Genus-Wide Characterization of Bumblebee Genomes Provides Insights into Their Evolution and Variation in Ecological and Behavioral Traits.

Bumblebees are a diverse group of globally important pollinators in natural ecosystems and for agricultural food production. With both eusocial and solitary life-cycle phases, and some social parasite species, they are especially interesting models to understand social evolution, behavior, and ecology. Reports of many species in decline point to pathogen transmission, habitat loss, pesticide usage, and global climate change, as interconnected causes. These threats to bumblebee diversity make our reliance on a handful of well-studied species for agricultural pollination particularly precarious. To broadly sample bumblebee genomic and phenotypic diversity, we de novo sequenced and assembled the genomes of 17 species, representing all 15 subgenera, producing the first genus-wide quantification of genetic and genomic variation potentially underlying key ecological and behavioral traits. The species phylogeny resolves subgenera relationships, whereas incomplete lineage sorting likely drives high levels of gene tree discordance. Five chromosome-level assemblies show a stable 18-chromosome karyotype, with major rearrangements creating 25 chromosomes in social parasites. Differential transposable element activity drives changes in genome sizes, with putative domestications of repetitive sequences influencing gene coding and regulatory potential. Dynamically evolving gene families and signatures of positive selection point to genus-wide variation in processes linked to foraging, diet and metabolism, immunity and detoxification, as well as adaptations for life at high altitudes. Our study reveals how bumblebee genes and genomes have evolved across the Bombus phylogeny and identifies variations potentially linked to key ecological and behavioral traits of these important pollinators.

Transposable Element Diversity Remains High in Gigantic Genomes.

Transposable elements (TEs) are repetitive sequences of DNA that replicate and proliferate throughout genomes. Taken together, all the TEs in a genome form a diverse community of sequences, which can be studied to draw conclusions about genome evolution. TE diversity can be measured using models for ecological community diversity that consider species richness and evenness. Several models predict TE diversity decreasing as genomes expand because of selection against ectopic recombination and/or competition among TEs to garner host replicative machinery and evade host silencing mechanisms. Salamanders have some of the largest vertebrate genomes and highest TE loads. Salamanders of the genus Plethodon, in particular, have genomes that range in size from 20 to 70 Gb. Here, we use Oxford Nanopore sequencing to generate low-coverage genomic sequences for four species of Plethodon that encompass two independent genome expansion events, one in the eastern clade (Plethodon cinereus, 29.3 Gb vs. Plethodon glutinosus, 38.9 Gb) and one in the western clade (Plethodon vehiculum, 46.4 Gb vs Plethodon idahoensis, 67.0 Gb). We classified the TEs in these genomes and found > 40 TE superfamilies, accounting for 22-27% of the genomes. We calculated Simpson's and Shannon's diversity indices to quantify overall TE diversity. In both pairwise comparisons, the diversity index values for the smaller and larger genome were almost identical. This result indicates that, when genomes reach extremely large sizes, they maintain high levels of TE diversity at the superfamily level, in contrast to predictions made by previous studies on smaller genomes.

Phylogenomics Reveals Ancient Gene Tree Discordance in the Amphibian Tree of Life.

Molecular phylogenies have yielded strong support for many parts of the amphibian Tree of Life, but poor support for the resolution of deeper nodes, including relationships among families and orders. To clarify these relationships, we provide a phylogenomic perspective on amphibian relationships by developing a taxon-specific Anchored Hybrid Enrichment protocol targeting hundreds of conserved exons which are effective across the class. After obtaining data from 220 loci for 286 species (representing 94% of the families and 44% of the genera), we estimate a phylogeny for extant amphibians and identify gene tree-species tree conflict across the deepest branches of the amphibian phylogeny. We perform locus-by-locus genealogical interrogation of alternative topological hypotheses for amphibian monophyly, focusing on interordinal relationships. We find that phylogenetic signal deep in the amphibian phylogeny varies greatly across loci in a manner that is consistent with incomplete lineage sorting in the ancestral lineage of extant amphibians. Our results overwhelmingly support amphibian monophyly and a sister relationship between frogs and salamanders, consistent with the Batrachia hypothesis. Species tree analyses converge on a small set of topological hypotheses for the relationships among extant amphibian families. These results clarify several contentious portions of the amphibian Tree of Life, which in conjunction with a set of vetted fossil calibrations, support a surprisingly younger timescale for crown and ordinal amphibian diversification than previously reported. More broadly, our study provides insight into the sources, magnitudes, and heterogeneity of support across loci in phylogenomic data sets.[AIC; Amphibia; Batrachia; Phylogeny; gene tree-species tree discordance; genomics; information theory.].

Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution.

The Asian tiger mosquito, Aedes albopictus, is a highly successful invasive species that transmits a number of human viral diseases, including dengue and Chikungunya fevers. This species has a large genome with significant population-based size variation. The complete genome sequence was determined for the Foshan strain, an established laboratory colony derived from wild mosquitoes from southeastern China, a region within the historical range of the origin of the species. The genome comprises 1,967 Mb, the largest mosquito genome sequenced to date, and its size results principally from an abundance of repetitive DNA classes. In addition, expansions of the numbers of members in gene families involved in insecticide-resistance mechanisms, diapause, sex determination, immunity, and olfaction also contribute to the larger size. Portions of integrated flavivirus-like genomes support a shared evolutionary history of association of these viruses with their vector. The large genome repertory may contribute to the adaptability and success of Ae. albopictus as an invasive species.

Polymorphic duplicate genes and persistent non-coding sequences reveal heterogeneous patterns of mitochondrial DNA loss in salamanders.

BACKGROUND: Mitochondria are the site of the citric acid cycle and oxidative phosphorylation (OXPHOS). In metazoans, the mitochondrial genome is a small, circular molecule averaging 16.5 kb in length. Despite evolutionarily conserved gene content, metazoan mitochondrial genomes show a diversity of gene orders most commonly explained by the duplication-random loss (DRL) model. In the DRL model, (1) a sequence of genes is duplicated in tandem, (2) one paralog sustains a loss-of-function mutation, resulting in selection to retain the other copy, and (3) the non-functional paralog is eventually deleted from the genome. Despite its apparent role in generating mitochondrial gene order diversity, little is known about the tempo and mode of random gene loss after duplication events. Here, we determine mitochondrial gene order across the salamander genus Aneides, which was previously shown to include at least two DRL-mediated rearrangement events. We then analyze these gene orders in a phylogenetic context to reveal patterns of DNA loss after mitochondrial gene duplication. RESULTS: We identified two separate duplication events that resulted in mitochondrial gene rearrangements in Aneides; one occurred at the base of the clade tens of millions of years ago, while the other occurred much more recently (i.e. within a single species), resulting in gene order polymorphism and paralogs that are readily identifiable. We demonstrate that near-complete removal of duplicate rRNA genes has occurred since the recent duplication event, whereas duplicate protein-coding genes persist as pseudogenes and duplicate tRNAs persist as functionally intact paralogs. In addition, we show that non-coding DNA duplicated at the base of the clade has persisted across species for tens of millions of years. CONCLUSIONS: The evolutionary history of the mitochondrial genome, from its inception as a bacterial endosymbiont, includes massive genomic reduction. Consistent with this overall trend, selection for efficiency of mitochondrial replication and transcription has been hypothesized to favor elimination of extra sequence. Our results, however, suggest that there may be no strong disadvantage to extraneous sequences in salamander mitochondrial genomes, although duplicate rRNA genes may be deleterious.

Small RNAs from a Big Genome: The piRNA Pathway and Transposable Elements in the Salamander Species Desmognathus fuscus.

Most of the largest vertebrate genomes are found in salamanders, a clade of amphibians that includes 686 species. Salamander genomes range in size from 14 to 120 Gb, reflecting the accumulation of large numbers of transposable element (TE) sequences from all three TE classes. Although DNA loss rates are slow in salamanders relative to other vertebrates, high levels of TE insertion are also likely required to explain such high TE loads. Across the Tree of Life, novel TE insertions are suppressed by several pathways involving small RNA molecules. In most known animals, TE activity in the germline is primarily regulated by the Piwi-interacting RNA (piRNA) pathway. In this study, we test the hypothesis that salamanders' unusually high TE loads reflect the loss of the ancestral piRNA-mediated TE-silencing machinery. We characterized the small RNA pool in the female and male adult gonads, testing for the presence of small RNA molecules that bear the characteristics of TE-targeting piRNAs. We also analyzed the amino acid sequences of piRNA pathway proteins from salamanders and other vertebrates, testing whether the overall patterns of sequence divergence are consistent with conserved pathway function across the vertebrate clade. Our results do not support the hypothesis of piRNA pathway loss; instead, they suggest that the piRNA pathway is expressed in salamanders. Given these results, we propose hypotheses to explain how the extraordinary TE loads in salamander genomes could have accumulated, despite the expression of TE-silencing machinery.

DNA transposons have colonized the genome of the giant virus Pandoravirus salinus.

BACKGROUND: Transposable elements are mobile DNA sequences that are widely distributed in prokaryotic and eukaryotic genomes, where they represent a major force in genome evolution. However, transposable elements have rarely been documented in viruses, and their contribution to viral genome evolution remains largely unexplored. Pandoraviruses are recently described DNA viruses with genome sizes that exceed those of some prokaryotes, rivaling parasitic eukaryotes. These large genomes appear to include substantial noncoding intergenic spaces, which provide potential locations for transposable element insertions. However, no mobile genetic elements have yet been reported in pandoravirus genomes. RESULTS: Here, we report a family of miniature inverted-repeat transposable elements (MITEs) in the Pandoravirus salinus genome, representing the first description of a virus populated with a canonical transposable element family that proliferated by transposition within the viral genome. The MITE family, which we name Submariner, includes 30 copies with all the hallmarks of MITEs: short length, terminal inverted repeats, TA target site duplication, and no coding capacity. Submariner elements show signs of transposition and are undetectable in the genome of Pandoravirus dulcis, the closest known relative Pandoravirus salinus. We identified a DNA transposon related to Submariner in the genome of Acanthamoeba castellanii, a species thought to host pandoraviruses, which contains remnants of coding sequence for a Tc1/mariner transposase. These observations suggest that the Submariner MITEs of P. salinus belong to the widespread Tc1/mariner superfamily and may have been mobilized by an amoebozoan host. Ten of the 30 MITEs in the P. salinus genome are located within coding regions of predicted genes, while others are close to genes, suggesting that these transposons may have contributed to viral genetic novelty. CONCLUSIONS: Our discovery highlights the remarkable ability of DNA transposons to colonize and shape genomes from all domains of life, as well as giant viruses. Our findings continue to blur the division between viral and cellular genomes, adhering to the emerging view that the content, dynamics, and evolution of the genomes of giant viruses do not substantially differ from those of cellular organisms.

Low levels of LTR retrotransposon deletion by ectopic recombination in the gigantic genomes of salamanders.

Across the tree of life, species vary dramatically in nuclear genome size. Mutations that add or remove sequences from genomes-insertions or deletions, or indels-are the ultimate source of this variation. Differences in the tempo and mode of insertion and deletion across taxa have been proposed to contribute to evolutionary diversity in genome size. Among vertebrates, most of the largest genomes are found within the salamanders, an amphibian clade with genome sizes ranging from ~14 to ~120 Gb. Salamander genomes have been shown to experience slower rates of DNA loss through small (i.e., <30 bp) deletions than do other vertebrate genomes. However, no studies have addressed DNA loss from salamander genomes resulting from larger deletions. Here, we focus on one type of large deletion-ectopic-recombination-mediated removal of LTR retrotransposon sequences. In ectopic recombination, double-strand breaks are repaired using a "wrong" (i.e., ectopic, or non-allelic) template sequence-typically another locus of similar sequence. When breaks occur within the LTR portions of LTR retrotransposons, ectopic-recombination-mediated repair can produce deletions that remove the internal transposon sequence and the equivalent of one of the two LTR sequences. These deletions leave a signature in the genome-a solo LTR sequence. We compared levels of solo LTRs in the genomes of four salamander species with levels present in five vertebrates with smaller genomes. Our results demonstrate that salamanders have low levels of solo LTRs, suggesting that ectopic-recombination-mediated deletion of LTR retrotransposons occurs more slowly than in other vertebrates with smaller genomes.

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development.

BACKGROUND: Chromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution--some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 - 75 Gb, 12-74 Gb of which are lost from pre-somatic cell lineages at germline--soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown. RESULTS: Here, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome. CONCLUSIONS: Our results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms.

Zebrafish tbx5 paralogs demonstrate independent essential requirements in cardiac and pectoral fin development.

BACKGROUND: T-box genes constitute a large family of transcriptional regulators involved in developmental patterning. Homozygous mutation of tbx5 leads to embryonic lethal cardiac phenotypes and forelimb malformations in vertebrate models. Haploinsufficiency of tbx5 results in Holt-Oram syndrome, a human congenital disease characterized by cardiac and forelimb defects. Homozygous mutation of zebrafish tbx5a leads to lethal defects in cardiac looping morphogenesis, blocks pectoral fin initiation, and impairs outgrowth. Recently, a second zebrafish tbx5 gene was described, termed tbx5b. RESULTS: Our phylogenetic analyses confirm tbx5b as a paralog that likely arose in the teleost-specific whole genome duplication ∼270 MYA. Using morpholino depletion studies, we find that tbx5b is required in the heart for embryonic survival, and influences the timing and morphogenesis of pectoral fin development. Because tbx5a hypomorphic mutations are embryonic lethal, tbx5a and tbx5b functions in the heart must not be completely redundant. Consistent with this hypothesis, simultaneous depletion of both tbx5 paralogs did not lead to more severe phenotypes, and injection of wild-type mRNA from one tbx5 paralog was not sufficient to cross-rescue phenotypes of the paralogous gene. CONCLUSIONS: Collectively, these data indicate that, despite similar spatio-temporal expression patterns, tbx5a and tbx5b have independent functions in heart and fin development.

Gigantic animal cells suggest organellar scaling mechanisms across a 50-fold range in cell volume.

The largest cells are orders of magnitude bigger than the smallest cells. Organelle content scales to maintain cell function, with different organelles increasing in volume, length, or number as cells increase in size. Scaling may also reflect functional demands placed on organelles by increased cell size. Amphibians exhibit exceptional diversity in cell size. Using transmission electron microscopy, we analyzed 3 species whose enterocyte cell volumes range from 228 to 10,593 µm3. We show that nuclear volume increases by an increase in radius while mitochondrial volume increases by an increase in total network length; the endoplasmic reticulum and Golgi apparatus, with their complex shapes, are intermediate. Notably, all 4 organelle types increase in total volume proportional to cell volume, despite variation in functional (i.e., metabolic, transport) demands. This pattern suggests that organellar building blocks are incorporated into more or larger organelles following the same rules across species that vary ~50-fold in cell sizes, consistent with a "limited precursor" model for organellar scaling that, in turn, assumes equivalent cytoplasmic concentrations of organellar building block proteins. Taken together, our results lead us to hypothesize that salamanders have evolved increased biosynthetic capacity to maintain functional protein concentrations despite huge cell volumes.

Transposable element and host silencing activity in gigantic genomes.

Transposable elements (TEs) and the silencing machinery of their hosts are engaged in a germline arms-race dynamic that shapes TE accumulation and, therefore, genome size. In animal species with extremely large genomes (>10 Gb), TE accumulation has been pushed to the extreme, prompting the question of whether TE silencing also deviates from typical conditions. To address this question, we characterize TE silencing via two pathways-the piRNA pathway and KRAB-ZFP transcriptional repression-in the male and female gonads of Ranodon sibiricus, a salamander species with a ∼21 Gb genome. We quantify 1) genomic TE diversity, 2) TE expression, and 3) small RNA expression and find a significant relationship between the expression of piRNAs and TEs they target for silencing in both ovaries and testes. We also quantified TE silencing pathway gene expression in R. sibiricus and 14 other vertebrates with genome sizes ranging from 1 to 130 Gb and find no association between pathway expression and genome size. Taken together, our results reveal that the gigantic R. sibiricus genome includes at least 19 putatively active TE superfamilies, all of which are targeted by the piRNA pathway in proportion to their expression levels, suggesting comprehensive piRNA-mediated silencing. Testes have higher TE expression than ovaries, suggesting that they may contribute more to the species' high genomic TE load. We posit that apparently conflicting interpretations of TE silencing and genomic gigantism in the literature, as well as the absence of a correlation between TE silencing pathway gene expression and genome size, can be reconciled by considering whether the TE community or the host is currently "on the attack" in the arms race dynamic.

Gigantic genomes of salamanders indicate that body temperature, not genome size, is the driver of global methylation and 5-methylcytosine deamination in vertebrates.

Transposable elements (TEs) are sequences that replicate and move throughout genomes, and they can be silenced through methylation of cytosines at CpG dinucleotides. TE abundance contributes to genome size, but TE silencing variation across genomes of different sizes remains underexplored. Salamanders include most of the largest C-values - 9 to 120 Gb. We measured CpG methylation levels in salamanders with genomes ranging from 2N = ∼58 Gb to 4N = ∼116 Gb. We compared these levels to results from endo- and ectothermic vertebrates with more typical genomes. Salamander methylation levels are approximately 90%, higher than all endotherms. However, salamander methylation does not differ from other ectotherms, despite an approximately 100-fold difference in nuclear DNA content. Because methylation affects the nucleotide compositional landscape through 5-methylcytosine deamination to thymine, we quantified salamander CpG dinucleotide levels and compared them to other vertebrates. Salamanders and other ectotherms have comparable CpG levels, and ectotherm levels are higher than endotherms. These data show no shift in global methylation at the base of salamanders, despite a dramatic increase in TE load and genome size. This result is reconcilable with previous studies that considered endothermy and ectothermy, which may be more important drivers of methylation in vertebrates than genome size.

Genome size drives morphological evolution in organ-specific ways.

Morphogenesis is an emergent property of biochemical and cellular interactions during development. Genome size and the correlated trait of cell size can influence these interactions through effects on developmental rate and tissue geometry, ultimately driving the evolution of morphology. We tested whether variation in genome and body size is related to morphological variation in the heart and liver using nine species of the salamander genus Plethodon (genome sizes 29-67 gigabases). Our results show that overall organ size is a function of body size, whereas tissue structure changes dramatically with evolutionary increases in genome size. In the heart, increased genome size is correlated with a reduction of myocardia in the ventricle, yielding proportionally less force-producing mass and greater intertrabecular space. In the liver, increased genome size is correlated with fewer and larger vascular structures, positioning hepatocytes farther from the circulatory vessels that transport key metabolites. Although these structural changes should have obvious impacts on organ function, their effects on organismal performance and fitness may be negligible because low metabolic rates in salamanders relax selective pressure on function of key metabolic organs. Overall, this study suggests large genome and cell size influence the developmental systems involved in heart and liver morphogenesis.

Gigantic Genomes Provide Empirical Tests of Transposable Element Dynamics Models.

Transposable elements (TEs) are a major determinant of eukaryotic genome size. The collective properties of a genomic TE community reveal the history of TE/host evolutionary dynamics and impact present-day host structure and function, from genome to organism levels. In rare cases, TE community/genome size has greatly expanded in animals, associated with increased cell size and changes to anatomy and physiology. Here, we characterize the TE landscape of the genome and transcriptome in an amphibian with a giant genome - the caecilianIchthyophis bannanicus, which we show has a genome size of 12.2 Gb. Amphibians are an important model system because the clade includes independent cases of genomic gigantism. The I. bannanicus genome differs compositionally from other giant amphibian genomes, but shares a low rate of ectopic recombination-mediated deletion. We examine TE activity using expression and divergence plots; TEs account for 15% of somatic transcription, and most superfamilies appear active. We quantify TE diversity in the caecilian, as well as other vertebrates with a range of genome sizes, using diversity indices commonly applied in community ecology. We synthesize previous models that integrate TE abundance, diversity, and activity, and test whether the caecilian meets model predictions for genomes with high TE abundance. We propose thorough, consistent characterization of TEs to strengthen future comparative analyses. Such analyses will ultimately be required to reveal whether the divergent TE assemblages found across convergent gigantic genomes reflect fundamental shared features of TE/host genome evolutionary dynamics.

Spatio-temporal regulation of Wnt and retinoic acid signaling by tbx16/spadetail during zebrafish mesoderm differentiation.

BACKGROUND: A complex network of signaling pathways and transcription factors regulates vertebrate mesoderm development. Zebrafish mutants provide a powerful tool for examining the roles of individual genes in such a network. spadetail (spt) is a mutant with a lesion in tbx16, a T-box transcription factor involved in mesoderm development; the mutant phenotype includes disrupted primitive red blood cell formation as well as disrupted somitogenesis. Despite much recent progress, the downstream targets of tbx16 remain incompletely understood. The current study was carried out to test whether any of the five major signaling pathways are regulated by tbx16 during two specific stages of mesoderm development: primitive red blood cell formation in the intermediate mesoderm and somite formation in the tail paraxial mesoderm. This test was performed using Gene Set Enrichment Analysis, which identifies coordinated changes in expression among a priori sets of genes associated with biological features or processes. RESULTS: Our Gene Set Enrichment Analysis results identify Wnt and retinoic acid signaling as likely downstream targets of tbx16 in the developing zebrafish intermediate mesoderm, the site of primitive red blood cell formation. In addition, such results identify retinoic acid signaling as a downstream target of tbx16 in the developing zebrafish posterior somites. Finally, using candidate gene identification and in situ hybridization, we provide expression domain information for 25 additional genes downstream of tbx16 that are outside of both pathways; 23 were previously unknown downstream targets of tbx16, and seven had previously uncharacterized expression in zebrafish. CONCLUSIONS: Our results suggest that (1) tbx16 regulates Wnt signaling in the developing zebrafish intermediate mesoderm, the site of primitive red blood cell formation, and (2) tbx16 regulates retinoic acid signaling at two distinct embryonic locations and developmental stages, which may imply ongoing spatio-temporal regulation throughout mesoderm development.

Limited effects of among-lineage rate variation on the phylogenetic performance of molecular markers.

Variation in substitution rates among evolutionary lineages (among-lineage rate variation or ALRV) has been reported to negatively affect the estimation of phylogenies. When the substitution processes underlying ALRV are modeled inadequately, non-sister taxa with similar substitution rates are estimated incorrectly as sister species due to long-branch attraction. Recent advances in modeling site-specific rate variation (heterotachy) have reduced the impacts of ALRV on phylogeny estimation in several empirical and simulated datasets. However, the addition of parameters to the substitution model reduces power to estimate each parameter correctly, which can also lead to incorrect phylogeny estimation. A potential solution to this problem is to identify the levels of ALRV that negatively impact phylogeny estimation such that molecular markers with non-deleterious levels of ALRV can be identified. To this end, we used analyses of empirical and simulated gene datasets to evaluate whether levels of ALRV identified in a mitochondrial genomic dataset for salamanders negatively impacted phylogeny estimation. We simulated data with and without ALRV, holding all other evolutionary parameters constant, and compared the phylogenetic performance of both simulated and empirical datasets. Overall, we found limited, positive effects of ALRV on phylogeny estimation in this dataset, the majority of which resulted from an increase in substitution rate on short branches. We conclude that ALRV does not always negatively impact phylogeny estimation. Therefore, ALRV can likely be disregarded as a criterion for marker selection in comparable phylogenetic studies.