Coalescent Species Delimitation Research Articles

The phylogenetic structure and coalescent species delimitation of an endemic trapdoor spider genus, Stasimopus (Araneae, Mygalomorphae, Stasimopidae) in the Karoo region of South Africa

The Karoo region of South Africa is a unique and sensitive ecosystem which is facing pressure for development due to economic incentives such as mining, farming and shale gas exploration. The species diversity of many taxa in the area is largely unknown. A phylogenetic analysis of the cork-lid trapdoor spider genus, Stasimopus (Stasimopidae) was undertaken in order to gain insight into the relationships between the species that may be present in the area. The species within Stasimopus are challenging to identify and define using traditional morphological methods due to a high degree of morphological conservatism within the genus. For this reason, multiple coalescent based species delimitation methods were used to attempt to determine the species present for Stasimopus in the region which was tested against the morphological identifications and genetic clades (based on CO1, 16S and EF-1ɣ). We tested single-locus methods Automatic Barcode Gap Discovery (ABGD), Bayesian implementation of Poisson Tree Processes (bPTP) and General Mixed Yule- Coalescent (GMYC), as well as multi-locus Brownie. The phylogenetic analysis of Stasimopus in the Karoo showed that there is a high degree of genetic diversity within the genus. The species delimitation results proved unfruitful for the genus, as they appear to delimit population structure rather than species for most methods. Alternative methods should be investigated to aid in the identification of the species in order truly understand the species diversity of the genus.

Taxonomic revision of maned sloths, subgenusBradypus(Scaeopus), Pilosa, Bradypodidae, with revalidation ofBradypus crinitusGray, 1850

AbstractWe present a taxonomic revision of maned sloths, subgenus Bradypus (Scaeopus), a taxon endemic to the Brazilian Atlantic Forest and currently composed of a single species, the vulnerable Bradypus torquatus. Our review is based on coalescent species delimitation analyses using mitochondrial and nuclear DNA, morphological analyses, and field observations. Our integrative approach demonstrates that two species of maned sloth can be recognized: the northern maned sloth (Bradypus torquatusIlliger, 1811) occurring in the Brazilian states of Bahia and Sergipe, and the southern maned sloth (Bradypus crinitusGray, 1850), occurring in Rio de Janeiro and Espirito Santo states. The two species diverged in the Early Pliocene and are allopatrically distributed. We discuss the biogeographic pattern of the two maned sloth species, comparing it with other Atlantic Forest mammals. We also suggest that the conservation status of both maned sloths needs to be reassessed after this taxonomic rearrangement.

Species limits and recent diversification of Cerradomys (Sigmodontinae: Oryzomyini) during the Pleistocene.

Cerradomys is a genus of the tribe Oryzomyini with eight species currently recognized, and a controversial taxonomy. These species are mainly distributed in the South America dry diagonal, but some species extend into Atlantic Forest, reaching the coastal sandy plains known as Restingas. This study aimed to address species limits and patterns of diversification of Cerradomys species. For this purpose, we performed cytogenetic and molecular analyses (phylogeny, coalescent species delimitation, barcoding, and divergence times estimation) using multiple mitochondrial and nuclear markers on a comprehensive sampling, representing all nominal taxa reported so far. Chromosomal information was a robust marker recognizing eight Cerradomys species. Reciprocal monophyly was recovered for all the species, except for C. subflavus. These results together with coalescent analyses recovered eight species as the most congruent species delimitation scenario for the genus (mean Ctax: 0.72). Divergence time estimates revealed that Cerradomys’ diversification occurred about 1.32 million years ago (Mya) during the Pleistocene. Although our results conservatively support the eight Cerradomys species described so far, different lines of evidence suggest that C. langguthi and C. subflavus could potentially be species-complexes. We discussed this scenario in the light of multiple evolutionary processes within and between species and populations, since Cerradomys comprises a species group with recent diversification affected by Pleistocene climatic changes and by the complex biogeographic history of South America dry diagonal. This work supports that the diversity of Cerradomys is underestimated and reiterates that interdisciplinary approaches are mandatory to identify small rodent species properly, and to unhide cryptic species.

Revalidation of Mazama rufa (Illiger 1815) (Artiodactyla: Cervidae) as a Distinct Species out of the Complex Mazama americana (Erxleben 1777).

The red brocket deer Mazama americana Erxleben, 1777 is considered a polyphyletic complex of cryptic species with wide chromosomal divergence. Evidence indicates that the observed chromosomal divergences result in reproductive isolation. The description of a neotype for M. americana allowed its genetic characterization and represented a comparative basis to resolve the taxonomic uncertainties of the group. Thus, we designated a neotype for the synonym Mazama rufa Illiger, 1815 and tested its recognition as a distinct species from the M. americana complex with the analysis of morphological, cytogenetic and molecular data. We also evaluated its distribution by sampling fecal DNA in the wild. Morphological data from craniometry and body biometry indicated an overlap of quantitative measurements between M. rufa and the entire M. americana complex. The phylogenetic hypothesis obtained through mtDNA confirmed the reciprocal monophyly relationship between M. americana and M. rufa, and both were identified as distinct molecular operational taxonomic units by the General Mixed Yule Coalescent species delimitation analysis. Finally, classic cytogenetic data and fluorescence in situ hybridization with whole chromosome painting probes showed M. rufa with a karyotype of 2n = 52, FN = 56. Comparative analysis indicate that at least fifteen rearrangements separate M. rufa and M. americana (sensu stricto) karyotypes, which confirmed their substantial chromosomal divergence. This divergence should represent an important reproductive barrier and allow its characterization as a distinct and valid species. Genetic analysis of fecal samples demonstrated a wide distribution of M. rufa in the South American continent through the Atlantic Forest, Cerrado and south region of Amazon. Thus, we conclude for the revalidation of M. rufa as a distinct species under the concept of biological isolation, with its karyotype as the main diagnostic character. The present work serves as a basis for the taxonomic review of the M. americana complex, which should be mainly based on cytogenetic characterization and directed towards a better sampling of the Amazon region, the evaluation of available names in the species synonymy and a multi-locus phylogenetic analysis.

Integrative taxonomy clarifies species limits in the hitherto monotypic passion‐vine butterfly genera Agraulis and Dryas (Lepidoptera, Nymphalidae, Heliconiinae)

AbstractThe Heliconiini genera Agraulis and Dryas are widely distributed throughout the Neotropics and into adjacent temperate regions, and although they are currently treated as monotypic, both show significant geographic phenotypic variation. In this work, we employ six genetic markers (4199 bp), two mitochondrial and four nuclear, to perform coalescent species delimitation analyses in Bayesian Phylogenetics and Phylogeography (BPP) and in integrated BPP (iBPP), the latter also includes linear measurements and wings landmarks. We also analyze cytochrome c oxidase I (COI) barcode sequences for each genus using genetic distances, haplotype networks and a character‐based approach. Based on the model testing results, complemented with data from previous studies, we performed morphometric analyses to compare fore and hindwing size, aspect ratio and shape among the new species. In addition, we compared the forewing spot pattern of hypothesizes species using the r package patternize and, for Dryas, compared the colour patterns of mature larvae. Model testing of the molecular species delimitation outputs favoured a seven species hypothesis for Agraulis and a four species hypothesis for Dryas. Average distances among COI barcode sequences of these groups were from 1.09 to 5.81% in Agraulis and from 1.09 to 3.44% in Dryas. Within‐group distances ranged from 0 to 1.11% and between 0 and 2.43%, respectively. NeighborNet haplotype networks showed that all but one of the species are monophyletic, and the character‐based approach found exclusive diagnostic positions for most species, while the rest can be recognized by unique combinations of the 44 informative nucleotide positions analysed. Morphometric analysis supported all species of Agraulis and Dryas based on wing shape, and also in several cases on wing sizes and aspect ratio (hindwing length–forewing length), including A. v. galapagensis, which was absent from the molecular study. The analysis of the forewing spot pattern also revealed differences among most species hypothesis. The colour pattern of the last instar larva is also diagnostic for each Dryas species hypothesis. Locality data for species in both genera show that most of them are allopatric albeit a few have contact zones being parapatric at some locations. Based on the observed genetic differences, which covary with morphology and geographical distribution, we recommend the recognition of eight species of Agraulis: A. incarnata, A. vanillae, A. forbesi new status, A. insularis reinstated status, A. maculosa new status, A. lucina reinstated status, A. galapagensis new status and one undescribed species, and four species of Dryas: D. iulia, D. dominicana revised status, D. lucia revised status and D. alcionea reinstated status. Further work is needed to investigate, which selective forces have led to the current configurations of venation and wing shape, the probable gene flow among species with a focus on Agraulis and gather more data on species ecology.LSIDurn: lsid: zoobank.org: pub: 9E8C9F6E‐91C3‐4FB2‐804D‐1A9C4BD2EABB.

Resolving pitfalls in pit viper systematics – A multi-criteria approach to species delimitation in pit vipers (Reptilia, Viperidae, Craspedocephalus) of Peninsular India reveals cryptic diversity

Abstract Asian pit vipers belonging to the genus Craspedocephalus are a complex group of vipers, distributed in South and Southeast Asia. Their taxonomy is unresolved in many lineages across their distributional range. Here, we reassess the taxonomy and systematics of pit vipers of the genus Craspedocephalus in Peninsular India based on extensive field sampling, in particular in the Western Ghats. We build and expand on the previous findings of genetic relatedness between the peninsular Indian lineages with the Sundaic clade (C. puniceus complex) with greater evidence, based on additional taxa sequenced herein. We reconstruct the phylogeny of the group using three mitochondrial genes and delineated lineages using coalescent species delimitation methods. We then used multiple criteria including genetic divergence and separation in morphological and geographic space to designate taxonomic units. Our work revealed the presence of a South Asian radiation of the clade Craspedocephalus, with a few Sundaic members. Our study reveals the systematic relationships of four Peninsular Indian species of Craspedocephalus, including Peltopelor macrolepis and C. strigatus, sequenced here for the first time, that are classified or confirmed as members of Craspedocephalus. Hence, we place the genus Peltopelor in the synonymy of Craspedocephalus. Using our multi-criteria approach, we delimit four new cryptic evolutionary lineages within the Western Ghats escarpment of Peninsular India. These cryptic lineages belong to the C. malabaricus, C. gramineus and C. macrolepis complexes and are geographically and/or ecologically (in terms of habitat association) distinct from their sister lineages across their distributional range, while others are separated in morphological space. Our new phylogenetic tree and delimitation analysis thus reveals the presence of multiple clades with several cryptic lineages separated by geographical barriers or habitat association.

Species delimitation with distinct methods based on molecular data to elucidate species boundaries in the Cycas taiwaniana complex (Cycadaceae)

AbstractAccurately delimiting species boundaries is of critical importance in many areas of biology including population genetics, conservation biology, biogeography, and evolutionary biology because inaccurate species delimitation may significantly affect downstream inferences. The Cycas taiwaniana complex consists of six morphologically similar taxa (C. changjiangensis, C. fairylakea, C. hainanensis, C. lingshuigensis, C. szechuanensis, C. taiwaniana) that are distributed throughout a narrow region of South China. The members of this complex and the taxonomic status of their names have long been debated. In this study, combining morphological characteristics, we employed three distinct approaches to delimit species: haplotype phylogeny delimitation, Bayesian coalescent species delimitation (BPP), and population cluster analyses. To delimitate the species boundaries within the C. taiwaniana complex, we used 4 plastid intergenic spacers (cpDNA), 4 nuclear genes (nDNA) and 10 microsatellites. All three approaches revealed the presence of two distinct species in the C. taiwaniana complex under the unified species concept, C. taiwaniana and C. szechuanensis, largely corresponding to morphological differentiation. Cycas fairylakea was a synonym for C. szechuanensis, and the other three taxa were synonyms for C. taiwaniana. Species delimitation using molecular data was consistent with our preliminary morphological inference. This study thus optimally resolved the species boundaries and taxonomic treatment of the C. taiwaniana complex from an integrated perspective using multiple sources of molecular data and distinct analytical approaches.

Shards, sequences, and shorelines: two new species of Bembidion from North America (Coleoptera, Carabidae).

Two new species of Bembidion are described from river shores in North America. One, Bembidion mimbressp. nov., from the Gila River watershed in the lands of the Mimbres culture in New Mexico and Arizona, is closely related to the widespread Bembidion levigatum. DNA sequences from several linkage groups and morphology provide evidence of the distinctiveness of B. mimbres. The second, Bembidion corgenomasp. nov., has been the subject of recent genomic and transcriptomic studies. It belongs in the Bembidion transversale subgroup, and occurs from California north to British Columbia, east to Montana and Nevada. The B. transversale subgroup as a whole is reviewed, and morphological characters that distinguish B. corgenoma from the similar and sympatric B. transversale and B. erosum are described and illustrated. DNA sequences of these three species show no consistent differences in 28S, COI, CAD, and Topoisomerase, and a coalescent species delimitation analysis reveals no notable structure within the complex. This is the first known trio of species within Bembidion for which those genes provide no clear signal of species boundaries. A neotype is designated for the one name in the group that lacks a primary type, Bembidium haplogonum Chaudoir. Chromosomes of the new species and their relatives are as is typical for Bembidion, with eleven pairs of autosomes and an XY/XX sex chromosome system.

Haemoproteus syrnii (Haemosporida: Haemoproteidae) in owls from Brazil: morphological and molecular characterization, potential cryptic species, and exo-erythrocytic stages.

Haemoproteus syrnii is a haemosporidian parasite found in owls. Although morphological and molecular data on the species is available, its exo-erythrocytic development was never researched. In this study, we provide the morphological, morphometric, and molecular characterization of H. syrnii populations found in owl species from Minas Gerais, southeast Brazil. We also characterized the coalescent species delimitation based on the molecular and histopathology data. Samples from 54 owls from six different species were analyzed, generating 11 sequences of the cyt b gene, from which six were new sequences. The overall prevalence of infection was high (72.22%). The H. syrnii sequences were grouped into two well-supported independent clades, which included other Haemoproteus (Parahaemoproteus) species. This was supported by both the coalescent species delimitation analysis and by the genetic divergence between lineages of these distinct clades. There were small morphological and morphometric differences within the population presented in this study. However, when compared with other studies, the molecular analysis demonstrated considerable intraspecific variation and suggests potential cryptic species. The histopathological analysis revealed, for the first time, that lungs and skeletal muscle are exo-erythrocytic stage location of H. syrnii, and that the parasite is linked to the histopathological changes found in owls. This study brings new data from Haemoproteus species biology and host infection, and improves host-parasite relationship understanding under an owl conservation perspective.

Poor performance of DNA barcoding and the impact of RAD loci filtering on the species delimitation of an Iberian ant-eating spider

Genomic data provide unprecedented power for species delimitation. However, current implementations are still time and resource consuming. In addition, bioinformatic processing is contentious and its impact on downstream analyses is insufficiently understood. Here we employ ddRAD sequencing and a thorough sampling for species delimitation in Zodarion styliferum, a widespread Iberian ant-eating spider. We explore the influence of the loci filtering strategy on the downstream phylogenetic analyses, genomic clustering and coalescent species delimitation. We also assess the accuracy of one mitochondrial (COI) and one nuclear (ITS) barcode for fast and inexpensive species delineation in the group. Our genomic data strongly support two morphologically cryptic but ecologically divergent lineages, mainly restricted to the central-eastern and western parts of the Iberian Peninsula, respectively. Larger matrices with more missing data showed increased genomic diversity, supporting that bioinformatic strategies to maximize matrix completion disproportionately exclude loci with the highest mutation rates. Moderate loci filtering gave the best results across analyses: although larger matrices returned concatenated phylogenies with higher support, middle-sized matrices performed better in genetic structure analyses. COI displayed high diversity and a conspicuous barcode gap, revealing 13 mitochondrial lineages. Mitonuclear discordance is consistent with ancestral isolation in multiple groups, probably in glacial refugia, followed by range expansion and secondary contact that produced genomic homogenization. Several apparently (unidirectionally) introgressed specimens further challenge the accuracy of species identification through mitochondrial barcodes in the group. Conversely, ITS failed to separate both lineages of Z. styliferum. This study shows an extreme case of mitonuclear discordance that highlights the limitations of single molecular barcodes for species delimitation, even in presence of distinct barcode gaps, and brings new light on the effects of parameterization on shallow-divergence studies using RAD data.

Molecular diversity and coalescent species delimitation of avian haemosporidian parasites in an endemic bird species of South America.

Haemoproteus spp. and Plasmodium spp. are blood parasites that occur in birds worldwide. Identifying the species within this group is complex, especially in wild birds that present low parasitemia when captured, making morphological identification very difficult. Thus, the use of alternative tools to identify species may be useful in the elucidation of the distribution of parasites that circulate in bird populations. The objectives of this study were to determine the prevalence and parasitemia of the genera Plasmodium and Haemoproteus in Tachyphonus coronatus in the Atlantic Forest, Brazil, and to evaluate the molecular diversity, geographic distribution, and specificity of these parasites based on coalescent species delimitation methods. Microscopic analysis, PCR, cyt b gene sequencing, phylogenetic analysis and coalescent species delimitation using single-locus algorithms were performed (Poisson tree process (PTP) and multi-rate Poisson tree process (MPTP) methods). The analyses were performed in 117 avian host individuals. The prevalence was 55.5% for Plasmodium and 1.7% for Haemoproteus, with a mean parasitemia of 0.06%. Twenty-five Plasmodium and two Haemoproteus lineages were recovered. The MPTP method recovered seven different evolutionarily significant units (ESUs) of Plasmodium and one of Haemoproteus, whereas PTP presented fourteen ESUs of Plasmodium and one of Haemoproteus. The MPTP was more consistent with current taxonomy, while PTP overestimated the number of lineages. These ESUs are widely distributed and have already been found in 22 orders of birds that, all together, inhabit every continent, except Antarctica. The computational methods of species delimitation proved to be effective in cases where the classification of Haemosporida based just on morphology is insufficient.

Cryptic Patterns of Speciation in Cryptic Primates: Microendemic Mouse Lemurs and the Multispecies Coalescent.

Mouse lemurs (Microcebus) are a radiation of morphologically cryptic primates distributed throughout Madagascar for which the number of recognized species has exploded in the past two decades. This taxonomic revision has prompted understandable concern that there has been substantial oversplitting in the mouse lemur clade. Here, we investigate mouse lemur diversity in a region in northeastern Madagascar with high levels of microendemism and predicted habitat loss. We analyzed RADseq data with multispecies coalescent (MSC) species delimitation methods for two pairs of sister lineages that include three named species and an undescribed lineage previously identified to have divergent mtDNA. Marked differences in effective population sizes, levels of gene flow, patterns of isolation-by-distance, and species delimitation results were found among the two pairs of lineages. Whereas all tests support the recognition of the presently undescribed lineage as a separate species, the species-level distinction of two previously described species, M. mittermeieri and M. lehilahytsara is not supported-a result that is particularly striking when using the genealogical discordance index (gdi). Nonsister lineages occur sympatrically in two of the localities sampled for this study, despite an estimated divergence time of less than 1 Ma. This suggests rapid evolution of reproductive isolation in the focal lineages and in the mouse lemur clade generally. The divergence time estimates reported here are based on the MSC calibrated with pedigree-based mutation rates and are considerably more recent than previously published fossil-calibrated relaxed-clock estimates. We discuss the possible explanations for this discrepancy, noting that there are theoretical justifications for preferring the MSC estimates in this case. [Cryptic species; effective population size; microendemism; multispecies coalescent; speciation; species delimitation.].

Outstanding diversity and microendemism in a clade of rare Atlantic Forest montane frogs

Distributed across topographically complex landscapes that vary from lowland to high elevation, the Atlantic Forest harbors one of the richest biotas worldwide. Atlantic Forest amphibians are particularly speciose, taxonomic accounts are rising and the group is used as model for biogeographic inference. Past climate-related habitat fragmentation is often invoked to explain diversification, with montane taxa expected to become more widespread during glacial times and restrained at interglacials. In this study we investigate diversification in Ischnocnema lactea and I. holti (Anura: Brachycephalidae), two rare frog species inhabiting Atlantic Forest montane regions in Southeastern Brazil. Previous phylogenetic accounts have suggested uncertain limits between these two sister species. We assembled a multilocus DNA dataset, delimited lineages in this clade, and used ecological niche modeling to explore past and future putative ranges. Assignment analyses and traditional and coalescent phylogenetic methods confirmed the existence of a species complex of Miocene origin comprising nine lineages, most of which show very narrow ranges. Lineages were fully supported as species based in coalescent species delimitation, but the phylogenetic relationships among lineages in higher elevation were unresolved. Models of past ranges suggest extensive suitable areas at the last glacial maximum which, along with phylogenetic uncertainty, are consistent with a hypothesis that climate driven vicariance at higher elevation areas resulted in hard polytomies. Species distribution models under future climates suggest narrower ranges of the lineages relative to now, but no species are currently considered endangered. Overall, our results argue in favor for the reassessment of the taxonomic and conservation status of the I. holti – I. lactea species complex.

Cryptic molecular diversity in the morphologically variable rotiferan Brachionus quadridentatus (Rotifera: Monogononta)

Brachionus quadridentatus is a morphologically variable species distributed worldwide. Its taxonomy is confusing due the numerous infrasubspecific variants described in the taxon. Here we explore genetic diversity of certain B. quadridentatus populations, using sequences of COI mtDNA and 18S rDNA genes. With traditional morphology (taxonomic keys), the specimens identified fell into three recognized variants: B. quadridentatus quadridentatus, B. quadridentatus f. brevispinus and B. quadridentatus f. cluniorbicularis. The coalescent species delimitation analysis highly supports the presence of at least three putative species within B. quadridentatus complex in agreement with the phylogenetic result and GMYC analysis with the 18S gene. Nevertheless, the morphological and mitochondrial information show that the variation within each of the three putative species is much more extensive, finding seven monophyletic and highly divergent genetic groups (>10%) that have congruence with the morphotypes found in this study and ABGD analysis, which display clear morphological differences mainly in the diagnostic characters.

Geographic structure of genetic variation in the Parachute Gecko Ptychozoon lionotum Annandale, 1905 across Indochina and Sundaland with descriptions of three new species.

An integrative taxonomic analysis of the Ptychozoon lionotum group across its range in Indochina and Sundaland recovers P. lionotum sensu lato Annandale, 1905 as paraphyletic with respect to P. popaense Grismer, Wood, Thura, Grismer, Brown, Stuart, 2018a and composed of four allopatric, genetically divergent, ND2 mitochondrial lineages. Multivariate and univariate analyses of continuous and discrete morphological and color pattern characters statistically and discretely diagnose each lineage from one another and together, with maximum likelihood and Bayesian inference analyses, provide the foundation for the recognition of each lineage as a new species-hypotheses corroborated with a Generalized Mixed Yule Coalescent species delimitation analysis. Ptychozoon cicakterbang sp. nov. ranges throughout Peninsular Malaysia to Pulau Natuna Besar, Indonesia; P. kabkaebin sp. nov. is endemic to northern and central Laos; and P. tokehos sp. nov. ranges from southern Thailand south of the Isthmus of Kra northward to Chiang Mai, fringing the Chao Phraya Basin and ranging southward through Cambodia to southern Vietnam. Ptychozoon lionotum sensu stricto ranges from northwestern Laos through southern Myanmar to eastern India. The phylogeographic structure within each species varies considerably with P. lionotum s.s. showing no genetic divergence across its 1,100 km range compared to P. cicakterbang sp. nov. showing upwards of 8.2% sequence divergence between syntopic individuals. Significant phylogeographic structure exists within P. tokehos sp. nov. and increased sampling throughout Thailand may require additional taxonomic changes within this species.

Genetic variation of the most abundant forest‐dwelling rodents in Central Africa (Praomys jacksoni complex): Evidence for Pleistocene refugia in both montane and lowland forests

AbstractAimWe investigate the Plio‐Pleistocene evolutionary history of one of the most abundant rodents in Afrotropical forests. Specifically, we ask how their diversification was influenced by climate change, topography and major rivers.LocationTropical Africa: Lower Guinea (including Cameroon volcanic line; CVL), Congolia, Albertine Rift (AR), Kenyan highlands (KH).TaxonMurine rodents of the Praomys jacksoni complex.MethodsWe used 849 genotyped individuals to describe the overall diversity and spatial genetic structure across a majority of their known distribution area. The combination of one mitochondrial and three nuclear markers was used to infer dated phylogenies using Bayesian and maximum likelihood approaches. Genetic structure was further assessed by multispecies coalescent species delimitation. Current and past distributions of particular taxa were predicted using environmental niche modelling.ResultsThe complex is composed of five major genetic clades (proposed species). Two of them are restricted to specific habitat types (either montane forests of AR or wetlands in lowland forests along the Congo River), three others have wide geographic distributions and lower levels of ecological specialization. The earliest divergence is dated to the Plio‐Pleistocene boundary and is in accordance with the separation of AR forests and Guineo‐Congolian forests. Further diversification of the complex is associated with Pleistocene climate changes. Relatively stable refugia of suitable climatic conditions were identified in lowland Congolia (for two species currently distributed only in lowland forests) as well as in montane forests of CVL, AR, KH (playing the role of reservoirs of diversity). Large rivers, especially the Congo River, are important barriers to gene flow for most taxa, but probably were not the primary cause of differentiation.Main conclusionsThe evolutionary history of the complex was primarily affected by Pleistocene climate changes and diversification in forest refugia. There is little support for ecological parapatric speciation or the riverine barrier hypothesis.

Phylogeny and species delimitation based on molecular approaches on the species of the Australoheros autrani group (Teleostei, Cichlidae), with biogeographic comments

Three distinct and independent molecular-based species delimitation analyses were performed among the species and populations included within theAustraloherosautranigroup, based on sequences of the mitochondrial gene Cytochrome b: a tree-based method proposed by Wiens and Penkrot (WP), a Character-based DNA Barcoding (CBB) and coalescent species delimitation method termed the Bayesian Implementation of the Poisson tree processes (bPTP). The congruence of WP and CBB delimited 11 independent lineages (species), while the bPTP delimited just nine lineages. We did not favour any of the methods, and we considered the possibility of two slightly variant scenarios. A time-calibrated phylogenetic analysis is proposed based on the predominant congruence of the results of these three species delimitation methods herein applied. The monophyly of theA.autranispecies group was highly supported with maximum node support value and diagnosed by 11 nucleotide substitutions. The sister clade of theA.autranispecies group is the clade comprisingA.sp. Timbé do Sul andA.minuano. The phylogenetic analysis supports three main clades within theA.autranispecies group, supported by maximum node support value, with the Southern Mata Atlântica clade as the most basal clade. Divergence time estimates indicate that the diversification of theAustraloherosoriginated during the early Neogene, but only in the late Neogene did the processes of diversification in the southeast and north regions occur. Diversification within theAustraloherosautranispecies group occurred synchronically for the three main clades during the beginning of the Quaternary. It is demonstrated that molecular characters are valuable tools for species recognition, particularly in speciose groups with inconspicuous or difficult to record morphological characters. The resulting phylogeny of theAustraloherosautranigroup is highly compatible with the geological and biogeographic scenarios proposed for the Neogene and Quarternary shaping of the extant river basins of eastern Brazil. Despite the origin of theA.autranigroup being dated to the late Miocene, species level diversification occurred in the Pleistocene and was probably driven by headwater capture events and sea-level fluctuations.

Integrating multilocus DNA data and 3D geometric morphometrics to elucidate species boundaries in the case of Pyrenaearia (Pulmonata: Hygromiidae)

To accurately delimit species the use of multiple character types is essential as all speciation processes are not equally reflected in different data (e.g. morphological, molecular or ecological characters). With the introduction of geometric morphometrics methods and advances in 3D technology, a comprehensive combination of molecular and morphological data has been enabled in groups where exhaustively quantifying and measuring morphological shape change was not possible before such as gastropod shells. In this study, we combined multilocus coalescent species delimitation methods with 3D geometric morphometrics of shell shape to delimit species within the land snail genus Pyrenaearia. A new taxonomic scheme was constructed for the genus identifying ten species. Two nominal species were synonymized and a hitherto unrecognized cryptic species was identified. Our findings support the importance of combining multiple lines of evidence as molecular and morphological data on their own do not yield the same information. Further, the integration of morphological and molecular data shows the importance of allometry in shell shape and suggests a combined effect of population history and selection in different environments on shells morphological variation. Our new taxonomy and phylogenetic reconstruction suggest that, besides the glacial cycles of the Pleistocene, passive dispersal and rock substrate complexity could also have been involved in the speciation of the genus.

Integrating coalescent species delimitation with analysis of host specificity reveals extensive cryptic diversity despite minimal mitochondrial divergence in the malaria parasite genus Leucocytozoon

BackgroundCoalescent methods that use multi-locus sequence data are powerful tools for identifying putatively reproductively isolated lineages, though this approach has rarely been used for the study of microbial groups that are likely to harbor many unrecognized species. Among microbial symbionts, integrating genetic species delimitation methods with trait data that could indicate reproductive isolation, such as host specificity data, has rarely been used despite its potential to inform species limits. Here we test the ability of an integrative approach combining genetic and host specificity data to delimit species within the avian malaria parasite genus Leucocytozoon in central Alaska.ResultsWe sequenced seven nuclear loci for 69 Leucocytozoon samples and used multiple species delimitation methods (GMYC and BPP models), tested for differences in host infection patterns among putative species based on 406 individual infections, and characterized parasite morphology. We found that cryptic morphology has masked a highly diverse Leucocytozoon assemblage, with most species delimitation methods recovering support for at least 21 separate species that occur sympatrically and have divergent host infection patterns. Reproductive isolation among putative species appears to have evolved despite low mtDNA divergence, and in one instance two Leucocytozoon cytb haplotypes that differed by a single base pair (~ 0.2% divergence) were supported as separate species. However, there was no consistent association between mtDNA divergence and species limits. Among cytb haplotypes that differed by one to three base pairs we observed idiosyncratic patterns of nuclear and ecological divergence, with cytb haplotype pairs found to be either conspecific, reproductively isolated with no divergence in host specificity, or reproductively isolated with divergent patterns of host specialization.ConclusionIntegrating multi-locus genetic species delimitation methods and non-traditional ecological data types such as host specificity provide a novel view of the diversity of avian malaria parasites that has been missed previously using morphology and mtDNA barcodes. Species delimitation methods show that Leucocytozoon is highly species-rich in Alaska, and the genus is likely to harbor extraordinary species-level diversity worldwide. Integrating genetic and ecological data will be an important approach for understanding the diversity and evolutionary history of microbial symbionts moving forward.

Monotypic status of the South American relictual marsupial Dromiciops gliroides (Microbiotheria)

The long-recognized monotypic status of the relictual marsupial genus Dromiciops was recently challenged by a controversial reanalysis of previously published mtDNA data that were combined with a new morphological study to conclude that the genus contains 3 species: D. bozinovici, D. mondaca, and D. gliroides. We present here new phylogenetic and coalescent species delimitation analyses to test the multispecific status of Dromiciops relative to the proposed 3 new species. Our molecular analysis is based on partial sequences of 4 nuclear (RAG1, ApoB, vWF, and IRBP) and 2 mitochondrial (12S RNA and Cytb) genes. Genetic distances showed low differentiation among the proposed Dromiciops species, consistent with typical levels of intraspecific variation. Phylogenetic analyses yielded only moderate support for monophyly of D. bozinovici, while depicting polyphyly in D. gliroides and D. mondaca. Species delimitation analyses did not recover the proposed 3-species scenario, and the taxonomic index of congruence among these methods (Ctax = 0.5–1.0) supported monotypic status for Dromiciops. Taking into consideration all the genetic analyses and previously reported morphological analyses, we conclude that all Dromiciops lineages belong to a single species.