Mitochondrial and nuclear DNA sequences reveal recent divergence in morphologically indistinguishable petrels

1 Department of Biology, University of Washington, Seattle, WA, USA 2 Department of Fisheries and Wildlife Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA 3 Department of Biology, Valdosta State University, Valdosta, GA, USA 4 New Mexico Museum of Natural History, Albuquerque, NM, USA 5 Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, CO, USA 6 School of Molecular and Life Sciences, University of Limpopo, Sovenga, South Africa

Seabirds are highly vagile and can disperse up to thousands of kilometers, making it difficult to identify the factors that promote isolation between populations. The endemic Hawaiian petrel (Pterodroma sandwichensis) is one such species. Today it is endangered, and known to breed only on the islands of Hawaii, Maui, Lanai and Kauai. Historical records indicate that a large population formerly bred on Molokai as well, but this population has recently been extirpated. Given the great dispersal potential of these petrels, it remains unclear if populations are genetically distinct and which factors may contribute to isolation between them. We sampled petrels from across their range, including individuals from the presumably extirpated Molokai population. We sequenced 524 bp of mitochondrial DNA, 741 bp from three nuclear introns, and genotyped 18 microsatellite loci in order to examine the patterns of divergence in this species and to investigate the potential underlying mechanisms. Both mitochondrial and nuclear data sets indicated significant genetic differentiation among all modern populations, but no differentiation was found between historic samples from Molokai and modern birds from Lanai. Population-specific nonbreeding distribution and strong natal philopatry may reduce gene flow between populations. However, the lack of population structure between extirpated Molokai birds and modern birds on Lanai indicates that there was substantial gene flow between these populations and that petrels may be able to overcome barriers to dispersal prior to complete extirpation. Hawaiian petrel populations could be considered distinct management units, however, the dwindling population on Hawaii may require translocation to prevent extirpation in the near future.

Through the first chapter, this study investigates the Calycopis bactra complex in Central America, integrating COI barcodes and male genitalia morphology to clarify species boundaries. An analysis of COI sequences from Costa Rican specimens confirmed that Calycopis susanna in Central America is comprised of two distinct species (Calycopis bactra and Calycopis janzeni, new species) identifying 24 fixed COI nucleotide differences separating Calycopis janzeni, new species from C. bactra. Nuclear DNA sequences further confirm the specific distinctness of these taxa, however, detailed nuclear analysis will be presented in chapter III. Calycopis janzeni, new species, ranges across North and Central America from Mexico to Panama, inhabiting wet lowland areas characterized by tropical evergreen rainforests and riparian zones. In South America, it ranges from Colombia to northern Brazil. A distribution map is provided to illustrate the geographic range of Calycopis janzeni, new species and the broader Calycopis bactra complex across North, Central and South America. The results demonstrate that the C. bactra complex in Central America consists of three closely related species with minimal morphological differentiation, likely due to recent evolutionary divergence. The adoption of molecular methods often highlights how traditional reliance on the biological species concept often underestimates biodiversity. In the second chapter, this study focuses on the Calycopis caulonia group, originally defined as the Calycopis janeirica group by Field in his work back in 1967. Using a maximum likelihood phylogenetic framework based on nuclear DNA sequences of all protein-coding genes from 45 specimens, we reevaluated the taxonomy of the group. Our findings reveal that four taxa previously recognized as distinct speciesC. caulonia, C. chacona, C. janeirica, and C. nicolayiare conspecific, representing a single species, C. caulonia. The inclusion of additional taxa, C. thama and C. mirna, further refined the monophyly of the group. This study also identified a misclassified species, previously referred to as C. bactra by Field in 1967, as a new species, Calycopis lanina. These results emphasize the limitations of morphology-based taxonomy and highlight the critical role of molecular data in resolving phylogenetic relationships and taxonomic ambiguities. The integration of molecular and morphological evidence offers a robust framework for reassessing species boundaries and understanding evolutionary processes within Calycopis. Future research on the ecological and life- history traits of these species will enhance our knowledge of their evolutionary adaptations in the Neotropics. At last, we perused a case of mito-nuclear discordance in the Calycopis cecrops lineage in North, Central and South America. The third chapter examines these discrepancies by integrating nuclear DNA data and morphological analyses to clarify the taxonomy within this group. To define the Calycopis cecrops lineage, we sequenced all protein-coding nuclear genes from 74 specimens identified as belonging to the genus Calycopis. These specimens were classified based on the morphological characters established by Field in 196 and refined by Duarte & Robbins back in 2010. We sequenced nuclear genes from specimens within the Calycopis cecrops lineage and conducted maximum likelihood phylogenetic analyses to confirm the monophyly of this lineage. We also performed phylogenetic analyses of mitochondrial sequences to detect incongruences with nuclear data, focusing on potential mtDNA introgression. Our findings indicate that each of the seven recognized species in the C. cecrops lineage forms a monophyletic group based on nuclear DNA sequences. However, significant discordance exists between mitochondrial and nuclear datasets, with only a few species consistently identified. This incongruence is likely due to evolutionary processes such as introgressive hybridization and incomplete lineage sorting. Previous research has documented mtDNA introgression in Calycopis species, suggesting that such genetic exchanges may contribute to the observed mito-nuclear discordance. Further investigation is necessary to elucidate the mechanisms driving mtDNA introgression and its implications for species delineation within the C. cecrops lineage.

Lygus pratensis (L.) is an important cotton pest in China, especially in the northwest region. Nymphs and adults cause serious quality and yield losses. However, the genetic structure and geographic distribution of L. pratensis is not well known. We analyzed genetic diversity, geographical structure, gene flow, and population dynamics of L. pratensis in northwest China using mitochondrial and nuclear sequence datasets to study phylogeographical patterns and demographic history. L. pratensis (n = 286) were collected at sites across an area spanning 2,180,000 km2, including the Xinjiang and Gansu-Ningxia regions. Populations in the two regions could be distinguished based on mitochondrial criteria but the overall genetic structure was weak. The nuclear dataset revealed a lack of diagnostic genetic structure across sample areas. Phylogenetic analysis indicated a lack of population level monophyly that may have been caused by incomplete lineage sorting. The Mantel test showed a significant correlation between genetic and geographic distances among the populations based on the mtDNA data. However the nuclear dataset did not show significant correlation. A high level of gene flow among populations was indicated by migration analysis; human activities may have also facilitated insect movement. The availability of irrigation water and ample cotton hosts makes the Xinjiang region well suited for L. pratensis reproduction. Bayesian skyline plot analysis, star-shaped network, and neutrality tests all indicated that L. pratensis has experienced recent population expansion. Climatic changes and extensive areas occupied by host plants have led to population expansion of L. pratensis. In conclusion, the present distribution and phylogeographic pattern of L. pratensis was influenced by climate, human activities, and availability of plant hosts.

BackgroundDuikers in the subfamily Cephalophinae are a group of tropical forest mammals believed to have first originated during the late Miocene. However, knowledge of phylogenetic relationships, pattern and timing of their subsequent radiation is poorly understood. Here we present the first multi-locus phylogeny of this threatened group of tropical artiodactyls and use a Bayesian uncorrelated molecular clock to estimate divergence times.ResultsA total of 4152 bp of sequence data was obtained from two mitochondrial genes and four nuclear introns. Phylogenies were estimated using maximum parsimony, maximum likelihood, and Bayesian analysis of concatenated mitochondrial, nuclear and combined datasets. A relaxed molecular clock with two fossil calibration points was used to estimate divergence times. The first was based on the age of the split between the two oldest subfamilies within the Bovidae whereas the second was based on the earliest known fossil appearance of the Cephalophinae and molecular divergence time estimates for the oldest lineages within this group. Findings indicate strong support for four major lineages within the subfamily, all of which date to the late Miocene/early Pliocene. The first of these to diverge was the dwarf duiker genus Philantomba, followed by the giant, eastern and western red duiker lineages, all within the genus Cephalophus. While these results uphold the recognition of Philantomba, they do not support the monotypic savanna-specialist genus Sylvicapra, which as sister to the giant duikers leaves Cephalophus paraphyletic. BEAST analyses indicate that most sister species pairs originated during the Pleistocene, suggesting that repeated glacial cycling may have played an important role in the recent diversification of this group. Furthermore, several red duiker sister species pairs appear to be either paraphyletic (C.callipygus/C. ogilbyi and C. harveyi/C. natalensis) or exhibit evidence of mitochondrial admixture (C. nigrifrons and C. rufilatus), consistent with their recent divergence and/or possible hybridization with each other.ConclusionsMolecular phylogenetic analyses suggest that Pleistocene-era climatic oscillations have played an important role in the speciation of this largely forest-dwelling group. Our results also reveal the most well supported species phylogeny for the subfamily to date, but also highlight several areas of inconsistency between our current understanding of duiker taxonomy and the evolutionary relationships depicted here. These findings may therefore prove particularly relevant to future conservation efforts, given that many species are presently regulated under the Convention for Trade in Endangered Species.

The multispecies network coalescent (MSNC) is a stochastic process that captures how gene trees grow within the branches of a phylogenetic network. Coupling the MSNC with a stochastic mutational process that operates along the branches of the gene trees gives rise to a generative model of how multiple loci from within and across species evolve in the presence of both incomplete lineage sorting (ILS) and reticulation (e.g., hybridization). We report on a Bayesian method for sampling the parameters of this generative model, including the species phylogeny, gene trees, divergence times, and population sizes, from DNA sequences of multiple independent loci. We demonstrate the utility of our method by analyzing simulated data and reanalyzing an empirical data set. Our results demonstrate the significance of not only coestimating species phylogenies and gene trees, but also accounting for reticulation and ILS simultaneously. In particular, we show that when gene flow occurs, our method accurately estimates the evolutionary histories, coalescence times, and divergence times. Tree inference methods, on the other hand, underestimate divergence times and overestimate coalescence times when the evolutionary history is reticulate. While the MSNC corresponds to an abstract model of "intermixture," we study the performance of the model and method on simulated data generated under a gene flow model. We show that the method accurately infers the most recent time at which gene flow occurs. Finally, we demonstrate the application of the new method to a 106-locus yeast data set.

Phylogenomic analysis of large genome-wide sequence data sets can resolve phylogenetic tree topologies for large species groups, help test the accuracy of and improve resolution for earlier multi-locus studies and reveal the level of agreement or concordance within partitions of the genome for various tree topologies. Here we used a target-capture approach to sequence 1088 single-copy exons for more than 200 labrid fishes together with more than 100 outgroup taxa to generate a new data-rich phylogeny for the family Labridae. Our time-calibrated phylogenetic analysis of exon-capture data pushes the root node age of the family Labridae back into the Cretaceous to about 79 Ma years ago. The monotypic Centrogenys vaigiensis, and the order Uranoscopiformes (stargazers) are identified as the sister lineages of Labridae. The phylogenetic relationships among major labrid subfamilies and within these clades were largely congruent with prior analyses of select mitochondrial and nuclear datasets. However, the position of the tribe Cirrhilabrini (fairy and flame wrasses) showed discordance, resolving either as the sister to a crown julidine clade or alternatively sister to a group formed by the labrines, cheilines and scarines. Exploration of this pattern using multiple approaches leads to slightly higher support for this latter hypothesis, highlighting the importance of genome-level data sets for resolving short internodes at key phylogenetic positions in a large, economically important groups of coral reef fishes. More broadly, we demonstrate how accounting for sources of biological variability from incomplete lineage sorting and exploring systematic error at conflicting nodes can aid in evaluating alternative phylogenetic hypotheses. [coral reefs; divergence time estimation; exon-capture; fossil calibration; incomplete lineage sorting.].

Tick mitogenomes: Structural characteristics, phylogenetic implications, and a critical synthesis from sequencing to systematics.

Both mitochondrial and nuclear gene sequences have been employed in efforts to reconstruct deep-level phylogenetic relationships. A fundamental question in molecular systematics concerns the efficacy of different types of sequences in recovering clades at different taxonomic levels. We compared the performance of four mitochondrial data sets (cytochrome b, cytochrome oxidase II, NADH dehydrogenase subunit I, 12S rRNA-tRNA-16S rRNA) and eight nuclear data sets (exonic regions of alpha-2B adrenergic receptor, aquaporin, ss-casein, gamma-fibrinogen, interphotoreceptor retinoid binding protein, kappa-casein, protamine, von Willebrand Factor) in recovering deep-level mammalian clades. We employed parsimony and minimum-evolution with a variety of distance corrections for superimposed substitutions. In 32 different pairwise comparisons between these mitochondrial and nuclear data sets, we used the maximum set of overlapping taxa. In each case, the variable-length bootstrap was used to resample at the size of the smaller data set. The nuclear exons consistently performed better than mitochondrial protein and rRNA-tRNA coding genes on a per-residue basis in recovering benchmark clades. We also concatenated nuclear genes for overlapping taxa and made comparisons with concatenated mitochondrial protein-coding genes from complete mitochondrial genomes. The variable-length bootstrap was used to score the recovery of benchmark clades as a function of the number of resampled base pairs. In every case, the nuclear concatenations were more efficient than the mitochondrial concatenations in recovering benchmark clades. Among genes included in our study, the nuclear genes were much less affected by superimposed substitutions. Nuclear genes having appropriate rates of substitution should receive strong consideration in efforts to reconstruct deep-level phylogenetic relationships.

Several morphologically dissimilar ascomycete fungi including Schizosaccharomyces, Taphrina, Saitoella, Pneumocystis, and Neolecta have been grouped into the taxon Taphrinomycotina (Archiascomycota or Archiascomycotina), originally based on rRNA phylogeny. These analyses lack statistically significant support for the monophyly of this grouping, and although confirmed by more recent multigene analyses, this topology is contradicted by mitochondrial phylogenies. To resolve this inconsistency, we have assembled phylogenomic mitochondrial and nuclear data sets from four distantly related taphrinomycotina taxa: Schizosaccharomyces pombe, Pneumocystis carinii, Saitoella complicata, and Taphrina deformans. Our phylogenomic analyses based on nuclear data (113 proteins) conclusively support the monophyly of Taphrinomycotina, diverging as a sister group to Saccharomycotina + Pezizomycotina. However, despite the improved taxon sampling, Taphrinomycotina continue to be paraphyletic with the mitochondrial data set (13 proteins): Schizosaccharomyces species associate with budding yeasts (Saccharomycotina) and the other Taphrinomycotina group as a sister group to Saccharomycotina + Pezizomycotina. Yet, as Schizosaccharomyces and Saccharomycotina species are fast evolving, the mitochondrial phylogeny may be influenced by a long-branch attraction (LBA) artifact. After removal of fast-evolving sequence positions from the mitochondrial data set, we recover the monophyly of Taphrinomycotina. Our combined results suggest that Taphrinomycotina is a legitimate taxon, that this group of species diverges as a sister group to Saccharomycotina + Pezizomycotina, and that phylogenetic positioning of yeasts and fission yeasts with mitochondrial data is plagued by a strong LBA artifact.

Deep phylogenetic incongruence in the angiosperm clade Rosidae

Reappraising adaptive radiation

Does gene flow destroy phylogenetic signal? The performance of three methods for estimating species phylogenies in the presence of gene flow

Inferring complex evolutionary history of the closely related East Asian wild roses in Rosa sect. Synstylae (Rosaceae) based on genomic evidence from conserved orthologues.

Speciation processes are not well resolved or agreed upon. They are however essential to our general understanding of the evolutionary processes that lead to diversification. Determining the juncture at which a genetically and/or morphologically divergent population becomes a unique species can be challenging, especially with respect to recent divergences and closely related taxa where issues such as incomplete lineage sorting may bring about confounding results. To complicate our theoretical disagreements on species definitions, different models inferring species boundaries may accordingly lump or split species. Using multiple lines of evidence to define species boundaries can greatly improve species inference and preclude erroneous taxonomic groupings. Taxa in the Cymopterus terebinthinus (Apiaceae) species complex have long puzzled botanists owing to their diverse morphologies that are defined as separate varieties. These varieties are often found in generally well-defined geographical subregions of varying habitat types. Additionally, previous phylogenetic studies were interpreted to show that varieties in Cymopterus terebinthinus were not monophyletic. I aim to clarify species boundaries and infer evolutionary relationships in the Cymopterus terebinthinus species complex using phylogenetic inference and species delimitation programs. Additionally, I will compare trends observed in ecological, morphological, and geographical evidence to clade groupings. I apply the genealogical species concept to guide my interpretations of species boundaries in this group. To further explore species boundaries in C. terebinthinus, I sampled from 8-12 populations of the four varieties of C. terebinthinus and 6 populations of C. petraeus. These taxa occur predominantly in the Western United States, ranging from the Rocky Mountains to the Pacific Coast. I then extracted DNA, prepared libraries, and performed target capture with the angiosperms353 bait kit. Libraries were then sent off for next generation sequencing with Illumina. I used HybPiper and HybPhaser to both assemble 353 target genes and filter poorly recovered loci and paralogs. To assemble chloroplast genomes for each sample, I used MITObim as it assembles circular genomes where other assembly methods do not. I used maximum likelihood (RAxML and IQtree) and coalescent based phylogenetic analyses (Astral) in addition to species delimitation analyses (SODA) to infer evolutionary relationships and taxonomic grouping in C. terebinthinus. I also performed analysis of ecological variables including soil and climatic properties to better understand environmental factors related to phylogenetic groupings. I find that in all phylogenetic analyses other than the chloroplast phylogeny, Cymopterus terebinthinus and its varietal infrataxa comprise a monophyletic clade that includes Cymopterus petraeus. The phylogenetic analysis using chloroplast data resulted in a polyphyletic C. terebinthinus and clade assignments that are inconsistent with other biological evidence. I suspect that the nuclear based phylogenies more accurately depict evolutionary relationships in C. terebinthinus. For the majority of samples analyzed, nuclear phylogenies infer clades that largely correspond to previous varietal assignments. Among all four nuclear phylogenetic estimates, six samples did not comprise a clade with their previously assigned taxa. I suspect that various evolutionary factors that often confound phylogenetic analyses, like incomplete lineage sorting and paralogous genes could explain why these six samples did not comprise a clade with their previously identified variety. Additionally, it is possible that these lineages’ recent divergence has resulted in incomplete barriers to gene flow, leading to clade groupings that are not unanimously aligned to previous taxonomic assignment. Overall, clear genetic differentiation is occurring among the currently recognized varieties and might be related to limited seed dispersal in C. terebinthinus. Limited dispersal mechanisms can cause occasional establishment of allopatric populations with restricted gene flow which can lead to diversification. While the phylogenies inferred in this analysis are generally congruent, and genetic structuring among named varieties suggest divergence is occurring, I suspect that speciation is ongoing in this complex.