical biogeography of the Neotropical frog genus Adenomera (Anura, Leptodactylidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecules, wing pattern and distribution: an approach to species delimitation in the "loxurina group" (Lepidoptera: Lycaenidae: Penaincisalia)

The wide range of morphological variations in the “loxurina group” makes taxa identification difficult, and despite several reviews, serious taxonomical confusion remains. We make use of DNA data in conjunction with morphological appearance and available information on species distribution to delimit the boundaries of the “loxurina” group species previously established based on morphology. A fragment of 635 base pairs within the mtDNA gene cytochrome oxidase I (COI) was analysed for seven species of the “loxurina group”. Phylogenetic relationships among the included taxa were inferred using maximum parsimony and maximum likelihood methods. Penaincisalia sigsiga (Bálint et al), P. cillutincarae (Draudt), P. atymna (Hewitson) and P. loxurina (C. Felder & R. Felder) were easily delimited as the morphological, geographic and molecular data were congruent. Penaincisalia ludovica (Bálint & Wojtusiak) and P. loxurina astillero (Johnson) represent the same entity and constitute a sub-species of P. loxurina. However, incongruence among morphological, genetic, and geographic data is shown in P. chachapoya (Bálint & Wojtusiak) and P. tegulina (Bálint et al). Our results highlight that an integrative approach is needed to clarify the taxonomy of these neotropical taxa, but more genetic and geographical studies are still required.

Species delimitation in sharpnose sharks (genus Rhizoprionodon) in the western Atlantic Ocean using mitochondrial DNA

Despite Springer’s (1964) revision of the sharpnose sharks (genus Rhizoprionodon), the taxonomic definition and ranges of Rhizoprionodon in the western Atlantic Ocean remains problematic. In particular, the distinction between Rhizoprionodon terraenovae and R. porosus, and the occurrence of R. terraenovae in South American waters are unresolved issues involving common and ecologically important species in need of fishery management in Caribbean and southwest Atlantic waters. In recent years, molecular markers have been used as efficient tools for the detection of cryptic species and to address controversial taxonomic issues. In this study 415 samples of the genus Rhizoprionodon captured in the western Atlantic Ocean from Florida to southern Brazil were examined for sequences of the COI gene and the D-loop and evaluated for nucleotide differences. The results on nucleotide composition, AMOVA tests, and relationship distances using Bayesian-likelihood method and haplotypes network, corroborates Springer’s (1964) morphometric and meristic finding and provide strong evidence that supports consideration of R. terraenovae and R. porosus as distinct species.

Cryptic species in Pagamea coriacea sensu lato (Rubiaceae): evidence from morphology, ecology and reproductive behavior in a sympatric context

In this study we explore morphological and ecological variation in sympatric populations of Pagamea coriacea s.l. - a species complex from white-sand vegetation in the Amazon. A total of 147 trees were sampled and monitored at three nearby sites in Central Amazon, Brazil. Multivariate analyses of morphology indicated two distinct groups (A and B), which also differed in bark type, each containing subgroups associated with sexual dimorphism. However, a single hermaphroditic individual was observed within group B. As expected, all pistillate plants produced fruits, but 23% of the staminate plants of group B, and 5% of group A also produced fruits. This variation suggests that the sexual systems of both groups are between dioecy and gynodioecy. There was an overlap in flowering phases between the two groups, but the pattern of floral maturation differed. Ecologically, plants of group B were found in more shaded habitats and over sandstone bedrocks, while group A was prevalent in deeper sandy soils as canopy plants. The significances of morphological and environmental differences were tested by a multivariate analysis of variance, and a canonical discriminant analysis assessed the importance of variables. The coexistence in sympatry of two discrete morphological groups in the P. coriacea s.l., with different habitat preferences and reproductive behaviors, indicates they represent distinct species.

Molecular taxonomy of the Formica rufa group (red wood ants) (Hymenoptera: Formicidae): a new cryptic species in the Swiss Alps?

Because of their beneficial impact on forest ecosystems, European red wood ants (Formica rufa group) are protected by law in many European countries and are considered to be among the most reliable bioindicators of forest stability. However, their taxonomy has been much debated and, unfortunately, it is too often neglected. This happens mainly because the morphology-based method for species delimitation requires lots of time and experience. We therefore employed 9 microsatellite loci and mitochondrial DNA (COI gene) to verify the power of genetic markers for red wood ant species delimitation and to investigate the cryptic diversity of these ants within the Eastern Swiss Alps. We analyzed 83 nests belonging to all red wood ant species that occur in the Swiss National Park area. Genetic data indicated that these species represent different genetic pools. Moreover, results showed that Formica aquilonia YARROW, 1955 and F. paralugubris SEIFERT, 1996 often hybridize within the Park, confirming that these two species are genetically very close and could have diverged only recently. Nevertheless, microsatellites also revealed that one entire population, located in the Minger Valley and morphologically identified as F. lugubris ZETTERSTEDT, 1838, is genetically different to all other analyzed F. lugubris populations found within the same area and to other red wood ant species. These findings, confirmed by mitochondrial DNA analyses, suggest the existence of a new cryptic species within the Eastern Swiss Alps. This putative cryptic species has been provisionally named F. lugubris-A2. These results have a great importance for future conservation plans, monitoring and evolutionary studies on these protected ants.

The small species of Belostoma Latreille (Heteroptera, Belostomatidae). III. A revision of oxyurum group, with a new species from Brazil and description of the male of B. noualhieri Montandon

Species of the oxyurum group (sensu Lauck) consist of five extant Neotropical small species, whose lengths range 15.0 to 20.0 mm. The anterior interocular width about 1.5 times the width of an eye and ventral diverticulum of phallus flattened, circular, and large are, in combination, diagnostic. The small species of the oxyurum group were included in the Lauck´s key to the identification of the species groups, without dealing with the species included in it because many of them are very similar in appearance. Therefore here we redescribe and key the Belostoma species of the oxyurum group. Belostoma oxyurum (Dufour) is newly recorded from Brazil (Paraná and Rio Grande do Sul). Holotype and lectotype are designated for B. oxyurum and B. sanctulum Montandon, respectively. The aspect of the prosternal keel, the ratio between the width of the ventral diverticulum of phallus and its length in ventral view, and the aspect of dorsal arms of ventral diverticulum have proven useful for better species delimitation. Based on specimens from Pará State (N. Brazil), Belostoma carajaensis Ribeiro & Estévez, sp. nov. is described and illustrated. This new species differs from B. sanctulum in having anteoculus shorter than interoculus and the dorsal arms of ventral diverticulum divergent and large. A male specimen of B. noualhieri Montandon was collected in São Paulo State and based mainly on features of male genitalia, this species is here also included under oxyurum group.

Cryptic species in the cosmopolitan Bugulan eritina complex (Bryozoa,Cheilostomata)

Previous analyses of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) and γ-proteobacterial endosymbiont diversity have suggested that the marine bryozoan Bugula neritina is a complex of three cryptic species, namely Types S, D and N. Types D and N were previously reported to have restricted distributions along California (western USA) and Delaware and Connecticut (eastern USA), respectively, whereas Type S is considered widespread in tropical, subtropical and temperate regions due to anthropogenic transport. Here, Bayesian species delimitation analysis of a data set composed of two mitochondrial (COI and large ribosomal RNA subunit [16S]) and two nuclear genes (dynein light chain roadblock type-2 protein [DYN] and voltage-dependent anion-selective channel protein [VDAC]) demonstrated that Types S, D and N correspond to three biological species. This finding was significantly supported, in spite of the combinations of priors applied for ancestral population size and root age. Furthermore, COI sequences were used to assess the introduction patterns of the cosmopolitan Type S species. Two COI haplotypes of Type S (S1a and S1d) were found occurring at a global scale. Mantel tests showed correlation between these haplotypes and local sea surface temperature tolerance. Accordingly, the distributions of Type S haplotypes may reflect intraspecific temperature tolerance variation, in addition to the role of introduction vectors. Finally, we show that the Type N may also have been introduced widely, as this species was found for the first time in Central California and north-eastern Australia.

Evidence of cryptic and pseudocryptic speciation in the Paracalanus parvus species complex (Crustacea, Copepoda, Calanoida), supplementary material

Introduction Many marine planktonic crustaceans such as copepods have been considered as widespread organisms. However, the growing evidence for cryptic and pseudo-cryptic speciation has emphasized the need of re-evaluating the status of copepod species complexes in molecular and morphological studies to get a clearer picture about pelagic marine species as evolutionary units and their distributions. This study analyses the molecular diversity of the ecologically important Paracalanus parvus species complex. Its seven currently recognized species are abundant and also often dominant in marine coastal regions worldwide from temperate to tropical oceans. Results COI and Cytochrome b sequences of 160 specimens of the Paracalanus parvus complex from all oceans were obtained. Furthermore, 42 COI sequences from GenBank were added for the genetic analyses. Thirteen distinct molecular operational taxonomic units (MOTU) and two single sequences were revealed with cladistic analyses (Maximum Likelihood, Bayesian Inference), of which seven were identical with results from species delimitation methods (barcode gaps, ABDG, GMYC, Rosenberg's P(AB)). In total, 10 to 12 putative species were detected and could be placed in three categories: (1) temperate geographically isolated, (2) warm-temperate to tropical wider spread and (3) circumglobal warm-water species. Conclusions The present study provides evidence of cryptic or pseudocryptic speciation in the Paracalanus parvus complex. One major insight is that the species Paracalanus parvus s.s. is not panmictic, but may be restricted in its distribution to the northeastern Atlantic.